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Immune Evasion by Mycobacterium tuberculosis: Living with the Enemy
Abstract
Mycobacterium tuberculosis is successful as a pathogen because of its ability to persist in an immunocompetent host. This bacterium lives within the macrophage, a cell whose function is the elimination of microbes. Recent advances have improved our understanding of how M. tuberculosis evades two major antimicrobial mechanisms of macrophages: phagolysosome fusion and the production of toxic reactive nitrogen intermediates. M. tuberculosis also modulates antigen presentation to prevent the detection of infected macrophages by CD4(+) T cells.
... The synonyms for Mycobacterium tuberculosis (MTB) include: Koch bacillus and tubercle bacillus. Recognized as the [4] world's leading bacterial cause of death, TB [1] remains a threat to the health of people worldwide. ...
... It can take 2 to 8 weeks after the initial TB infection for the body's immune system to be able to react to tuberculin and for the infection to be detected by the TST or IGRA. The formation of granuloma walls off the bacilli from the rest of the lung tissue, but within the granuloma, cells of the immune system continue with efforts to clear the bacteria but some resistant bacteria escape killing and enter a state of dormancy and persist by [4,14,15] avoiding elimination by the immune system. During latent tuberculosis infection (LTBI) there is persistent immune response to stimulation by Mycobacterium tuberculosis antigen without clinical manifestations of active TB and it can be maintained for a life time. ...
Tuberculosis (TB) is an infectious disease of bacterial origin caused by Mycobacterium tuberculosis (MTB), which is a member of the Mycobacterium tuberculosis complex (MTBC). Mycobacterium tuberculosis (MTB) is also known as Koch bacillus or tubercle bacillus. The burden of TB has remained a problem due to factors that have promoted and fostered transmission, including increasing resistance of the disease to the most effective first-line anti-TB drugs. There is the need for cases to be diagnosed early and managed appropriately by skilled and knowledgeable health care workers (HCWs). Reports of low level of knowledge concerning TB among some HCWs during the 2016 hajj prompted this review which was prepared using articles on TB searched on various websites of international institutions like the world health organization (WHO), the United States center for disease control and prevention (CDC) and PubMed. Here we provide a brief history of tuberculosis and an overview of the current literature on, basic classification of, immunology, public health concerns and treatment guidelines of TB. The information provided will be a useful guide for HCWs and the general public.
... After infection with Mycobacterium tuberculosis, host cytokine responses influence disease manifestations. 3 Differences in cytokine expression may determine whether TB worsens, resolves, or becomes latent. In particular, the balance between Th1 and Th2 cytokine responses may influence disease expression in individuals with pulmonary TB. ...
... Th2 cytokines are usually involved in antibody-mediated humoral immunity with limited protective effects in intracellular M. tuberculosis infection. 3 During primary infection, IFN-γproducing cells are continuously activated. ...
Background: Tuberculosis (TB) is a mycobacterial infection that usually attacks the lungs and triggers lung damage and dysfunction related to the reléase of IL-17, Erythrocyte Sedimentation Rate (ESR), and C-Reactive Protein (CRP). This study aims to evaluate the correlation between serum IL-17 levels with ESR and CRP levels in new cases of pulmonary tuberculosis at Sanglah Central General Hospital, Bali, Indonesia. Methods: A cross-sectional study was conducted among 56 TB patients treated as new pulmonary TB cases in Outpatient and Inpatient Internal Medicine at Sanglah Hospital, Bali, Indonesia. IL-17 levels in this study were the results of IL-17 examination from patient serum using the ELISA method from R&Dsystems with units of pg/mL. The Erythrocyte Sedimentation Rate (ESR) in this study is the rate of blood deposition in mm/hour using a Westergreen tube. Meanwhile, the C-Reactive Protein (CRP) examination was carried out using a Roche 6000 analyzer in mg/dL. Data were analyzed with SPSS version 16 for Windows. Results: Most respondents are female (75.0%) and the 30-34 years old group (26.8%). The mean value of ESR is 40.39±25.86 mm/hour, followed by CRP (9.95±10.01 mg/dL), and IL-17 (15.21±1.20 pg/mL). There is no significant correlation between ESR to CRP (r=0.069; p=0.613) and IL-17 (r=0.135; p=0.323). In addition, there is no significant correlation between CRP and IL-17 (r=0.168; p=0.215). Conclusion: This study's results indicate no significant correlation between IL-17 levels with CRP and ESR or between CRP and ESR.
... This allows the surviving bacilli to replicate extracellularly and spread to other parts of the body besides the lungs through the lymphatic and blood system (40). In latent tuberculosis, Mtb bacilli are able to evade the intense host immune defenses, survive the stressful microenvironment and progress to a dormancy state while resisting the eradication from the immune system (41). The bacilli remain dormant until there are opportunities for reactivation which in most cases are due to immunosuppression or weaken immune system due to infections including HIV and COVID-19, malnutrition, tobacco smoke, air pollution, alcoholism, diabetes, kidney failure and malignancy (15,41,42). ...
... In latent tuberculosis, Mtb bacilli are able to evade the intense host immune defenses, survive the stressful microenvironment and progress to a dormancy state while resisting the eradication from the immune system (41). The bacilli remain dormant until there are opportunities for reactivation which in most cases are due to immunosuppression or weaken immune system due to infections including HIV and COVID-19, malnutrition, tobacco smoke, air pollution, alcoholism, diabetes, kidney failure and malignancy (15,41,42). ...
2020 will be marked in history for the dreadful implications of the COVID-19 pandemic that shook the world globally. The pandemic has reshaped the normality of life and affected mankind in the aspects of mental and physical health, financial, economy, growth, and development. The focus shift to COVID-19 has indirectly impacted an existing air-borne disease, Tuberculosis. In addition to the decrease in TB diagnosis, the emergence of the TB/COVID-19 syndemic and its serious implications (possible reactivation of latent TB post-COVID-19, aggravation of an existing active TB condition, or escalation of the severity of a COVID-19 during TB-COVID-19 coinfection), serve as primary reasons to equally prioritize TB. On a different note, the valuable lessons learnt for the COVID-19 pandemic provide useful knowledge for enhancing TB diagnostics and therapeutics. In this review, the crucial need to focus on TB amid the COVID-19 pandemic has been discussed. Besides, a general comparison between COVID-19 and TB in the aspects of pathogenesis, diagnostics, symptoms, and treatment options with importance given to antibody therapy were presented. Lastly, the lessons learnt from the COVID-19 pandemic and how it is applicable to enhance the antibody-based immunotherapy for TB have been presented.
... Macrophages can also produce reactive nitrogen intermediates (RNI) (Flynn & Chan 2003) or reactive oxygen intermediates (ROI) (Rosenvasser et al. 2006); the two effector systems cooperate to destroy the pathogen (Tomioka et al. 1997). Cytokines increase the acidification and maturation o f phagosomes containing mycobacteria, promoting the intracellular killing o f bacteria (Hostetter et al. 2002). ...
... tuberculosis and several genes, such as M. tb noxRl and noxR3, encode for the enzymes responsible for this effect (Flynn & Chan 2003). ...
Background: The development of new treatments for tuberculosis is a public health priority. We have shown that a South African native remedy for respiratory ailments, based on extracts of Pelargonium reniforme and P. sidoides, contain fatty acids with antimycobacterial activity. In order to further assess their potential as a supplement to conventional therapy, we examined root extracts of these plants for the capacity to stimulate the uptake and intracellular killing of mycobacteria by macrophages. Methods: Murine peritoneal macrophages were obtained by lavage and incubated with Mycobacterium fortuitum. Extract-mediated stimulation of bacterial uptake was determined by enumeration of stained intracellular bacteria; the effect of extracts on intracellular killing was evaluated by spread plate counting of bacteria within macrophages. Bioactive extracts, obtained by sequential extraction with solvents of increasing polarity, were subjected to bioassay-guided fractionation using size exclusion and ion exchange chromatography. HPLC, NMR and mass spectroscopy were used to identify compounds of interest. Active compounds were examined for their capacity to stimulate macrophage uptake of M. tuberculosis. Results: Root powders were sequentially extracted with hexane, ethyl acetate, ethanol, 50% ethanol/water and water; aqueous fractions had a greater capacity to stimulate uptake and killing of M. fortuitum compared to other fractions. P. reniforme extracts possessed significantly greater stimulating activity than P. sidoides extracts. Fractions obtained by bioassay-guided fractionation of P. reniforme aqueous extracts contained a small number of chemically distinct structures, the most prominent being gallic acid, methyl gallate, myricetin and quercetin 3-D-glucoside. Highly pure preparations of these compounds (2-25 μg/ml) stimulated macrophage killing of M fortuitum and M tuberculosis. Conclusion: Representatives of the plant genus Pelargonium elaborate metabolites that increase the capacity of mice peritoneal macrophages to internalize and kill mycobacteria, including M. tuberculosis.
... Much of our knowledge of the function of cytokines has been derived from studies wherein homeostasis has been dis-rupted by infection and the absence of specific cytokines results in a failure to control the disease process. In this context, infection with Mycobacterium tuberculosis has proven to be very informative and has highlighted the role of cytokines in controlling infection without promoting uncontrolled and damaging inflammatory responses (2)(3)(4). Herein, we focus on the key cytokine and chemokines that have been studied in the context of human TB using experimental medicine as well as M. tuberculosis infection of various animal models, including non-human primates (NHPs), mice, and rabbits. Perhaps the most important message of this review is that in a complex disease such as TB the role of any one cytokine cannot be designated either "good" or "bad" but rather that cytokines can elicit both protective and pathologic consequences depending on context. ...
... IL-23 also plays a chemokine-dependent role in the efficient expression of vaccine-induced mucosal immunity. This role was first highlighted in mice subcutaneously vaccinated with an adjuvant-paired I-A b -restricted ESAT6 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) peptide, which induces both IFNγ-and IL-17-producing antigen-specific CD4 + T cell responses (157). Critically, the improved kinetics of the vaccineinduced IFNγ-producing T cells is lost in the absence of IL-23, because this cytokine is required for the generation of lung resident IL-17 producing CD4 + memory T cells that generate a chemokine gradient facilitating the accelerated IFNγ response. ...
... Several hypotheses have been suggested to explain the extraordinary capacity of Mtb to cause disease in susceptible individuals and great emphasis has been given to its capacity to evade host immune responses [20,21]. ...
... Mtb that infects a single individual [22]. Thus, a variety of alternative mechanisms have been proposed and in vitro proven to explain the survival of Mtb within the macrophage and its escape from immunorecognition, such as the inhibition of phagosome-lysosome fusion, phagosome acidification and the resistance to killing by oxygenated metabolites [11,21]. Thus, by interfering with its intracellular degradation, Mtb would substantially block the processing of its antigens, the loading of immuno-dominant peptides onto MHC class II molecules, and/or the transport of MHC-peptide complexes to the cell surface [12] resulting hidden to T cell recognition. ...
... However, certain pathogens hijack the alveolar macrophages by inhibiting the acidification of the phagosome. For example, Mycobacterium tuberculosis (Mtb) secretes tyrosine phosphatase PtpA, that inactivates the host vacuolar ATPase, and consequently phagosomal acidification, creating a place for the bacteria to reside and persists [72]. Aryl-based synthetic mimics of antimicrobial peptides (SMAMPs) with antimicrobial activity were found to perform immunomodulatory activities in macrophages both in the presence and in the absence of lipopolysaccharide. ...
Skin is the primary and largest protective organ of the human body. It produces a number of highly evolved arsenal of factors to counter the continuous assault of foreign materials and pathogens from the environment. One such potent factor is the repertoire of Antimicrobial Peptides (AMPs) that not only directly destroys invading pathogens, but also optimally modulate the immune functions of the body to counter the establishment and spread of infections. The canonical direct antimicrobial functions of these AMPs have been in focus for a long time to design principles for enhanced therapeutics, especially against the multi-drug resistant pathogens. However, in recent times the immunomodulatory functions performed by these peptides at sub-microbicidal concentrations have been a point of major focus in the field of host-directed therapeutics. Such strategies have the added benefit of not having the pathogens develop resistance against the immunomodulatory pathways, since the pathogens exploit these signaling pathways to obtain and survive within the host. Thus, this review summarizes the potent immunomodulatory effect of these AMPs on, specifically, the different host immune cells with the view of providing a platform of information that might help in designing studies to exploit and formulate effective host-directed adjunct therapeutic strategies that would synergies with drug regimens to counter the current diversity of drug-resistant skin opportunistic pathogens.
