[Show abstract][Hide abstract] ABSTRACT: Background:
Bronchial epithelial ciliary dysfunction is an important feature of asthma. We sought to determine the role in asthma of neutrophilic inflammation and NADPH oxidases in ciliary dysfunction.
Bronchial epithelial ciliary function was assessed by video-microscopy in fresh ex vivo epithelial strips from asthmatics stratified by their sputum cell differentials and in cultures from healthy controls and asthmatics. Bronchial epithelial oxidative damage was determined by 8-oxo-dG expression. NOX/DUOX expression was assessed in bronchial epithelial cells by microarrays, with NOX4, DUOX1/2 expression assessed in bronchial biopsies. Ciliary dysfunction following NADPH oxidase inhibition, using GKT137831, was evaluated in fresh epithelial strips from asthmatics and a murine model of ovalbumin sensitisation and challenge.
Ciliary beat frequency was impaired in asthmatics with sputum neutrophilia (n=11) versus those without (n=10) (5.8 [0.6] versus 8.8 [0.5]Hz; P=0.003) and was correlated with sputum neutrophil count (r=-0.70; P<0.001). Primary bronchial epithelial cells expressed DUOX1/2 and NOX4. 8-oxo-dG and NOX4, were elevated in neutrophilic versus non-neutrophilic asthmatics, DUOX1 was elevated in both, and DUOX2 was elevated in non-neutrophilic asthma in vivo. In primary epithelial cultures ciliary dysfunction did not persist, although NOX4 expression and reactive oxygen species generation was increased from subjects with neutrophilic asthma. GKT137831 both improved ciliary function in ex vivo epithelial strips (n=13), relative to the intensity of neutrophilic inflammation, and abolished ciliary dysfunction in the murine asthma model without a reduction in inflammation.
Ciliary dysfunction is increased in neutrophilic asthma associated with increased NOX4 expression and is attenuated by NADPH oxidase inhibition.
[Show abstract][Hide abstract] ABSTRACT: Inflammation is a crucial part of innate immune responses but if imbalanced can lead to serious clinical conditions or even death. Cytokines regulate inflammation and studies report their impact on clinical outcome. However, host and pathogen genetic background influence cytokine production making it difficult to evaluate, which inflammatory profiles (if any), relates to improved prognosis.
is a common human pathogen associated with asymptomatic nasopharyngeal carriage. Infrequently it can lead to a wide range of diseases with a high morbidity and mortality rate. Studies show that both pneumococcal serotype and host genetic background affect development of the disease and contribute to variation in inflammatory responses.
In this study we investigated impact of the host and pneumococcal genetic background on pulmonary cytokine responses and their relationship to animal survival. Two inbred mouse strains, BALB/c and CBA/Ca, were infected with ten pneumococcal strains and the concentration of six pulmonary cytokines was measured at 6h and 24h post-infection. Collected data were analysed by Principal Component Analysis to identify whether there is any pattern in the observed cytokine variation. Our results show that each of the host-pneumococcus combination resulted in a unique pattern of cytokine response and although, host and pneumococcal background was at the core of the observed variation the resulting cytokine profile was associated with animal survival. Therefore our results indicate that although alternative inflammatory profiles are generated during pneumococcal infection, a common pattern emerged that determined clinical outcome of pneumococcal infections.
Preview · Article · Jan 2016 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: For generation of energy the important human pathogen
relies on host derived sugars, including β-glucoside analogs. The catabolism of these nutrients involves the action of 6-phospho-β-glucosidase to convert them into usable monosaccharaides. In this study, we characterized a 6-phospho-β-glucosidase (BglA3), encoded by SPD_0247. We found that this enzyme has a cell membrane localization and is active only against a phosphorylated substrate. A mutated pneumococcal ΔSPD0247 strain had reduced 6-phospho-glucosidase activity, and was attenuated in growth on cellobiose and hyaluronic acid compared to the wild type D39. ΔSPD0247-infected mice survived significantly longer than the wild type infected cohort, and the colony counts of the mutant were less than those of the wild type in the lungs. The expression of SPD_0247 in
harvested from infected tissues was significantly increased relative to its expression
on glucose. Additionally, ΔSPD0247 is severely impaired in its attachment to an abiotic surface. These results indicate the importance of β-glucoside metabolism in pneumococcal survival and virulence.