... The Ure gene is also expressed in the bacteria which maintains the phagosome in a deacidified state [16] . Production of diterpene isotuberculosinol and neutralization of reactive nitrogen intermediates are some other mechanisms involved in their survival [17] . ...
The objective of this research project is to carry out the characterization of the Fic protein present
in Mycobacterium smegmatis. Through the study of its gene sequence it was possible to identify,
with established reference, a toxin-antitoxin system. Upon identifying the toxin as well as antitoxin
domains, we were now capable of identifying key residues and wished to see their role in the
protein function. By causing intentional mutagenesis (site directed mutagenesis by single primer
method) we constructed mutants which were structurally identical to the wild type protein but were
hindered functionally. We cloned these inserts into a suitable vector that was driven by T7RNA
polymerase and was capable of showing expression in E. coli as well as M. smegmatis. By
overexpression of the Fic gene (and the relevant mutants) in a suitable bacterial strain and by
optimizing the protein purification process, we were able to facilitate maximum protein
production. Growth curve studies were carried out to check the effect of this Fic protein on the
growth of the E. coli cells. These clones were transformed into M. smegmatis to check for the
protein’s influence on its growth as well as morphology but we were incapable of achieving
overexpression of the protein within the cell. However, our attempts to crystallize the wild type
protein proved successful. Having concentrated the protein in a few different buffers we were able
to carry out CS1 and CS2 set ups for crystallization. We obtained hits in specific buffer conditions.
Additionally, Circular dichroism (CD) studies showed us the distribution of the alpha and beta
forms in the protein. We carried out Thermal Shift assays (TSA) which showed us the stability of
the wild type and mutant proteins. Using autoradiography, the wild type protein was checked for
the process of AMPylation, a post transcriptional modification it is originally famous for. By not
providing the substrate necessary for this reaction we noticed that the protein was capable of
addition of AMP on of its own amino acids. This auto-AMPylation reinstates that the protein is
functionally sound. Additionally, our attempts to crystallize the wild type protein proved
successful.
... M. tb is an obligate human pathogen that survives and replicates in host macrophages inside a modified phagosomal compartment, thus evading macrophage killing by neutralizing RNIs [62,63]. As previously described, infection with M. tb begins with phagocytosis of the pathogen by antigen-presenting cells such as dendritic cells [64]. ...
Tuberculosis (TB) is a leading cause of mortality due to infectious disease and rates have increased during the emergence of COVID-19, but many of the factors determining disease severity and progression remain unclear. Type I Interferons (IFNs) have diverse effector functions that regulate innate and adaptive immunity during infection with microorganisms. There is well-documented literature on type I IFNs providing host defense against viruses; however, in this review, we explore the growing body of work that indicates high levels of type I IFNs can have detrimental effects to a host fighting TB infection. We report findings that increased type I IFNs can affect alveolar macrophage and myeloid function, promote pathological neutrophil extracellular trap responses, inhibit production of protective prostaglandin 2, and promote cytosolic cyclic GMP synthase inflammation pathways, and discuss many other relevant findings.
... Most such studies are focussed on M.tb and macrophage interaction. The bacteria interfere with the trafficking pathways within the macrophage to inhibit its degradation via lysosomal fusion and autophagy (Flynn and Chan, 2003). Macrophages secrete various pro-inflammatory cytokines and chemokines that attract various mononuclear cells including T cells to contain the bacteria within the granuloma. ...
Mycobacterium tuberculosis (M.tb) is an intracellular pathogen that predominantly affects the alveolar macrophages in the respiratory tract. Upon infection, the activation of TLR2 and TLR4- mediated signaling pathways leads to lysosomal degradation of the bacteria. However, bacterium counteracts the host immune cells and utilizes them as a cellular niche for its survival. One distinctive mechanism of M.tb to limit the host stress responses such as hypoxia and nutrient starvation is induction of dormancy. As the environmental conditions become favorable, the bacteria resuscitate, resulting in a relapse of clinical symptoms. Different bacterial proteins play a critical role in maintaining the state of dormancy and resuscitation, namely, DevR (DosS), Hrp1, DATIN and RpfA-D, RipA, etc., respectively. Existing knowledge regarding the key proteins associated with dormancy and resuscitation can be employed to develop novel therapies. In this review we aim to highlight the current knowledge of bacterial progression from dormancy to resuscitation and the gaps in understanding the transition from dormant to active state. We have also focused on elucidating a few therapeutic strategies employed to prevent M.tb resuscitation.
... In another study, a greater phagocytic activity of macrophages was reported in female wall lizards compared with males (25). The ability of Mycobacterium species to survive in macrophages allow the bacteria to avoid detection and escape the immune system of the host (26). We could then speculate that female lizards, due to their increased capacity to phagocytize mycobacteria, could harbor more dormant organisms than males and be more prone to a systemic infection under stressful conditions that suppress immunity. ...
A commercial breeding colony of bearded dragons (Pogona vitticeps) experienced an increase in mortality that affected females only. Before death, the animals had lost appetite and weight, were dehydrated, and some had labored breathing. Necropsy revealed granulomas in many organs (ovaries, lungs, liver, kidneys, heart, bone marrow) in which numerous acid-fast bacteria were identified. Bacterial isolation confirmed Mycobacterium spp., which was identified by whole genome sequencing as closely related to the Mycobacterium ulcerans-marinum complex. Due to the zoonotic potential of this bacterium and the poor prognosis for the remaining sick animals, the entire colony was culled and 7 animals were evaluated. The possible routes for introduction of this bacterium, the female predisposition to the disease, as well as the zoonotic potential of this microorganism are discussed. Key clinical message: An atypical Mycobacterium species closely related to Mycobacterium ulcerans-marinum complex can cause high female morality in captive bearded dragons.
... As intracellular bacteria, M. bovis BCG are phagocytosed by macrophages, and the mycobacterial antigens subsequently processed and presented via a MHC class II-restricted pathway, to favor the activation of the CD4 T cell arm of immunity. Such CD4 T-cell responses are associated with the production and secretion of T-helper type 1 (Th1) cytokines, which may have bactericidal effects by activation of nitric oxide (NO) production in Mtb infected cells (17)(18)(19). It has also been shown that both Mtb and M. bovis BCG can arrest phagosome maturation, which again will prevent antigen presentation and immunity (20)(21)(22)(23). ...
Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis . The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1β in vivo . Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.
... Mycobacterium tuberculosis (M. tuberculosis), the causative pathogen of tuberculosis, has developed a complex mechanism to combating immune reaction (Flynn and Chan, 2003). The unique composition of M. tuberculosis cell wall is a key factor for its survival in the infected host. ...
Ser/Thr phosphorylation by serine/threonine protein kinases (STPKs) plays significant roles in molecular regulation, which allows Mycobacteria to adapt their cell wall structure in response to the environment changes. Identifying direct targets of STPKs and determining their activities are therefore critical to revealing their function in Mycobacteria , for example, in cell wall formation and virulence. Herein, we reported that RmlA, a crucial L -rhamnose biosynthesis enzyme, is a substrate of STPK PknB in Mycobacterium tuberculosis ( M. tuberculosis ). Mass spectrometry analysis revealed that RmlA is phosphorylated at Thr-12, Thr-54, Thr-197, and Thr-12 is located close to the catalytic triad of RmlA. Biochemical and phenotypic analysis of two RmlA mutants, T12A/T12D, showed that their activities were reduced, and cell wall formation was negatively affected. Moreover, virulence of RmlA T12D mutant was attenuated in a macrophage model. Overall, these results provide the first evidence for the role of PknB-dependent RmlA phosphorylation in regulating cell wall formation in Mycobacteria , with significant implications for pathogenicity.
... A single cavitation sputum holds about 1 million bacilli per milliliter. The problem in cavitation is that it affects the healthy tissue and progresses the destruction process [18,19]. So, direct drug delivery to the lungs is a successful possibility for TB treatment. ...
The Mycobacterium tuberculosis bacterium causes pulmonary tuberculosis (TB), and it is the second-deadliest infectious agent after HIV. Several antitubercular drugs are used for treatment. The frequent utilization of various drugs and their combination action is the reason for the development of drug-resistant tuberculosis. So there is a need for an effective drug-delivery system to control drug-resistant strains while also reducing the treatment regimen and side effects. A nano drug-delivery regimen is more effective at controlling TB while also being a promising delivery system for drug administration in various routes. It also controlled the release, reduced the dosage, increased the half-life, had a higher drug concentration at target sites, reduced toxicity, and lessened side effects. This chapter emphasizes various nanotechnology-based therapies that can be used for antitubercular drug delivery.
... The Mtb secretes bacterial proteins ATP1/2 (secretion ATPase1/2, secreted secA1/2 protein) and early secretory antigen-6/culture filtrate protein to inhibit the accumulation of vacuolar ATP and GTP enzymes, therefore decreasing intracellular pH, which results in the suppression of phagocyte maturation (Rohde et al., 2007;Zhai et al., 2019). Mtb also supresses the formation of lysosomes by inducing an increase in the expression of the coronin 1 protein on the host phagocyte membrane; the extent of the recruitment process and the amount of coronin 1 are proportional to the amount of activated Mtb in the microsomes (Schuller et al., 2001;Flynn and Chan, 2003;Zhai et al., 2019). ...
The major causative agent of tuberculosis (TB), i.e., Mycobacterium tuberculosis (Mtb) , has developed mechanisms to evade host defense responses and persist within host cells for prolonged periods of time. Mtb is also increasingly resistant to existing anti-TB drugs. There is therefore an urgent need to develop new therapeutics for TB and host directed therapies (HDTs) hold potential as effective therapeutics for TB. There is growing interest in the induction of autophagy in Mtb host cells using autophagy inducing compounds (AICs). Nanoparticles (NPs) can enhance the effect of AICs, thus improving stability, enabling cell targeting and providing opportunities for multimodal therapy. In this review, we focus on the macrophage responses to Mtb infection, in particular, the mechanistic aspects of autophagy and the evasion of autophagy by intracellular Mtb . Due to the overlap between the onset of autophagy and apoptosis; we also focus on the relationship between apoptosis and autophagy. We will also review known AICs in the context of Mtb infection. Finally, we discuss the applications of NPs in inducing autophagy with the intention of sharing insights to encourage further research and development of nanomedicine HDTs for TB therapy.
... Latent individuals will develop into active tuberculosis patients due to weak immune system when aged or threatened by other disease, such as AIDS virus infection. As a intracellular pathogen, M.tb has evolved specific strategies to escape being killed and survive inside host macrophages [4,5], such as resistance to reactive oxygen species (ROS) and reactive nitrogen species (RNS) [6,7], inhibition of host cell apoptotic pathways [8], and limiting of phagosome maturation and phago-lysosome fusion [9]. Above all, blocking the fusion of phagosomes with late endosomes or lysosomes is a main reason for M.tb survival within macrophages [10]. ...
The survivability of Mycobacterium tuberculosis (M.tb) in macrophages in granuloma is a predominant cause for tuberculosis (TB) infection and recurrence. However, the mechanism of mycobacterial clearance in macrophages still needs further study. Here, we explored a novel role of B and T lymphocyte Attenuator (BTLA) in macrophage-mediated host defense against mycobacterial infection. We found that the surface expression of BTLA was increased in CD14⁺ monocytes from active TB patients. The mRNA levels of BTLA were induced in human and mice monocytes/macrophages during Mycobacterium bovis BCG or M.tb H37Rv infection, as well as spleen and lung of H37Rv-infected mice. Furthermore, silencing of BTLA promoted the intracellular survival of BCG and H37Rv by suppressing the autophagy in macrophages but not effecting phagocytosis, reactive oxygen species (ROS) and apoptosis. Silence of BTLA reduced bacterial-autophagosome and bacterial-lysosome colocalization. Moreover, BTLA inhibited AKT and mTOR signaling substrates S6K and 4EBP1 phosphorylation in BCG and H37Rv infected macrophages, and BTLA-mediated AKT-mTOR signaling and intracellular BCG survival were reversed by PI3K inhibitors in macrophages. Finally, treatment with BTLA agonist ameliorated lung pathology and promoted autophagy and mycobacterial clearance during mycobacterial infection in vivo. These results demonstrate that BTLA promotes host defense against mycobacteria by enhancing autophagy, which may provide potential therapeutic interventions against tuberculosis.
... M2 phenotype is associated with TB, leprosy and other chronic inflammatory diseases such as asthma and parasitic infections, but also with resolution of non-infectious inflammatory lesions. Mtb virulence factors such as ESAT-6 of ESX-1 system apparently drive M2 polarisation (Flynn and Chan, 2003;Russel et al., 2009;Gupta et al., 2012). A critical macrophage polarisation factor is transcription factor Kruppel-like factor 4 (KLF4), which belongs to zinc-finger family proteins (Liao et al., 2011) (Fig. 1). ...
... When M. TB enters the macrophage, it becomes confined in a membrane-bound vesicle, known as phagosome [5]. M. TB, a highly successful bacillus, has developed numerous strategies to overcome critical innateeffector immune responses of macrophages such as fusion of phagosome with the lysosome, presentation of the antigen, autophagy, and inhibition of reactive oxygen and nitrogen species (ROS and RNS) to ensure its longer survival inside the macrophage [3,[15][16]. For instance, this phagosome maturation arrest increases the life span of the respective macrophages [5]. ...
Tuberculosis (TB) is a chronic infection caused by Mycobacterium tuberculosis (M. TB). It is transmitted through respiratory droplets. Increased cholesterol level is a predisposing factor for TB. M. TB uses cholesterol in the host macrophage membranes to bind and enter the macrophages. Statins are the drugs that are prescribed to hyperlipidemic patients to maintain their lipid levels in the normal range, thereby reducing the risk of stroke and cardiovascular events. Moreover, statins aid in reducing the levels of cholesterol in human macrophages. Therefore, a reduction in the membrane cholesterol minimizes the entry of TB pathogen inside macrophages. Furthermore, acting as vitamin D3 analogs and positively influencing pancreatic beta-cell function in a chronic diabetic state, statins minimize the occurrence of M. TB infection among diabetic population as well. This review aims to provide a comprehensive detail of all in vitro, in vivo, and retrospective studies that investigated the effects of statins in relation to the prevention or treatment of TB infection.
... Mycobacteria have evolved to escape from macrophage by using elaborate evasion mechanisms [6] . Although, the healthy immune system is control the acute infection, a subclinical latent or chronic infection may persist [7] . ...
Background: Tuberculosis (TB), an infectious
disease caused by Mycobacterium Tuberculosis
(MTB). Toll-like receptors (TLRs) are the beststudied class of PRRs, and they recognize specific
pathogen-associated molecular patterns (PAMPs)
from various microorganisms.
Objectives: This study aimed to determine and
measure cellular TLR2 and TLR4 in active TB,
latent TB and healthy individuals. Also aimed to
define correlation between gene expression and
present of TLR4 and TLR2 in study volunteersMaterials & Methods: This Multi-site, prospective
unmatched age and sex case-control, hospital and
community-based study. 244 Consented
volunteers or patients diagnosed as active
tuberculosis, diagnosed latent TBI and a community
controls volunteers with negative both TST and INF
γ release assay were enrolled. Three-color Flow
cytometry using Heparinized blood to study surface
expression of TLR4 (CD284) and TLR2 (CD282),
by Mean channel fluorescence intensity (MFI). EDTA blood was used for mRNA expression of
both receptors using Quantitative RT-PCR.
Results: TLR2, TLR4 cell surface expression by
Flow cytometry was decreased in all study
volunteers with no significance differences (p.value
0.24).mRNA expression of TLR2 were increased in
active TB compared to Latent TBI and healthy
individuals,while mRNA TLR4 decreased in active
TB and showed increased pattern in latent and low
level of expression in healthy volunteers, associated
with no significant difference in the expression of
TLR-2 mRNA between patients and close contacts
with LTBI (p. value 0.045), compared with
significance correlation between cellular TLR2 and
expression and BCG status (p. value 0.00)
respectively. There was highly significance
correlation between TST indurations and level of
both TLR2 and TLR4 (p. value 0.00). Also there
was significant correlation between mRNA
expression of TLR2 and disease types (p value
0.01). Single Nucleotide Polymorphisms (SNPs) in
TLR2and TLR4,in active Tb Patients Showed three
alleles,group one were heterozygous allele,
homozygous wild type in group two, while
homozygous mutant present in group three with
statistical significant with disease stages, (P.value
0.02), while LTBI volunteers had only homozygous
mutant present and group two had undetermined
alleles, and four of them develop active TB
infection, while healthy controls had similar alleles.
Conclusion: Up-regulation of TLR-2 and TLR-4
due to SNPs may be involved in the process of
tuberculosis infection in Sudanese individuals.
Keywords: Toll like receptor; Latent TB; Active
... The switch from the latent infection state to active disease is the motivation for this work. A lot of theories and models have been developed to explain the different outcomes of individuals after exposure with MTB [5][6][7][8][9][10] . The control of MTB infection is normally based on the dynamics of the human immune response. ...
The use of mathematical tools to study biological processes is of necessity in determining the effects of these biological processes occurring at different levels. In this paper, we study the immune system's response to infection with the bacteria Mycobacterium tuberculosis (the causative agent of tuberculosis). The response by the immune system is either global (lymph node, thymus, and blood) or local (at the site of infection). The response by the immune system against tuberculosis (TB) at the site of infection leads to the formation of spherical structures which comprised of cells, bacteria, and effector molecules known as granuloma. We developed a deterministic model capturing the dynamics of the immune system, macrophages, cytokines and bacteria. The hallmark of Mycobacterium tuberculosis (MTB) infection in the early stages requires a strong protective cell-mediated naive T cells differentiation which is characterised by antigen-specific interferon gamma (IFN-γ). The host immune response is believed to be regulated by the interleukin-10 cytokine by playing the critical role of orchestrating the T helper1 and T helper2 dominance during disease progression. The basic reproduction number is computed and a stability analysis of the equilibrium points is also performed. Through the computation of the reproduction number, we predict disease progression scenario including the latency state. The occurrence of latent infection is shown to depend on a number of effector function and the bacterial load for R0<1. The model predicts that endemically there is no steady state behaviour; rather it depicts the existence of MTB to be a continuous process progressing over a differing time period. Simulations of the model predict the time at which the activated macrophages overcome the infected macrophages (switching time) and observed that the activation rate (ω) correlates negatively with it. The efficacy of potential host-directed therapies was determined by the use of the model.
... Once exposed to MTB, the bacteria can remain dormant in the infected person without any symptoms. However, MTB can be reactivated years later when the patient's immunologic responsiveness weakens because of old age or immunosuppressive disease, causing the latent disease to become active TB later in life [7]. In theory, one droplet nucleus may be enough to establish infection. ...
... The atypical cell wall of M. tuberculosis defends it from the digestion of macrophages and gives it an innate resistance to many antibiotics (2,3). Multiple drug resistance TB (MDR-TB), resistant to the first line combination therapy of antibiotics is becoming fairly obvious with more than 500 000 people in the world. ...
Introduction: The development of multi drug resistant (MDR) tuberculosis (TB) and
extensively drug resistant (XDR) TB, increased the interest in the usage of medicinal plants
that are complementary to antibiotics to improve anti-TB efficacy. The present study aimed
to confirm the anti-TB efficacy of volatile oils (VOs) isolated from different parts of Premna
odorata in vivo, and moreover, to test the possible involvement of TLR4/NF-κB signaling
pathway in its anti-TB efficacy.
Methods: Thirty mice were divided into six equal groups. Group 1: healthy mice (negative
control). Groups 2-6 were injected intravenously with a positive TB solution of purified
MeDiPro Mycobacterium tuberculosis (MTB) antigen for 7 days to induce tuberculosis. Group
3-6: TB-injected mice treated respectively with leaves VO (300 μL/d), young stems VO (300
μL/d), flowers VO and a combination of the three essential VOs (1:1:1). Various immunologic
factors and antioxidant activity were evaluated and compared in the groups.
Results: TB-infected mice showed a significant increase in the serum levels of tumor necrosis
factor-α (TNF-α), interferon-gamma (IFN-γ), interleukin (IL) 1-β and the mRNA expression
levels of toll-like receptor 4 (TLR-4) & nuclear factor-κB (NF-κB) and a decrease in IL-10 & total
antioxidant capacity (TAC). While pretreatment with VOs extracted from leaves, flowers, young
stems and a combination of the three oils reversed these effects.
Conclusion: The immunomodulatory effects of VOs extracted from different parts of P. odorata
against TB infection involve the TLR-4/NFκB signaling pathway as well as, antioxidant effects,
recommending that the use of this plant may help TB infected patients.
... mononuclear phagocytes, has developed specific mechanisms to evade the host innate immune response which facilitate its long-term survival [1,2]. Phagosome maturation and phago-lysosome fusion block, interference with antigen presentation, resistance to reactive oxygen and nitrogen intermediates [3,4], alteration of host cell apoptotic pathways [5] and inhibition of autophagy in host cells [6] count among the strategies which enhance M.tb survival inside the macrophages. ...
Mycobacterium indicus pranii (MIP) known for its immunotherapeutic potential against leprosy and tuberculosis is undergoing various clinical trials and also simultaneously being studied in animal models to get insight into the mechanistic details contributing to its protective efficacy as a vaccine candidate. Studies have shown potential immunomodulatory properties of MIP, the most significant being the ability to induce strong Th1 type of response, enhanced expression of pro-inflammatory cytokines, activation of APCs and lymphocytes, elicitation of M.tb specific poly-functional T cells. All of these form crucial components of host-immune response during M.tb infection. Also, MIP was found to be potent inducer of autophagy in macrophages which resulted in enhanced clearance of M.tb from MIP and M.tb co-infected cells. Hence, we further examined the component/s of MIP responsible for autophagy induction. Interestingly, we found that MIP lipids and DNA were able to induce autophagy but not the protein fraction. LAM being one of the crucial components of mycobacterial cell-wall lipids and possessing the ability of immunomodulation; we isolated LAM from MIP and did a comparative study with M.tb-LAM. Stimulation with MIP-LAM resulted in significantly high secretion of pro-inflammatory cytokines and displayed high autophagy inducing potential in macrophages as compared to M.tb-LAM. Treatment with MIP-LAM enhanced the co-localization of M.tb within the phago-lysosomes and increased the clearance of M.tb from the infected macrophages. This study describes LAM to be a crucial component of MIP which has significant contribution to its immunotherapeutic efficacy against TB.
... Though, persister pathogens are difficult to eradicate with standard antimicrobial therapy as persister bacteria often establish a protected environment to survive. For example biofilm formation in Salmonella typhi [9], macrophage colonization of Mycobacterium tuberculosis [10] and similar mechanism in other pathogens maintain their survival in the intracellular environment. This physical protection provides an additional barrier to hostile factors such as antibodies, complementary factors, and antibiotics. ...
More than half of the world's population is infected with persistent bacterial infections, consequently, persisters are gradually becoming a major public health concern. During the persistent phase, bacterial pathogens deploy many regulatory strategies to compensate unfavorable host environmental conditions. The stringent response is one of such gene regulatory mechanisms which is stimulated by nutrient starvation. It is regulated by the synthesis of highly phosphorylated signaling nucleotides, (p)ppGpp or alarmone. (p)ppGpp is synthesized by ppGpp synthetases, and these proteins are classified as RelA/SpoT homolog (RSH) proteins. Subsequently, (p)ppGpp modulate several molecular and biochemical processes ranging from transcription to metabolism. Imperativeness of (p)ppGpp synthetases has been investigated by numerous approaches including microbiology and animal studies, thereby establishing that Rel enzyme deleted strains of pathogenic bacteria were unable to transform in persister form. In this review, we summarize recent findings to corroborate the rationality to consider (p)ppGpp synthetase as a potential target in discovering a novel class of antimicrobial agents to combat persistent infections. Moreover, inhibition studies on Mycobacterium tuberculosis (p)ppGpp synthetase shows that these inhibitors prevent dormant state transition and biofilm formation. Also, we have highlighted the structural biology of (p)ppGpp synthetases, which may provide significant information that could be used in structure-based inhibitor design.