Full-text · Article · Nov 2015 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: Pneumolysin is a cholesterol-dependent cytolysin (CDC) and virulence factor of Streptococcus pneumoniae. It kills cells by forming pores assembled from oligomeric rings in cholesterol-containing membranes. Cryo-EM has revealed the structures of the membrane-surface bound pre-pore and inserted-pore oligomers, however the molecular contacts that mediate these oligomers are unknown because high-resolution information is not available. Here we have determined the crystal structure of full-length pneumolysin at 1.98 Å resolution. In the structure, crystal contacts demonstrate the likely interactions that enable polymerisation on the cell membrane and the molecular packing of the pre-pore complex. The hemolytic activity is abrogated in mutants that disrupt these intermolecular contacts, highlighting their importance during pore formation. An additional crystal structure of the membrane-binding domain alone suggests that changes in the conformation of a tryptophan rich-loop at the base of the toxin promote monomer-monomer interactions upon membrane binding by creating new contacts. Notably, residues at the interface are conserved in other members of the CDC family, suggesting a common mechanism for pore and pre-pore assembly.
[Show abstract][Hide abstract] ABSTRACT: The human pathogen Streptococcus pneumoniae is a strictly fermentative organism that relies on glycolytic metabolism to obtain energy. In the human nasopharynx S. pneumoniae encounters glycoconjugates composed of a variety of monosaccharides, which can potentially be used as nutrients once depolymerized by glycosidases. Therefore, it is reasonable to hypothesise that the pneumococcus would rely on these glycan-derived sugars to grow. Here, we identified the sugar-specific catabolic pathways used by S. pneumoniae during growth on mucin. Transcriptome analysis of cells grown on mucin showed specific upregu-lation of genes likely to be involved in deglycosylation, transport and catabolism of galac-tose, mannose and N acetylglucosamine. In contrast to growth on mannose and N-acetylglucosamine, S. pneumoniae grown on galactose reroute their metabolic pathway from homolactic fermentation to a truly mixed acid fermentation regime. By measuring intra-cellular metabolites, enzymatic activities and mutant analysis, we provide an accurate map of the biochemical pathways for galactose, mannose and N-acetylglucosamine catabolism in S. pneumoniae. Intranasal mouse infection models of pneumococcal colonisation and disease showed that only mutants in galactose catabolic genes were attenuated. Our data pinpoint galactose as a key nutrient for growth in the respiratory tract and highlights the importance of central carbon metabolism for pneumococcal pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM) and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.
[Show abstract][Hide abstract] ABSTRACT: Dairy products, in particular soft cheeses, pose a major concern to the dairy industry and public health authorities as they are the leading source of listeriosis outbreaks, a severe foodborne infection affecting pregnant women, children, elderly and immunocompromised people, with a high (20–30%) mortality rate. Cheeses offer a suitable environment for the survival and growth of Listeria monocytogenes, allowing this pathogen to display tolerance responses that can favour its presence in cheese and persistence in dairy processing plants. Extensive food safety regulations in the EU towards prevention of contamination of dairy products with L. monocytogenes have been implemented. However due to the specific abilities of this pathogen to overcome the processing hurdles, its control remains a challenge. Compliance with the Good Manufacturing Practices, observation of Hazard Analysis Critical Control Points (HACCP) and the surveillance of the pathogen in the cheese processing environment are crucial to provide consumers with a safe product. This review aims to provide an overview on the current knowledge about the potential for the transmission of L. monocytogenes in cheese and its abilities to overcome the challenging processing conditions and implications for the behaviour of the pathogen in the host.