... Therefore, a better understanding of the interaction between Mtb and macrophages may contribute to the better control of TBC [15]. Despite this response, in early stages of the innate immune response, Mtb can avoid the macrophage defense functions and actively grow and replicate in the host [16]. In addition to Figure 2. Correlation between expression of TNF-α and IL-6 genes. ...
Introduction:
Tuberculosis (TBC) is a contagious chronic respiratory disease which despite the known cause, Mycobacterium tuberculosis (Mtb), and many decades of successful therapy, remains one of the leading global health problems. Immune responses against Mtb infection involve both of types of immunity, but cellular immunity, in which certain cytokines and Th1 cells play a key role, is crucial. A better understanding of the functions of the cytokine network involved in the state and progression of TBC could identify specific molecular markers for monitoring of disease activity as well as therapy outcomes in TBC patients.
Methodology:
We investigated expression of TNF-α, IL-6 and IRAK1 genes using an RT-qPCR technique in peripheral blood mononuclear cells of 33 TBC patients and 10 healthy individuals.
Results:
Comparison between TBC patients and healthy individuals revealed statistically significant differences for all analyzed genes. The levels of expression of TNF-α and IL-6 mRNA were higher, while the level of IRAK1 mRNA was lower in the TBC group compared to controls. Moreover, a strong positive correlation was observed between TNF-α and IL-6 gene expression. When clinical parameters were analyzed, increased levels of TNF-α mRNA were detected in patients with a longer duration of therapy (>2 months) compared to those with a shorter therapy duration (< 2 months), and in patients without anemia.
Conclusions:
Our results indicate that the inflammatory genes we examined play a crucial role in the pathogenesis of tuberculosis, and that the expression of the TNF-α gene could be a marker for monitoring the clinical effect of the ant-tuberculosis drugs during therapy.
... 7 However, mycobacteria have evolved various defensive strategies to interfere with antigen presentation and associated protective immunoresponses. 8 Therefore, for appropriate processing of antigens, efficient delivery mechanisms to antigen-presenting cells (APCs) like macrophages and dendritic cells is of utmost importance. ...
Background:
Bacillus Calmette-Guérin, the attenuated strain of Mycobacterium bovis, remains the only available vaccine against tuberculosis (TB). However, its ineffectiveness in adults against pulmonary TB and varied protective efficacy (0-80%) speak to an urgent need for the development of an improved and efficient TB vaccine. In this milieu, poly(lactic-co-glycolic acid) (PLGA), is a preferential candidate, due to such properties as biocompatibility, targeted delivery, sustained antigen release, and atoxic by-products.
Methods:
In this study, we formulated PLGA nanoparticles (NPs) encapsulating the bivalent H1 antigen, a fusion of Mycobacterium tuberculosis (Mtb) Ag85B and ESAT6 proteins, and investigated its role in immunomodulation and protection against Mtb challenge. Using the classical water-oil-water solvent-evaporation method, H1-NPs were prepared, with encapsulation efficiency of 86.1%±3.2%. These spherical NPs were ~244.4±32.6 nm in diameter, with a negatively charged surface (ζ-potential -4±0.6 mV).
Results:
Under physiological conditions, NPs degraded slowly and the encapsulated H1 antigen was released over a period of weeks. As a proof-of-concept vaccine candidate, H1 NPs were efficiently internalized by the THP-1 human macrophages. Six weeks after a single-dose vaccination, H1 NP-immunized C57BL/6J mice showed significant increase in the production of total serum IgG (P<0.0001) and its isotypes compared to H1 alone, IgG2a being the predominant one, followed by IgG1. Further, the cytokine-release profile of antigen-stimulated splenocyteculture supernatant indicated a strong TH1-biased immunoresponse in H1 NP-vaccinated mice, with ~6.03- and ~2.8-fold increase in IFNγ and TNFα cytokine levels, and ~twofold and 1.6 fold increase in IL4 and IL10 cytokines, respectively, compared to H1 alone-immunized mice. In protection studies, H1 NP-vaccinated mice displayed significant reductions in lung and spleen bacillary load (P<0.05) at 5-week post-Mtb H37Rv challenge and prolonged survival, with a mean survival time of 177 days, compared to H1 alone-vaccinated mice (mean survival time 80 days).
Conclusion:
Altogether, our findings highlight the significance of the H1-PLGA nanoformulation in terms of providing long-term protection in mice with a single dose.
... Another protein, initially termed tryptophan aspartate rich coat protein and now known as coronin 1, is recruited to phagosomes containing active bacilli but is rapidly released from phagosomes containing inactive mycobacteria [18]. MTB inhibits the formation of lysosomes by raising the coronin 1 expression on the host phagocyte membrane; the duration of the recruitment process and the amount of coronin 1 are positively correlated with the amount and activity of activated MTB in the microsomes [19,20]. Besides, the cytokine interferon (IFN)-α inhibits maturation by inducing the production of interleukin (IL)-10 in a STAT1-dependent manner. ...
Epidemiological data from the Center of Disease Control (CDC) and the World Health Organization (WHO) statistics in 2017 show that 10.0 million people around the world became sick with tuberculosis. Mycobacterium tuberculosis (MTB) is an intracellular parasite that mainly attacks macrophages and inhibits their apoptosis. It can become a long-term infection in humans, causing a series of pathological changes and clinical manifestations. In this review, we summarize innate immunity including the inhibition of antioxidants, the maturation and acidification of phagolysosomes and especially the apoptosis and autophagy of macrophages. Besides, we also elaborate on the adaptive immune response and the formation of granulomas. A thorough understanding of these escape mechanisms is of major importance for the prevention, diagnosis and treatment of tuberculosis.
... The atypical cell wall of M. tuberculosis defends it from the digestion of macrophages and gives it an innate resistance to many antibiotics (2,3). Multiple drug resistance TB (MDR-TB), resistant to the first line combination therapy of antibiotics is becoming fairly obvious with more than 500 000 people in the world. ...
extensively drug resistant (XDR) TB, increased the interest in the usage of medicinal plants that are complementary to antibiotics to improve anti-TB efficacy. The present study aimed to confirm the anti-TB efficacy of volatile oils (VOs) isolated from different parts of Premna odorata in vivo, and moreover, to test the possible involvement of TLR4/NF-κB signaling pathway in its anti-TB efficacy.
Methods: Thirty mice were divided into six equal groups. Group 1: healthy mice (negative control). Groups 2-6 were injected intravenously with a positive TB solution of purified MeDiPro Mycobacterium tuberculosis (MTB) antigen for 7 days to induce tuberculosis. Group 3-6: TB-injected mice treated respectively with leaves VO (300 μL/d), young stems VO (300 μL/d), flowers VO and a combination of the three essential VOs (1:1:1). Various immunologic factors and antioxidant activity were evaluated and compared in the groups.
Results: TB-infected mice showed a significant increase in the serum levels of tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), interleukin (IL) 1-β and the mRNA expression levels of toll-like receptor 4 (TLR-4) & nuclear factor-κB (NF-κB) and a decrease in IL-10 & total antioxidant capacity (TAC). While pretreatment with VOs extracted from leaves, flowers, young stems and a combination of the three oils reversed these effects.
Conclusion: The immunomodulatory effects of VOs extracted from different parts of P. odorata against TB infection involve the TLR-4/NFκB signaling pathway as well as, antioxidant effects, recommending that the use of this plant may help TB infected patients.
Tuberculosis, a lung disease caused by Mycobacterium tuberculosis ( Mtb ), remains a major global health problem ranking as the second leading cause of death from a single infectious agent. One of the major factors contributing toward Mtb’s success as a pathogen is its unique cell wall and its ability to counteract various arms of the host’s immune response. A recent genome-scale study profiled a list of candidate genes that are predicted to be essential for Mtb survival of host-mediated responses. One candidate was FtsEX, a protein complex composed of an ATP-binding domain, FtsE, and a transmembrane domain, FtsX. FtsEX functions through interaction with a periplasmic hydrolase, RipC. Homologs of FtsEX exist in other bacteria and have been linked with playing a key role in regulating peptidoglycan hydrolysis during cell elongation and division. Here, we report on Mycobacterium smegmatis, FtsE, FtsX, and RipC and their protective roles in stressful conditions. We demonstrate that the individual genes of FtsEX complex and RipC are not essential for survival in normal growth conditions but conditionally essential in low-salt media and antibiotic-treated media. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. Our results suggest that FtsE, FtsX, and RipC are required for both normal cell elongation and division and ultimately for survival in stressful conditions.
IMPORTANCE
Mycobacterial cell growth and division are coordinated with regulated peptidoglycan hydrolysis. Understanding cell wall gene complexes that govern normal cell division and elongation will aid in the development of tools to disarm the ability of mycobacteria to survive immune-like and antibiotic stresses. We combined genetic analyses and scanning electron microscopy to analyze morphological changes of mycobacterial FtsEX and RipC mutants in stressful conditions. We demonstrate that FtsE, FtsX, FtsEX, and RipC are conditionally required for the survival of Mycobacterium smegmatis during rifampicin treatment and in low-salt conditions. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. We also show that the FtsEX-RipC interaction is essential for the survival of M. smegmatis in rifampicin. Our results suggest that FtsE, FtsX, and RipC are required for normal cell wall regulation and ultimately for survival in stressful conditions.
Introduction:
In an historical era dominated by the Sars-Cov-2 pandemic, a fact of growing interest emerges regarding co-infection with Mycobacterium Tuberculosis (M.Tuberculosis). This represents today an important clinical and diagnostic challenge, as the two pathogens are capable, through specific immunopathological mechanisms, of interacting with each other, determining a severe respiratory condition with a severe prognosis.
Areas covered:
With this review we wanted to collect and analyze the latest scientific evidence concerning the main immunopathogenetic mechanisms shared by these two respiratory pathogens, with particular interest in the possible iatrogenic factors favoring coinfection and the need to define a multidisciplinary and standardized screening tools aimed to identify coinfection early, ensuring the best clinical and therapeutic management.
Expert opinion:
The existence of a direct immunopathogenetic link between COVID-19 and TB indirectly contributes to mutual morbidity and mortality. The identification and application of early and standardized screening tools aimed at the identification of this condition is essential, in addition to vaccine prevention.
Tuberculosis is one of the most common and oldest human afflictions caused by the deadly pathogen, Mycobacterium tuberculosis. Its infection drastically increasing worldwide with time despite the application of various antibiotics and live attenuated vaccines. The major risk factor associated with tuberculosis is the long-term malfunction of the respiratory system that increases the deaths whereas the continuous emergence of drug-resistant MTB strains (MDR/TDR/XDR) acts as a driving force to accelerate additional obstacles to humankind. Researchers are effortlessly involved in a systematic examination of tuberculosis and drug designing against it but still, we could not find a permanent cure for tuberculosis. Therefore, it is extremely necessary to analyse pathogenesis, epidemiology and associated risk factors to plan an overall strategy against this deadly disease. In the present study, we have gone through a comprehensive literature survey to provide all related information that may assist us understanding this disease and designing strategic planning. This study would fill the gap created due to lack of knowledge on MTB infection and molecular mechanism which is the biggest hurdle in finding out a therapeutic lead against tuberculosis.
Male dairy calf welfare is a key issue in the Canadian cattle industry. The welfare of male dairy calves can be explored through the aspects of health and biological functioning, affective states, and natural living. Presently, the main welfare issues associated with the production of male dairy calves include morbidity and mortality, colostrum and feeding management, transportation, isolation, castration and disbudding, and euthanasia. Opportunities to improve male dairy calf welfare include improving accepted industry practices, enhancing education and compliance with industry codes of practice, and increasing veterinarian involvement in on-farm animal welfare. The benefits of improving male dairy calf welfare include maintenance of the cattle industry's social license and improved producer mental health and occupational satisfaction. The main barriers to improving male dairy calf welfare are economics and cultural attitudes within the industry towards male dairy calves.
Inflammasome signaling results in cell death and release of cytokines from the IL-1 family, which facilitates control over an infection. However, some pathogens such as Salmonella typhimurium (ST) activate various innate immune signaling pathways, including inflammasomes, yet evade these cell death mechanisms, resulting in a chronic infection. Here we investigated inflammasome signaling induced by acute and chronic isolates of ST obtained from different organs. We show that ST isolated from infected mice during the acute phase display an increased potential to activate inflammasome signaling, which then undergoes a protracted decline during the chronic phase of infection. This decline in inflammasome signaling was associated with reduced expression of virulence factors, including flagella and the salmonella pathogenicity island I genes. This reduction in cell death of macrophages induced by chronic isolates had the greatest impact on the NLRP3 inflammasome, which correlated with a reduction in caspase-1 activation. Furthermore, rapid cell death induced by Casp-1/11 by ST in macrophages limited the subsequent activation of cell death cascade proteins Casp-8, RipK1, RipK3, and MLKL to prevent the activation of alternative forms of cell death. We observed that the lack of the ability to induce cell death conferred a competitive fitness advantage to ST only during the acute phase of infection. Finally, we show that the chronic isolates displayed a significant attenuation in their ability to infect mice through the oral route. These results reveal that ST adapts during chronic infection by circumventing inflammasome-recognition to promote the survival of both the host and the pathogen.
To identify sequences with a role in microbial pathogenesis, we assessed the adequacy of their annotation by existing controlled vocabularies and sequence databases. Our goal was to regularize descriptions of microbial pathogenesis for improved integration with bioinformatic applications. Here we review the challenges of annotating sequences for pathogenic activity. We relate the categorization of more than 2750 sequences of pathogenic microbes through a controlled vocabulary called Functions of Sequences of Concern (FunSoCs). These allow for an ease of description by both humans and machines. We provide a subset of 220 fully annotated sequences in the supplementary material as examples. The use of this compact (∼30 terms) controlled vocabulary has potential benefits for research in microbial genomics, public health, biosecurity, biosurveillance, and the characterization of new and emerging pathogens.
Tuberculosis is a highly contagious infectious disease triggered by Mycobacterium tuberculosis, which is widely spread by aerosol. The major site of infection is usually the lungs however the disease can attack any extra-pulmonary site as well, which is further diagnosis by necrotizing granulomatous inflammation. World Health Organization reported almost 8.9–10 million people are suffering from tuberculosis in 2019, including 56% men and 32% women, and 12% children. Multidrug-resistant tuberculosis (MDR-TB) is a medical condition in which Mycobacterium tuberculosis strains resistant to at least isoniazid and rifampicin. In-vitro studies suggest that several bioactive compounds and their synthetic derivatives obtained from plants, fungi, and marine organism possesses antimycobacterial affinity. Phenolic compounds such as dihydrocubebin, hinokinin, ethoxycubebin possess the antimycobacterial activity. Mycobacterial cell envelope antagonists have been shown to obstruct the synthesis of mycolic acids, arabinogalactan, and peptidoglycan, essential components of the mycobacterial cell wall. The paramount antituberculous drugs hamper the development of mycolic acids or the aid mechanism which links them to the cell membrane. Medicines targeting RNA synthesis encompass those that restrict the assembly of bacterial DNA-dependent RNA polymerases, that are indispensable enzymes for RNA synthesis. Various molecular pathways for the target to cure tuberculosis entail the targets of M. tuberculosis cell wall synthesis, energy metabolism, folate metabolism, DNA replication, and RNA synthesis. Interestingly, in preserving the health of patients diagnosed with tuberculosis, medicinal plants have tremendous advantages with limited side effects as compare to the standard drugs.
TNF signalling through TNFRp55 and TNFRp75, and receptor shedding is important for immune activation and regulation. TNFRp75 deficiency leads to improved control of Mycobacterium tuberculosis (M.tuberculosis) infection, but the effects of early innate immune events in this process are unclear. We investigated the role of TNFRp75 on cell activation and apoptosis of alveolar macrophages and neutrophils during M.tuberculosis and M.bovis BCG infection. We found increased microbicidal activity against M.tuberculosis occurred independently of IFNy and NO generation, and displayed an inverse correlation with alveolar macrophages (AMs) apoptosis. Both M.tuberculosis and M.bovis BCG induced higher expression of MHC‐II in TNFRp75−/− AMs; however, M bovis BCG infection did not alter AM apoptosis in the absence of TNFRp75. Pulmonary concentrations of CCL2, CCL3 and IL‐1β were increased in TNFRp75−/− mice during M,bovis BCG infection, but had no effect on neutrophil responses. Thus, TNFRp75‐dependent regulation of mycobacterial replication is virulence dependent and occurs independently of early alveolar macrophage apoptosis and neutrophil responses.
In this chapter, we will understand the role of Mycobacterium tuberculosis for initiating tuberculosis disease in human body. After a basic introduction to the mycobacteria and its scientific classification, we discussed on some general ideas about various factors, which are responsible for transmission of bacteria. Then we focused into different biological mechanisms, morphological advantages, and defense mechanisms showed by the mycobacterium in order to survive within the host cell for a very long period of time while not showing any quite symptoms of its presence. We have showcased two case studies, which provides an important evidence toward the involvement of bacteriological factors in spreading of TB epidemic. We concluded the pathogenesis section by discussing on some ongoing research topics like mycobacterium capsules, which strengthens the defense mechanisms of mycobacterium against the host cell. Then we moved to the role of immune system in controlling the Mycobacterium tuberculosis infection. First, we explained the defenses of the upper respiratory tract that prevent the inhalation of droplets containing Mycobacterium tuberculosis into alveoli. If the droplets manage to reach the alveoli despite the defenses of the upper respiratory tract, then innate immune system kicks in. Therefore, we explained various mechanisms of innate immune system in recognition and control of Mycobacterium tuberculosis growth in alveoli. Then we explained the role of inflammatory responses in activating the adaptive immune responses. These adaptive immune responses control the growth of Mycobacterium tuberculosis.
Мета роботи — установити порушення в системі імунітету, які обтяжують перебіг та знижують результативність лікування хворих на мультирезистентний туберкульоз легень.Матеріали та методи. Порівняльний аналіз порушень у ланці неспецифічної та системної клітинної імунної відповіді проведено у 44 хворих на вперше діагностований деструктивний мультирезистентний туберкульоз легень (ВДД МРТБЛ) з ефективною, малоефективною та неефективною антимікобактеріальною терапією.Результати та обговорення. У хворих на ВДД МРТБЛ з неефективною та малоефективною терапією порівняно з ефективно лікованими наявний різко виражений гуморальний тип імунної відповіді: підвищений у середньому на 20—25 % вміст ІgА (р < 0,05), ІgМ (р < 0,05), ІgЕ (р < 0,05) та рівень циркулюючих імунних комплексів (р < 0,05); знижений вміст катіонних лізосомальних білків (КЛБ) гранулоцитарних лейкоцитів (р < 0,001), пригнічена проліферативна активність Т-лімфоцитів на неспецифічний (ФГА; р < 0,05) та специфічний (ППД-Л; р < 0,05) мітоген. У неефективно лікованих пацієнтів у 2,0 разу частіше виявляли пригнічення на 30—50,0 % і більше проліферативної активності Т-лімфоцитів, в 1,7 разу зростання частки осіб з ознаками туберкулінової анергії специфічних Т-лімфоцитів та у 1,5 разу питомої ваги хворих зі зниженим вмістом КЛБ гранулоцитів. Суттєві порушення в системі імунітету констатувалися у 70 % хворих з неефективним і у 29,4 % з ефективним лікуванням.Висновки. Прогресуванню специфічного процесу та зниженню ефективності антимікобактеріальної терапії мультирезистентного туберкульозу легень сприяють: наявність Т-клітинної імунодепресії у вигляді зменшення більше ніж на 30,0 % Т-лімфоцитарного пулу та пригнічення їхньої проліферативної активності; туберкулінова толерантність специфічних Т-лімфоцитів, фагоцитарна недостатність, зумовлена зниженням на 30—50,0 % вмісту КЛБ гранулоцитарних лейкоцитів.
“Infection resisters” are broadly defined as individuals who despite significant exposure to Mycobacterium tuberculosis remain persistently unreactive to conventional detection assays, suggesting that they remain uninfected or rapidly clear their infection early on following exposure. In this review, we highlight recent studies that point to underlying host immune mechanisms that could mediate this natural resistance. We also illustrate some additional avenues that are likely to be differently modulated in resisters and possess the potential to be targeted, ranging from early mycobacterial sensing leading up to subsequent killing. Emerging research in this area can be harnessed to provide valuable insights into the development of novel therapeutic and vaccine strategies against M. tuberculosis .
Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Despite an urgent need for new therapies targeting persistent bacteria, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. Here we report that persistence of M. tuberculosis in mice is facilitated by isocitrate lyase (ICL), an enzyme essential for the metabolism of fatty acids. Disruption of the icl gene attenuated bacterial persistence and virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. A link between the requirement for ICL and the immune status of the host was established by the restored virulence of delta icl bacteria in interferon-gamma knockout mice. This link was apparent at the level of the infected macrophage: Activation of infected macrophages increased expression of ICL, and the delta icl mutant was markedly attenuated for survival in activated but not resting macrophages. These data suggest that the metabolism of M. tuberculosis in vivo is profoundly influenced by the host response to infection, an observation with important implications for the treatment of chronic tuberculosis.
The biogenesis and maturation of phagosomes is an area of study which has been employing aspects of proteomic analyses and variations on that theme by identifying components on isolated organelles and following their dynamic changes and interactions with the endocytic pathway. In the case of Mycobacterium tuberculosis phagosome, the arrest of its maturation in infected macrophages, referred to in classical texts as the inhibition of phagosome-lysosome fusion, represents a phenomenon that is used to functionally dissect the phagosomal maturation pathway. In this review, we summarize the recent studies on regulators of membrane trafficking and other organelle components in the context of phagosomal biogenesis and mycobacterial phagosome maturation arrest.
Vaccination of mice with Mycobacterium vaccae orM. smegmatis induces some protection against M. tuberculosis challenge. The 19-kDa lipoprotein of M. tuberculosis, expressed in M. vaccae or M. smegmatis (M. smeg19kDa), abrogates this protective immunity. To investigate the mechanism of this suppression of immunity, human monocyte-derived
macrophages (MDM) were infected with M. smeg19kDa. Infection resulted in reduced production of tumor necrosis factor alpha (TNF-α) (P < 0.01), interleukin-12 (IL-12) (P < 0.05), IL-6 (P < 0.05), and IL-10 (P < 0.05), compared to infection with M. smegmatis vector (M. smegV). Infection with M. smeg19kDa and with M. smegV had no differential effect on expression of costimulatory molecules on MDM, nor did it affect the proliferation of presensitized
T cells cocultured with infected MDM. When MDM were infected withM. smegmatis expressing mutated forms of the 19-kDa lipoprotein, including non-O-glycosylated (M. smeg19NOG), nonsecreted (M. smeg19NS), and nonacylated (M. smeg19NA) variants, the reduced production of TNF-α or IL-12 was not observed. When the purified 19-kDa lipoprotein was added
directly to cultures of infected monocytes, there was little effect on either induction of cytokine production or its inhibition.
Thus, the immunosuppressive effect is dependent on glycosylated and acylated 19-kDa lipoprotein present in the phagosome containing
the mycobacterium. These results suggest that the diminished protection against challenge with M. tuberculosis seen in mice vaccinated with M. smegmatis expressing the 19-kDa lipoprotein is the result of reduced TNF-α and IL-12 production, possibly leading to reduced induction
of T-cell activation.
The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.
A 19-kDa lipoprotein from Mycobacterium tuberculosis was expressed as a recombinant antigen in the nonpathogenic mycobacterial host strain M. vaccae. Immunization of mice with the recombinant M. vaccae resulted in induction of a strong type 1 immune response to the 19-kDa antigen, characterized by immunoglobulin G2a (IgG2a) antibodies and gamma interferon (IFN-gamma) production by splenocytes. Immunization with the same antigen in incomplete Freund's adjuvant induced a strong IgG1 response with only low levels of IFN-gamma. Subsequent intravenous and aerosol challenges of immunized mice with virulent M. tuberculosis demonstrated no evidence of protection associated with the response to the 19-kDa antigen; in fact, the presence of the recombinant 19-kDa antigen abrogated the limited protection conferred by M. vaccae (vector control). The recombinant M. vaccae system is a convenient approach to induction of type 1 responses to M. tuberculosis antigens. However, the unexpected reduction in protective efficacy of M. vaccae expressing the 19-kDa antigen highlights the complexity of testing recombinant subunit vaccines and the need for a better understanding of the immune mechanisms required for effective vaccination against tuberculosis.
Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2(-/-) mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-L-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.
Mycobacterium tuberculosis and the closely related organism Mycobacterium bovis can survive and replicate inside macrophages. Intracellular survival is at least in part attributed to the failure of mycobacterial phagosomes to undergo fusion with lysosomes. The transformation of phagosomes into phagolysosomes involves gradual acquisition of markers from the endosomal compartment. Members of the rab family of small GTPases which confer fusion competence in the endocytic pathway are exchanged sequentially onto the phagosomal membranes in the course of their maturation. To identify the step at which the fusion capability of phagosomes containing mycobacteria is compromised, we purified green fluorescent protein-labeled M. bovis BCG phagosomal compartments (MPC) and compared GTP-binding protein profiles of these vesicles with latex bead phagosomal compartments (LBC). We report that the MPC do not acquire rab7, specific for late endosomes, even 7 days postinfection, whereas this GTP-binding protein is present on the LBC within hours after phagocytosis. By contrast, rab5 is retained and enriched with time on the MPC, suggesting fusion competence with an early endosomal compartment. Prior infection of macrophages with M. bovis BCG also affected the dynamics of rab5 and rab7 acquisition by subsequently formed LBC. Selective exclusion of rab7, coupled with the retention of rab5 on the mycobacterial phagosome, may allow organisms from the M. tuberculosis complex to avert the usual physiological destination of phagocytosed material.
Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.
A unique feature of Mycobacterium tuberculosis is its ability to establish latent infection in the human host, which can reactivate to cause disease years later. In the present study, the mechanisms involved in the control of latent tuberculous infection were examined using two murine experimental tuberculosis models. Analysis of the model involving infection of mice with a relatively low inoculum of the virulent Erdman strain of M. tuberculosis indicated that in vivo inhibition of reactive nitrogen intermediate (RNI) production by the nitric oxide synthase inhibitor aminoguanidine resulted in reactivation. This reactivation was evidenced by hepatosplenomegaly, a robust tissue granulomatous reaction, and increased bacillary load. IFN-gamma, TNF-alpha, and inducible nitric oxide synthase were all expressed throughout the latent phase of infection. Reactivation of latent tuberculous infection by aminoguanidine treatment was confirmed using a second murine tuberculosis model based on treatment with antimycobacterial drugs. Results obtained using this drug-based model also suggested the existence of an RNI-independent antimycobacterial mechanism(s) operative in the latent phase of infection. Together, these data suggest that both RNI-dependent and -independent mechanisms contribute to the prevention of tuberculous reactivation.
Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) produced by activated macrophages participate in host defense against the facultative intracellular pathogens Mycobacterium tuberculosis and Salmonella typhimurium. To survive within macrophages, such pathogens may have evolved ROI and RNI resistance mechanisms. ROI resistance pathways have been intensively studied. Much less is known about the mechanisms of resistance to RNI. To identify possible RNI resistance genes in M. tuberculosis, a mycobacterial library was expressed in S. typhimurium and subjected to selection by exposure to the NO donor S-nitrosoglutathione (GSNO) in concentrations sufficient to kill the vast majority of nontransformed salmonellae. Among the rare surviving recombinants was a clone expressing noxR3, a novel and previously anonymous M. tuberculosis gene predicted to encode a small, basic protein. Expression of noxR3 protected S. typhimurium not only from GSNO and acidified nitrite but also from H2O2. noxR3 is the third gene cloned from M. tuberculosis that has been shown to protect heterologous cells from both RNI and ROI. This suggests diversity in the repertoire of mechanisms that help pathogens resist the oxidative and nitrosative defenses of the host.
The natural resistance associated macrophage protein 1 (Nramp1) gene determines the ability of murine macrophages to control infection with a group of intracellular pathogens, including Salmonella typhimurium, Leishmania donovani, and Mycobacterium bovis bacillus Calmette-Guérin (BCG). The expression of the resistant allele of the Nramp1 gene in murine macrophages is associated with a more efficient expression of several macrophage activation-associated genes, including class II MHC loci. In this study, we investigated the molecular mechanisms involved in IFN-gamma-induced MHC class II expression in three types of macrophages: those expressing a wild-type allele of the Nramp1 gene (B10R and 129/Mphi), those carrying a susceptible form of the Nramp1 gene (B10S), and those derived from 129-Nramp1-knockout mice (129/Nramp1-KO). Previously, we published results showing that Ia protein expression is significantly higher in the IFN-gamma-induced B10R macrophages, compared with its susceptible counterpart. In this paper, we also show that the higher expression of Ia protein in B10R cells is associated with higher I-Abeta mRNA expression, which correlates with a higher level of IFN-gamma-induced phosphorylation of the STAT1-alpha protein and subsequently with elevated expression of class II transactivator (CIITA) mRNA, compared with B10S. Furthermore, we demonstrate that the infection of macrophages with M. bovis BCG results in a down-regulation of CIITA mRNA expression and, consequently, in the inhibition of Ia induction. Therefore, our data explain, at least in part, the molecular mechanism involved in the inhibition of I-Abeta gene expression in M. bovis BCG-infected macrophages activated with IFN-gamma.
Human alveolar macrophages (AMphi) undergo apoptosis following infection with Mycobacterium tuberculosis in vitro. Apoptosis of cells infected with intracellular pathogens may benefit the host by eliminating a supportive environment for bacterial growth. The present study compared AMphi apoptosis following infection by M. tuberculosis complex strains of differing virulence and by Mycobacterium kansasii. Avirulent or attenuated bacilli (M. tuberculosis H37Ra, Mycobacterium bovis bacillus Calmette-Guérin, and M. kansasii) induced significantly more AMphi apoptosis than virulent strains (M. tuberculosis H37Rv, Erdman, M. tuberculosis clinical isolate BMC 96.1, and M. bovis wild type). Increased apoptosis was not due to greater intracellular bacterial replication because virulent strains grew more rapidly in AMphi than attenuated strains despite causing less apoptosis. These findings suggest the existence of mycobacterial virulence determinants that modulate the apoptotic response of AMphi to intracellular infection and support the hypothesis that macrophage apoptosis contributes to innate host defense in tuberculosis.
The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosisphagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating
membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of
Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both
pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages
and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant
Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c
Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant
vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas
Rab5 is absent from wild-type L. pneumophilaphagosomes, functional Rab5 persists on M. tuberculosisphagosomes. The absence of Rab5 on the L. pneumophilaphagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.
Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Phagosomes
containing inert particles have also been shown to transiently acquire Rab7 as they mature. Disruption in the pathway prior
to the acquisition of Rab7 has been suggested as playing a role in the altered maturation of Mycobacterium bovis BCG phagosomes. As a first step to determine whether disruption in the delivery or function of Rab7 could play a role in
the altered maturation of Legionella pneumophila and M. tuberculosis phagosomes, we have examined the distribution of wild-type Rab7 and the GTPase-deficient, constitutively active mutant form
of Rab7 in HeLa cells infected withL. pneumophila or M. tuberculosis. We have found that the majority of L. pneumophila and M. tuberculosis phagosomes acquire relatively abundant staining for Rab7 and for the constitutively active mutant Rab7 in HeLa cells that
overexpress these proteins. Nevertheless, despite acquisition of wild-type or constitutively active Rab7, both the L. pneumophila and the M. tuberculosis phagosomes continue to exhibit altered maturation as manifested by a failure to acquire lysosome-associated membrane glycoprotein
1. These results demonstrate that L. pneumophila and M. tuberculosis phagosomes have receptors for Rab7 and that the altered maturation of these phagosomes is not due to a failure to acquire
Rab7.
The interaction of microbes with dendritic cells (DCs) is likely to have an enormous impact on the initiation of the immune
response against a pathogen. In this study, we compared the interaction ofMycobacterium tuberculosis with murine bone marrow-derived DCs and macrophages (Mφ) in vitro. M. tuberculosis grew equally well within nonactivated DCs and Mφ. Activation of DCs and Mφ with gamma interferon and lipopolysaccharide inhibited
the growth of the intracellular bacteria in a nitric oxide synthase-dependent fashion. However, while this activation enabled
Mφ to kill the intracellular bacteria, the M. tuberculosis bacilli within activated DCs were not killed. Thus, DCs could restrict the growth of the intracellular mycobacteria but were
less efficient than Mφ at eliminating the infection. These results may have implications for priming immune responses to M. tuberculosis. In addition, they suggest that DCs may serve as a reservoir for M. tuberculosis in tissues, including the lymph nodes and lungs.
Mycobacterium tuberculosis (MTB) induces vigorous immune responses, yet persists inside macrophages, evading host immunity. MTB bacilli or lysate was found to inhibit macrophage expression of class II MHC (MHC-II) molecules and MHC-II Ag processing. This report characterizes and identifies a specific component of MTB that mediates these inhibitory effects. The inhibitor was extracted from MTB lysate with Triton X-114, isolated by gel electroelution, and identified with Abs to be MTB 19-kDa lipoprotein. Electroelution- or immunoaffinity-purified MTB 19-kDa lipoprotein inhibited MHC-II expression and processing of both soluble Ags and Ag 85B from intact MTB bacilli. Inhibition of MHC-II Ag processing by either MTB bacilli or purified MTB 19-kDa lipoprotein was dependent on Toll-like receptor (TLR) 2 and independent of TLR 4. Synthetic analogs of lipopeptides from Treponema pallidum also inhibited Ag processing. Despite the ability of MTB 19-kDa lipoprotein to activate microbicidal and innate immune functions early in infection, TLR 2-dependent inhibition of MHC-II expression and Ag processing by MTB 19-kDa lipoprotein during later phases of macrophage infection may prevent presentation of MTB Ags and decrease recognition by T cells. This mechanism may allow intracellular MTB to evade immune surveillance and maintain chronic infection.
Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC ) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met residues when these RNI cannot themselves oxidize Met. However, under anaerobic conditions, neither nitrite nor GSNO was bactericidal. Nitrite and GSNO can both give rise to NO, which may react with superoxide produced by bacteria during aerobic metabolism, forming peroxynitrite, a known oxidant of Met to Met-O. Thus, the findings are consistent with the hypotheses that nitrite and GSNO kill E. coli by intracellular conversion to peroxynitrite, that intracellular Met residues in proteins constitute a critical target for peroxynitrite, and that MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage.
Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.
Murine macrophages effect potent antimycobacterial function via the production of nitric oxide by the inducible isoform of
the enzyme nitric oxide synthase (NOS2). The protective role of reactive nitrogen intermediates (RNI) against Mycobacterium tuberculosisinfection has been well established in various murine experimental tuberculosis models using laboratory strains of the tubercle
bacillus to establish infection by the intravenous route. However, important questions remain about the in vivo importance
of RNI in host defense against M. tuberculosis. There is some evidence that RNI play a lesser role following aerogenic, rather than intravenous,M. tuberculosis infection of mice. Furthermore, in vitro studies have demonstrated that different strains of M. tuberculosis, including clinical isolates, vary widely in their susceptibility to the antimycobacterial effects of RNI. Thus, we sought
to test rigorously the protective role of RNI against infection with recent clinical isolates of M. tuberculosis following both aerogenic and intravenous challenges. Three recently isolated and unique M. tuberculosis strains were used to infect both wild-type (wt) C57BL/6 and NOS2 gene-disrupted mice. Regardless of the route of infection, NOS2−/− mice were much more susceptible than wt mice to any of the clinical isolates or to either the Erdman or H37Rv laboratory
strain of M. tuberculosis. Mycobacteria replicated to much higher levels in the organs of NOS2−/− mice than in those of wt mice. Although the clinical isolates all exhibited enhanced virulence in NOS2−/− mice, they displayed distinct growth rates in vivo. The present study has provided results indicating that RNI are required
for the control of murine tuberculous infection caused by both laboratory and clinical strains of M. tuberculosis. This protective role of RNI is essential for the control of infection established by either intravenous or aerogenic challenge.