Full-text · Article · Nov 2014 · Food Research International
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans including otitis media, bacteremia, meningitis and pneumonia. However, the mechanisms enabling pneumococcal survival in host, and cause disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end-products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo NMR measurements of glucose metabolism in non-growing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection, and leads to a significant reduction in virulence in pneumonia model that develops after intranasal infection, likely due to decrease in energy generation and virulence gene expression.
Full-text · Article · Sep 2014 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine
infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate
RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added
oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest
that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux
pumps can be used to eliminate non-growing mycobacteria.
[Show abstract][Hide abstract] ABSTRACT: Rationale: Respiratory syncytial virus (RSV) and Streptococcus pneumoniae are major respiratory pathogens. Co-infection with RSV and S. pneumoniae is associated with severe and often fatal pneumonia but the molecular basis for this remains unclear. Objectives: To determine if the interaction between RSV and pneumococci enhances pneumococcal virulence. Methods: We used confocal microscopy and western blot to identify the receptors involved in direct binding of RSV and pneumococci, the effects of which were studied in both in vivo and in vitro models of infection. Human ciliated respiratory epithelial cell cultures were infected with RSV for 72h and then challenged with pneumococci. Pneumococci were collected after 2h exposure and changes in gene expression determined using qRT-PCR. Results Following incubation with RSV or purified G protein, pneumococci demonstrated a significant increase in the inflammatory response and bacterial adherence to human ciliated epithelial cultures and markedly increased virulence in a pneumonia model in mice. This was associated with extensive changes in the pneumococcal transcriptome and significant upregulation in the expression of key pneumococcal virulence genes, including the gene for the pneumococcal toxin, pneumolysin. We show that mechanistically this is due to RSV G glycoprotein binding to penicillin binding protein 1a. Conclusion The direct interaction between a respiratory virus protein and the pneumococcus resulting in increased bacterial virulence and worsening disease outcome is a new paradigm in respiratory infection.
Full-text · Article · Jun 2014 · American Journal of Respiratory and Critical Care Medicine
[Show abstract][Hide abstract] ABSTRACT: Chronic obstructive pulmonary disease (COPD) is characterized by long periods of stable symptoms, but exacerbations occur,
which result in a permanent worsening of symptoms. Previous studies have shown a link between bacterial colonization of the
lower airways of COPD sufferers and an increase in exacerbation frequency. One of the most frequent bacterial colonizers is
Streptococcus pneumoniae. To mimic this aspect of COPD, a murine model of low-level pneumococcal colonization in the lung has been developed, in which
S. pneumoniae persisted in the lungs for at least 28 days. From day 14 postinfection, bacterial numbers remained constant until at least
28 days postinfection, and animals showed no outward signs of disease. The bacterial presence correlated with a low-level
inflammatory response that was localized to small foci across the left and inferior lobes of the lung. The cellular response
was predominantly monocytic, and focal fibroplasia was observed at the airway transitional zones. Physiological changes in
the lungs were investigated with a Forced Maneuvers system. This new model provides a means of study of a long-term pulmonary
infection with a human pathogen in a rodent system. This is an excellent tool for the development of future models that mimic
complex respiratory diseases such as COPD and asthma.
Preview · Article · May 2014 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae and Listeria monocytogenes, pathogens which can cause severe infectious disease in human, were used to infect properdin-deficient and wildtype mice. The aim was to deduce a role for properdin, positive regulator of the alternative pathway of complement activation, by comparing and contrasting the immune response of the two genotypes in vivo. We show that properdin-deficient and wildtype mice mounted antipneumococcal serotype-specific IgM antibodies, which were protective. Properdin-deficient mice, however, had increased survival in the model of streptococcal pneumonia and sepsis. Low activity of the classical pathway of complement and modulation of FcγR2b expression appear to be pathogenically involved. In listeriosis, however, properdin-deficient mice had reduced survival and a dendritic cell population that was impaired in maturation and activity. In vitro analyses of splenocytes and bone marrow-derived myeloid cells support the view that the opposing outcomes of properdin-deficient and wildtype mice in these two infection models is likely to be due to a skewing of macrophage activity to an M2 phenotype in the properdin-deficient mice. The phenotypes observed thus appear to reflect the extent to which M2- or M1-polarised macrophages are involved in the immune responses to S. pneumoniae and L. monocytogenes. We conclude that properdin controls the strength of immune responses by affecting humoral as well as cellular phenotypes during acute bacterial infection and ensuing inflammation.