Mycobacterium tuberculosis (MTB) inhibits phagosomal maturation to promote its survival inside macrophages. Control of MTB infection requires CD4 T cell responses and major histocompatibility complex (MHC) class II (MHC-II) processing of MTB antigens (Ags). To investigate phagosomal processing of MTB Ags, phagosomes containing heat-killed (HK) or live MTB were purified from interferon-gamma (IFN-gamma)-activated macrophages by differential centrifugation and Percoll density gradient subcellular fractionation. Flow organellometry and Western blot analysis showed that MTB phagosomes acquired lysosome-associated membrane protein-1 (LAMP-1), MHC-II, and H2-DM. T hybridoma cells were used to detect MTB Ag 85B(241-256)-I-A(b) complexes in isolated phagosomes and other subcellular fractions. These complexes appeared initially (within 20 min) in phagosomes and subsequently (>20 min) on the plasma membrane, but never within late endocytic compartments. Macrophages processed HK MTB more rapidly and efficiently than live MTB; phagosomes containing live MTB expressed fewer Ag 85B(241-256)-I-A(b) complexes than phagosomes containing HK MTB. This is the first study of bacterial Ag processing to directly show that peptide-MHC-II complexes are formed within phagosomes and not after export of bacterial Ags from phagosomes to endocytic Ag processing compartments. Live MTB can alter phagosome maturation and decrease MHC-II Ag processing, providing a mechanism for MTB to evade immune surveillance and enhance its survival within the host.
Mycobacterium tuberculosis (Mtb) mounts a stubborn defense against oxidative and nitrosative components of the immune response. Dihydrolipoamide dehydrogenase
(Lpd) and dihydrolipoamide succinyltransferase (SucB) are components of α-ketoacid dehydrogenase complexes that are central
to intermediary metabolism. We find that Lpd and SucB support Mtb's antioxidant defense. The peroxiredoxin alkyl hydroperoxide
reductase (AhpC) is linked to Lpd and SucB by an adaptor protein, AhpD. The 2.0 angstrom AhpD crystal structure reveals a
thioredoxin-like active site that is responsive to lipoamide. We propose that Lpd, SucB (the only lipoyl protein detected
in Mtb), AhpD, and AhpC together constitute a nicotinamide adenine dinucleotide (reduced)–dependent peroxidase and peroxynitrite
reductase. AhpD thus represents a class of thioredoxin-like molecules that enables an antioxidant defense.
Particles such as microorganisms that are taken up by the cell into phagosomes are usually ultimately degraded in phagolysosomes. However, despite its importance, phagolysosome biogenesis is poorly understood. This article presents a model for phagosome maturation into phagolysosomes that involves multiple transient fusion-fission interactions of phagosomes with endocytic organelles via a fusion-pore-like structure. This dynamic process may be modulated by the sequential appearance and disappearance of key phagosome proteins.
We have recently demonstrated that the macrophage L-arginine-dependent cytotoxic pathway effectively kills the virulent Erdman strain of Mycobacterium tuberculosis in vitro via the generation of toxic reactive nitrogen intermediates by the enzyme nitric oxide synthase. This report demonstrates that two distinct inhibitors of nitric oxide synthase (aminoguanidine and NG-monomethyl-L-arginine) render similar deleterious effects on tuberculous infection in mice, as assessed by mortality, bacterial burden, and pathological tissue damage, thus confirming the importance of reactive nitrogen intermediates in resistance against M. tuberculosis.
Despite the potential role of the macrophage in the eradication of invading microbes, Mycobacterium species have evolved mechanisms to ensure their survival and replication inside the macrophage. Particles phagocytosed by macrophages normally will be delivered into acid lysosomal compartments for degradation. Mycobacterium must, in some way, avoid this fate by modulation of their phagosome. Immunoelectron microscopy of macrophages infected with Mycobacterium avium or Mycobacterium tuberculosis indicates that the vacuolar membrane surrounding the bacilli possesses the late endosomal/lysosomal marker, LAMP-1 (lysosomal-associated membrane protein-1), but lacks the vesicular proton-ATPase. Analysis of the intersection of the bacteria-containing vacuoles with the endocytic network of the macrophage supports previous studies indicating that these bacilli restrict the fusion capability of their intracellular compartments. The occurrence of vesicles containing lipoarabinomannan, discrete from those containing Mycobacterium, indicate that material does traffic out from the mycobacterial vacuole. To compensate for this loss of membrane, the vacuole must remain dynamic and fuse with LAMP-1-containing vesicles to maintain the density of this marker.
At the interface between the innate and adaptive immune systems lies the high-output isoform of nitric oxide synthase (NOS2 or iNOS). This remarkable molecular machine requires at least 17 binding reactions to assemble a functional dimer. Sustained catalysis results from the ability of NOS2 to attach calmodulin without dependence on elevated Ca2+. Expression of NOS2 in macrophages is controlled by cytokines and microbial products, primarily by transcriptional induction. NOS2 has been documented in macrophages from human, horse, cow, goat, sheep, rat, mouse, and chicken. Human NOS2 is most readily observed in monocytes or macrophages from patients with infectious or inflammatory diseases. Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, fungi, protozoa, helminths, and tumor cells. The antimicrobial and cytotoxic actions of NO are enhanced by other macrophage products such as acid, glutathione, cysteine, hydrogen peroxide, or superoxide. Although the high-output NO pathway probably evolved to protect the host from infection, suppressive effects on lymphocyte proliferation and damage to other normal host cells confer upon NOS2 the same protective/destructive duality inherent in every other major component of the immune response.
Dendritic cells (DC) play an essential role in the initiation of primary T cell responses to foreign Ag. It is likely that these potent APC are critical in the initiation of immune responses to pathogens, such as bacteria or parasites. However, little is known about the interaction of these important APC with pathogens. To address this issue, the interaction of the bacterium Mycobacterium tuberculosis with human DC was studied. DC generated from human peripheral blood by short term culture in medium containing recombinant human cytokines granulocyte-macrophage-CSF and IL-4 were capable of phagocytosing M. tuberculosis. Infection of DC with live M. tuberculosis bacilli resulted in increased APC surface expression of the costimulatory molecules CD54, CD40, and B7.1, as well as MHC class I molecules. In addition, infected DC secreted elevated levels of inflammatory cytokines, including TNF-alpha, IL-1, and IL-12. M. tuberculosis-infected human monocytes also secreted inflammatory cytokines, but exhibited no enhancement of costimulatory or MHC class I molecule expression. These data indicate that infection with M. tuberculosis results in the direct activation and maturation of these DC. In vivo, such activation may facilitate migration to the lymph nodes, and enhance presentation of Ag to T cells, thereby facilitating the induction of the immune response against this pathogen.
In Salmonella typhimurium, ahpC encodes subunit C of alkyl hydroperoxide reductase, an enzyme that reduces organic peroxides. Here, we asked if ahpC could protect cells from reactive nitrogen intermediates (RNI). Salmonella disrupted in ahpC became hypersusceptible to RNI. ahpC from either Mycobacterium tuberculosis or S. typhimurium fully complemented the defect. Unlike protection against cumene hydroperoxide, protection afforded by ahpC against RNI was independent of the reducing flavoprotein, AhpF. Mycobacterial ahpC protected human cells from necrosis and apoptosis caused by RNI delivered exogenously or produced endogenously by transfected nitric oxide synthase. Resistance to RNI appears to be a physiologic function of ahpC. ahpC is the most widely distributed gene known that protects cells directly from RNI, and provides an enzymatic defense against an element of antitubercular immunity.
MHC class II expression was examined in macrophages infected with Mycobacterium tuberculosis. IFN-gamma increased the surface expression of class II molecules in THP-1 cells and this was markedly reduced in cells infected with M. tuberculosis. Despite this effect, steady state levels of HLA-DRalpha, HLA-DRbeta, and invariant (Ii) chains were equivalent in control and infected cells. Metabolic labeling combined with pulse-chase experiments and biochemical analysis showed that the majority of class II molecules in infected cells became resistant to endoglycosidase H, consistent with normal Golgi processing. However, results of intracellular staining and dual color confocal microscopy revealed a significant defect in transport of newly synthesized class II molecules through the endocytic compartment. Thus, compared with findings in control cells, class II molecules in infected cells colocalized to a minimal extent with a lysosomal-associated membrane protein-1+ endosomal compartment. In addition, in contrast to control cells, class II molecules in infected cells failed to colocalize with endocytosed BSA under conditions where this marker is known to label late endosomes, lysosomes, and the MHC class II compartment. Consistent with defective transport along the endocytic pathway, the maturation of SDS-stable class II alphabeta dimers--dependent upon removal of Ii chain and peptide loading of class II dimers in the MHC class II compartment--was markedly impaired in M. tuberculosis-infected cells. These findings indicate that defective transport and processing of class II molecules through the endosomal/lysosomal system is responsible for diminished cell surface expression of MHC class II molecules in cells infected with M. tuberculosis.
Mycobacteria are intracellular pathogens that can survive within macrophage phagosomes, thereby evading host defense strategies by largely unknown mechanisms. We have identified a WD repeat host protein that was recruited to and actively retained on phagosomes by living, but not dead, mycobacteria. This protein, termed TACO, represents a component of the phagosome coat that is normally released prior to phagosome fusion with or maturation into lysosomes. In macrophages lacking TACO, mycobacteria were readily transported to lysosomes followed by their degradation. Expression of TACO in nonmacrophages prevented lysosomal delivery of mycobacteria and prolonged their intracellular survival. Active retention of TACO on phagosomes by living mycobacteria thus represents a mechanism preventing cargo delivery to lysosomes, allowing mycobacteria to survive within macrophages.
The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12),
a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL-12 production by macrophages
and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators
of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins
stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal
pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.
Studies of various membrane trafficking steps over the past year indicate that membranes are tethered together prior to the interaction of v-SNAREs and t-SNAREs across the membrane junction. The tethering proteins identified to date are quite large, being either fibrous proteins or multimeric protein complexes. The tethering factors employed at different steps are evolutionarily unrelated, yet their function seems to be closely tied to the more highly conserved Rab GTPases. Tethering factors may collaborate with Rabs and SNAREs to generate targeting specificity in the secretory pathway.
Recent studies have implicated a family of mammalian Toll-like receptors (TLR) in the activation of macrophages by Gram-negative and Gram-positive bacterial products. We have previously shown that different TLR proteins mediate cellular activation by the distinct CD14 ligands Gram-negative bacterial LPS and mycobacterial glycolipid lipoarabinomannan (LAM). Here we show that viable Mycobacterium tuberculosis bacilli activated both Chinese hamster ovary cells and murine macrophages that overexpressed either TLR2 or TLR4. This contrasted with Gram-positive bacteria and Mycobacterium avium, which activated cells via TLR2 but not TLR4. Both virulent and attenuated strains of M. tuberculosis could activate the cells in a TLR-dependent manner. Neither membrane-bound nor soluble CD14 was required for bacilli to activate cells in a TLR-dependent manner. We also assessed whether LAM was the mycobacterial cell wall component responsible for TLR-dependent cellular activation by M. tuberculosis. We found that TLR2, but not TLR4, could confer responsiveness to LAM isolated from rapidly growing mycobacteria. In contrast, LAM isolated from M. tuberculosis or Mycobacterium bovis bacillus Calmette-Guérin failed to induce TLR-dependent activation. Lastly, both soluble and cell wall-associated mycobacterial factors were capable of mediating activation via distinct TLR proteins. A soluble heat-stable and protease-resistant factor was found to mediate TLR2-dependent activation, whereas a heat-sensitive cell-associated mycobacterial factor mediated TLR4-dependent activation. Together, our data demonstrate that Toll-like receptors can mediate cellular activation by M. tuberculosis via CD14-independent ligands that are distinct from the mycobacterial cell wall glycolipid LAM.
IFN-gamma activates macrophages to kill diverse intracellular pathogens, but does not activate human macrophages to kill virulent Mycobacterium tuberculosis. We tested the hypothesis that this is due to inhibition of IFN-gamma signaling by M. tuberculosis and found that M. tuberculosis infection of human macrophages blocks several responses to IFN-gamma, including killing of Toxoplasma gondii and induction of FcgammaRI. The inhibitory effect of M. tuberculosis is directed at transcription of IFN-gamma-responsive genes, but does not affect proximal steps in the Janus kinase-STAT pathway, as STAT1alpha tyrosine and serine phosphorylation, dimerization, nuclear translocation, and DNA binding are intact in M. tuberculosis-infected cells. In contrast, there is a marked decrease in IFN-gamma-induced association of STAT1 with the transcriptional coactivators CREB binding protein and p300 in M. tuberculosis-infected macrophages, indicating that M. tuberculosis directly or indirectly disrupts this protein-protein interaction that is essential for transcriptional responses to IFN-gamma. Gamma-irradiated M. tuberculosis and isolated cell walls reproduce the effects of live bacteria, indicating that the bacterial component(s) that initiates inhibition of IFN-gamma responses is constitutively expressed. Although lipoarabinomannan has been found to exert effects on macrophages, it does not account for the inhibitory effects of cell walls. These results indicate that one mechanism for M. tuberculosis to evade the human immune response is to inhibit the IFN-gamma signaling pathway, and that the mechanism of inhibition is distinct from that reported for Leishmania donovani or CMV, in that it targets the interaction of STAT1 with the basal transcriptional apparatus.
Macrophages orchestrate innate immunity by phagocytosing pathogens and coordinating inflammatory responses. Effective defence requires the host to discriminate between different pathogens. The specificity of innate immune recognition in Drosophila is mediated by the Toll family of receptors; Toll mediates anti-fungal responses, whereas 18-wheeler mediates anti-bacterial defence. A large number of Toll homologues have been identified in mammals, and Toll-like receptor 4 is critical in responses to Gram-negative bacteria. Here we show that Toll-like receptor 2 is recruited specifically to macrophage phagosomes containing yeast, and that a point mutation in the receptor abrogates inflammatory responses to yeast and Gram-positive bacteria, but not to Gram-negative bacteria. Thus, during the phagocytosis of pathogens, two classes of innate immune receptors cooperate to mediate host defence: phagocytic receptors, such as the mannose receptor, signal particle internalization, and the Toll-like receptors sample the contents of the vacuole and trigger an inflammatory response appropriate to defence against the specific organism.
To test the toxicity of reactive nitrogen intermediates (RNI), including authentic nitric oxide (NO), nitrogen dioxide (NO2), and peroxynitrite anion (ONOO-), a potent oxidant derived from NO and superoxide anion, on various mycobacterial strains including M. tuberculosis.
Relatively avirulent mycobacteria including M. smegmatis and BCG, as well as the pathogenic M. Bovis Ravenel and M. tuberculosis Erdman and the clinical isolate M160 (also known as the C strain) were tested for their susceptibility to the toxic effects of NO, NO2, and ONOO-, Deaerated, NO-saturated solutions as well as an anaerobic in vitro system in which mycobacteria can be exposed to desired concentrations of authentic NO or NO2, were employed in these studies. An in vitro ONOO- killing assay was used to examine the adverse effects of this NO-derived oxidant on the various strains of mycobacteria.
Both NO and NO2 exhibit antimycobacterial activity, with the former being more potent. Results obtained using ONOO- killing assay revealed that while avirulent mycobacteria including BCG and M. smegmatis are susceptible to this NO-derived oxidant, the virulent Erdman strain of M. tuberculosis and M. bovis, as well as the clinical tuberculous isolate M160, are remarkably resistant.
These results suggest that the interactions between RNI and various species of mycobacteria could be highly specific. And since activated macrophages produce peroxynitrite, the significance of the ONOO- resistance of M. tuberculosis strains in relation to intracellular survival deserves further investigation.
The 19-kD antigen is a cell wall-associated lipoprotein present in Mycobacterium tuberculosis and in bacille Calmette-Guérin (BCG) vaccine strains. Expression of the 19-kD antigen as a recombinant protein in two saprophytic mycobacteria-M. vaccae and M. smegmatis-resulted in abrogation of their ability to confer protection against M. tuberculosis in a murine challenge model, and in their ability to prime a DTH response to cross-reactive mycobacterial antigens. Induction of an immune response to the 19-kD antigen by an alternative approach of DNA vaccination had no effect on subsequent M. tuberculosis challenge. These results are consistent with a model in which the presence of the 19-kD protein has a detrimental effect on the efficacy of vaccination with live mycobacteria. Targeted inactivation of genes encoding selected antigens represents a potential route towards development of improved vaccine candidates.
Infection of murine bone-marrow-derived macrophages with viable Mycobacterium tuberculosis (MTB) H37Ra inhibited surface expression of MHC class II (MHC-II) molecules and processing of exogenous antigens for presentation to CD4(+) T hybridoma cells. The inhibition was not dependent on bacterial viability, since it was also produced by exposure to dead bacilli and MTB cytosol preparations, suggesting that it was initiated by a constitutively expressed bacterial component. Northern blot analysis demonstrated that MTB bacilli or cytosol decreased MHC-II mRNA, and immunoprecipitation of biosynthetically labeled molecules confirmed that MHC-II protein synthesis was diminished. Exposure to MTB or MTB cytosol also decreased expression of H2-DM, but H2-DM expression was still sufficient to catalyze conversion of MHC-II to SDS-stable dimers, a measure of MHC-II peptide loading. Thus, infection with MTB decreased both MHC-II and H2-DM expression, but diminished MHC-II synthesis provided the major limitation to antigen processing.
Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.
Nitric oxide (NO) is present in soil and air, and is produced by bacteria, animals and plants. Superoxide (O2-) arises in all organisms inhabiting aerobic environments. Thus, many organisms are likely to encounter peroxynitrite (OONO-), a product of NO and O2- that forms at near diffusion-limited rates, and rapidly decomposes upon protonation through isomerization to nitrate (NO3-; ref. 1) while generating hydroxyl radical (*OH) and nitrogen dioxide radical (*NO2) (refs 2, 3), both more reactive than peroxynitrite's precursors. The oxidative, inflammatory, mutagenic and cytotoxic potential (ref. 4) of peroxynitrite contrasts with the antioxidant, anti-inflammatory and tissue-protective properties ascribed to NO itself. Thus, the ability of cells to cope with peroxynitrite is central in determining the biological consequences of NO production. We considered whether cells might be equipped with enzymes to detoxify peroxynitrite. Peroxiredoxins have been identified in most genomes sequenced, but their functions are only partly understood. Here we show that the peroxiredoxin alkylhydroperoxide reductase subunit C (AhpC) from Salmonella typhimurium catalytically detoxifies peroxynitrite to nitrite fast enough to forestall the oxidation of bystander molecules such as DNA. Results are similar with peroxiredoxins from Mycobacterium tuberculosis and Helicobacter pylori. Thus, peroxynitrite reductase activity may be widespread among bacterial genera.
The ability of dendritic cells (DC) to initiate immune responses in naive T cells is dependent upon a maturation process that allows the cells to develop their potent Ag-presenting capacity. Although immature DC can be derived in vitro by treatment of peripheral blood monocytes with GM-CSF and IL-4, additional signals such as those provided by TNF-alpha, CD40 ligand, or LPS are required for complete maturation and maximum APC function. Because we recently found that microbial lipoproteins can activate monocytes and DC through Toll-like receptor (TLR) 2, we also investigated whether lipoproteins can drive DC maturation. Immature DC were cultured with or without lipoproteins and were monitored for expression of cell surface markers indicative of maturation. Stimulation with lipopeptides increased expression of CD83, MHC class II, CD80, CD86, CD54, and CD58, and decreased CD32 expression and endocytic activity; these lipopeptide-matured DC also displayed enhanced T cell stimulatory capacity in MLR, as measured by T cell proliferation and IFN-gamma secretion. The lipid moiety of the lipopeptide was found to be essential for induction of maturation. Preincubation of maturing DC with an anti-TLR2 blocking Ab before addition of lipopeptide blocked the phenotypic and functional changes associated with DC maturation. These results demonstrate that lipopeptides can stimulate DC maturation via TLR2, providing a mechanism by which products of bacteria can participate in the initiation of an immune response.
Expression of the Mycobacterium tuberculosis 19kDa lipoprotein in saprophytic mycobacteria has been found to reduce their ability to prime a protective response to subsequent virulent challenge in the mouse model. The present study was designed to test whether 19kDa expression has an analogous detrimental effect on the efficacy of BCG vaccination. In contrast to the results in saprophytes, neither overexpression of the 19kDa antigen, nor deletion of the endogenous 19kDa gene altered the ability of BCG to protect against M. tuberculosis challenge in a mouse model.
Cellular organelles in the exocytic and endocytic pathways have a distinctive
spatial distribution and communicate through an elaborate system of vesiculo-tubular
transport. Rab proteins and their effectors coordinate consecutive stages
of transport, such as vesicle formation, vesicle and organelle motility, and
tethering of vesicles to their target compartment. These molecules are highly
compartmentalized in organelle membranes, making them excellent candidates
for determining transport specificity and organelle identity.
Mycobacterium tuberculosis is a highly successful pathogen that parasitizes the macrophages of its host. Its success can be attributed directly to its ability to manipulate the phagosome that it resides in and to prevent the normal maturation of this organelle into an acidic, hydrolytic compartment. As the macrophage is key to clearing the infection, the interplay between the pathogen and its host cell reflects a constant battle for control.
By applying density gradient electrophoresis (DGE) to human macrophages infected with Mycobacterium bovis BCG, we were able to separate three different bacterial fractions representing arrested phagosomes, phagolysosomes and mycobacterial clumps. After further purification of the phagosomal population, we found that isolated phagosomes containing live BCG were arrested in maturation as they exhibited only low amounts of the lysosomal glycoprotein LAMP-1 and processing of the lysosomal hydrolase cathepsin D was blocked. In addition, low amounts of MHC class I and class II molecules and the absence of HLA-DM suggest sequestration of mycobacterial phagosomes from antigen-processing pathways. We further investigated the involvement of the actin-binding protein coronin in intracellular survival of mycobacteria and showed that human coronin, as well as F-actin, were associated with early stages of mycobacterial phagocytosis but not with phagosome maintenance. Therefore, we conclude that the unique DGE migration pattern of arrested phagosomes is not as a result of retention of coronin, but that there are other proteins or lipids responsible for the block in maturation in human macrophages.
The control of acute and chronic Mycobacterium tuberculosis infection is dependent on CD4(+) T cells. In a variety of systems CD8(+) T cell effector responses are dependent on CD4(+) T cell help. The development of CD8(+) T cell-mediated immune responses in the absence of CD4(+) T cells was investigated in a murine model of acute tuberculosis. In vitro and in vivo, priming of mycobacteria-specific CD8(+) T cells was unaffected by the absence of CD4(+) T cells. Infiltration of CD8(+) T cells into infected lungs of CD4(-/-) or wild-type mice was similar. IFN-gamma production by lung CD8(+) T cells in CD4(-/-) and wild-type mice was also comparable, suggesting that emergence of IFN-gamma-producing mycobacteria-specific CD8(+) T cells in the lungs was independent of CD4(+) T cell help. In contrast, cytotoxic activity of CD8(+) T cells from lungs of M. tuberculosis-infected mice was impaired in CD4(-/-) mice. Expression of mRNA for IL-2 and IL-15, cytokines critical for the development of cytotoxic effector cells, was diminished in the lungs of M. tuberculosis-infected CD4(-/-) mice. As tuberculosis is frequently associated with HIV infection and a subsequent loss of CD4(+) T cells, understanding the interaction between CD4(+) and CD8(+) T cell subsets during the immune response to M. tuberculosis is imperative for the design of successful vaccination strategies.
In the present study, we investigated in vivo the infection and APC functions of dendritic cells (DC) and macrophages (Mphi) after administration of live mycobacteria to mice. Experiments were conducted with Mycobacterium bovis bacillus Calmette-Guerin (BCG) or a rBCG expressing a reporter Ag. Following infection of mice, DC and Mphi were purified and the presence of immunogenic peptide/MHC class II complexes was detected ex vivo on sorted cells, as was the secretion of IL-12 p40. We show in this study that DC is a host cell for mycobacteria, and we provide an in vivo detailed picture of the role of Mphi and DC in the mobilization of immunity during the early stages of a bacterial infection. Strikingly, BCG bacilli survive but remain stable in number in the DC leukocyte subset during the first 2 wk of infection. As Ag presentation by DC is rapidly lost, this suggests that DC may represent a hidden reservoir for mycobacteria.