Electronic supplementary material
The online version of this article (doi:10.1007/s00430-013-0324-z) contains supplementary material, which is available to authorized users.
Full-text · Article · Apr 2014 · Medical Microbiology and Immunology
[Show abstract][Hide abstract] ABSTRACT: Modern medicine has established three central antimicrobial therapeutic concepts: vaccination, antibiotics, and, recently, the use of active immunotherapy to enhance the immune response toward specific pathogens. The efficacy of vaccination and antibiotics is limited by the emergence of new pathogen strains and the increased incidence of antibiotic resistance. To date, immunotherapy development has focused mainly on cytokines. Here we report the successful therapeutic application of a complement component, a recombinant form of properdin (Pn), with significantly higher activity than native properdin, which promotes complement activation via the alternative pathway, affording protection against N. menigitidis and S. pneumoniae. In a mouse model of infection, we challenged C57BL/6 WT mice with N. menigitidis B-MC58 6 h after i.p. administration of Pn (100 µg/mouse) or buffer alone. Twelve hours later, all control mice showed clear symptoms of infectious disease while the Pn treated group looked healthy. After 16 hours, all control mice developed sepsis and had to be culled, while only 10% of Pn treated mice presented with sepsis and recoverable levels of live Meningococci. In a parallel experiment, mice were challenged intranasally with a lethal dose of S. pneumoniae D39. Mice that received a single i.p. dose of Pn at the time of infection showed no signs of bacteremia at 12 h postinfection and had prolonged survival times compared with the saline-treated control group (P < 0.0001). Our findings show a significant therapeutic benefit of Pn administration and suggest that its antimicrobial activity could open new avenues for fighting infections caused by multidrug-resistant neisserial or streptococcal strains.
Full-text · Article · Mar 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae is an important human pathogen responsible for high mortality and morbidity worldwide. The susceptibility to pneumococcal infections is controlled by as yet unknown genetic factors. To elucidate these factors could help to develop new medical treatments and tools to identify those most at risk. In recent years genome wide association studies (GWAS) in mice and humans have proved successful in identification of causal genes involved in many complex diseases for example diabetes, systemic lupus or cholesterol metabolism. In this study a GWAS approach was used to map genetic loci associated with susceptibility to pneumococcal infection in 26 inbred mouse strains. As a result four candidate QTLs were identified on chromosomes 7, 13, 18 and 19. Interestingly, the QTL on chromosome 7 was located within S. pneumoniae resistance QTL (Spir1) identified previously in a linkage study of BALB/cOlaHsd and CBA/CaOlaHsd F2 intercrosses. We showed that only a limited number of genes encoded within the QTLs carried phenotype-associated polymorphisms (22 genes out of several hundred located within the QTLs). These candidate genes are known to regulate TGFβ signalling, smooth muscle and immune cells functions. Interestingly, our pulmonary histopathology and gene expression data demonstrated, lung vasculature plays an important role in resistance to pneumococcal infection. Therefore we concluded that the cumulative effect of these candidate genes on vasculature and immune cells functions as contributory factors in the observed differences in susceptibility to pneumococcal infection. We also propose that TGFβ-mediated regulation of fibroblast differentiation plays an important role in development of invasive pneumococcal disease. Gene expression data submitted to the NCBI Gene Expression Omnibus Accession No: GSE49533
SNP data submitted to NCBI dbSNP Short Genetic Variation http://www.ncbi.nlm.nih.gov/projects/SNP/snp_viewTable.cgi?handle=MUSPNEUMONIA.
[Show abstract][Hide abstract] ABSTRACT: Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically,
we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator
of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression
of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial
death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination
therapies for persistent infectious diseases, such as tuberculosis.
Full-text · Article · Mar 2014 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.