[Show abstract][Hide abstract] ABSTRACT: Autophagy plays a crucial role in the control of bacterial burden during Mycobacterium tuberculosis infection. MicroRNAs (miRNAs) are small non-coding RNAs which regulate immune signaling and inflammation in response to challenge by pathogens. Appreciating the potential of host-directed therapies designed to control autophagy during mycobacterial infection, we focused on the role of miRNAs in regulating M. tuberculosis-induced autophagy in macrophages. Here we demonstrate that M. tuberculosis infection leads to downregulation of miR-17 and concomitant upregulation of its targets Mcl-1 and STAT3, a transcriptional activator of Mcl-1. Forced expression of miR-17 reduces expression of Mcl-1 and STAT3, and also the interaction between Mcl-1 and Beclin-1. This is directly linked to enhanced autophagy, since Mcl-1 overexpression attenuates the effects of miR-17. At the same time, transfection with a kinase-inactive mutant of protein kinase C δ (PKCδ) (an activator of STAT3), augments M. tuberculosis-induced autophagy, and miR-17 overexpression diminishes phosphorylation of PKCδ, suggesting that a miR-17/PKC δ/STAT3 axis regulates autophagy during M. tuberculosis infection.
Full-text · Article · Oct 2015 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Coordinated bacterial cell septation and cell wall biosynthesis require formation of protein complexes at the sites of division and elongation, in a temporally controlled manner. The protein players in these complexes remain incompletely understood in mycobacteria. Using in vitro and in vivo assays, we show that Rv2147c (or SepF) of Mycobacterium tuberculosis (Mtb), interacts with the principal driver of cytokinesis, FtsZ. SepF also interacts with itself both in vitro and in the mycobacterial environment. Amino acid residues 189A, 190K and 215F are required for FtsZ-SepF interaction, and are conserved across Gram-positive bacteria. SepF enhances FtsZ polymer formation in vitro. Using M. smegmatis (Msmeg) as a surrogate system, we observed that sepFMSMEG is essential. Knock down of SepF led to cell elongation, defective growth and failure of FtsZ to localize to the site of division, suggesting that SepF assists FtsZ localization at the site of divison. Further, SepF interacts with MurG, a peptidoglycan-synthesizing enzyme, both in vitro and in vivo. The failure of MurG to localize at mid-cell when sepF is knocked down, suggests that SepF could serve as a link between cell division and peptidoglycan synthesis. SepF emerges as a newly identified essential player in cell division in mycobacteria.
[Show abstract][Hide abstract] ABSTRACT: The resuscitation promoting factors (Rpfs) of Mycobacterium tuberculosis are hydrolytic enzymes which are required for resuscitation of dormant cells. RpfB, a peptidoglycan remodelling enzyme similar to the lytic transglycosylase of Escherichia coli, is required for reactivation of M. tuberculosis from chronic infection in vivo, underscoring the need to understand its transcriptional regulation. Here we identify the transcriptional and translational start points of rpfB, and suggest from rpf promoter driven GFP expression and in vitro transcription assays that its transcription possibly occurs in a SigB-dependent manner. We further demonstrate that rpfB transcription is regulated by MtrA, the response regulator of the essential two-component system, MtrAB. Association of MtrA with the rpfB promoter region in vivo, was confirmed by chromatin immunoprecipitation analysis. Electrophoretic mobility shift assays (EMSAs) revealed a loose direct repeat sequence associated with MtrA binding. Binding of MtrA was enhanced upon phosphorylation. MtrA could be pulled down from lysates of M. tuberculosis using a biotinylated DNA fragment encompassing the MtrA binding site on the rpfB promoter, confirming that MtrA binds to the rpfB promoter. Enhanced GFP fluorescence driven by the rpfB promoter, upon deletion of the MtrA binding site, and repression of rpfB expression upon overexpression of MtrA, suggested that MtrA functions as a repressor of rpfB transcription. This was corroborated by EMSA showing diminished association of RNA polymerase with the rpfB promoter in the presence of MtrA. In vitro transcription assay confirmed that MtrA inhibits RNAP-driven rpfB transcription.
[Show abstract][Hide abstract] ABSTRACT: The resuscitation-promoting factors of Mycobacterium tuberculosis are hydrolytic enzymes,
which are required for resuscitation of dormant cells. RpfB, a peptidoglycan remodelling
enzyme similar to the lytic transglycosylase of Escherichia coli, is required for reactivation of
M. tuberculosis from chronic infection in vivo, underscoring the need to understand its
transcriptional regulation. Here, we identified the transcriptional and translational start points
of rpfB, and suggested from rpf promoter-driven GFP expression and in vitro transcription
assays that its transcription possibly occurs in a SigB-dependent manner. We further
demonstrated that rpfB transcription is regulated by MtrA – the response regulator of the
essential two-component system MtrAB. Association of MtrA with the rpfB promoter region
in vivo was confirmed by chromatin immunoprecipitation analysis. Electrophoretic mobility
shift assays (EMSAs) revealed a loose direct repeat sequence associated with MtrA
binding. Binding of MtrA was enhanced upon phosphorylation. MtrA could be pulled down
from lysates of M. tuberculosis using a biotinylated DNA fragment encompassing the
MtrA-binding site on the rpfB promoter, confirming that MtrA binds to the rpfB promoter.
Enhanced GFP fluorescence driven by the rpfB promoter, upon deletion of the
MtrA-binding site, and repression of rpfB expression, upon overexpression of MtrA,
suggested that MtrA functions as a repressor of rpfB transcription. This was corroborated
by EMSAs showing diminished association of RNA polymerase (RNAP) with the rpfB
promoter in the presence of MtrA. In vitro transcription assays confirmed that MtrA inhibits
RNAP-driven rpfB transcription.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is an intracellular catabolic process which is required to maintain cellular homeostasis. Pathogen-elicited host cell autophagy may favour containment of infection or may help in bacterial survival. Pathogens have developed the ability to modulate host autophagy. The secreted antigen HP0175, a peptidyl prolyl cis,trans isomerase of Helicobacter pylori has moonlighting functions with reference to host cells. Here we show that it executes autophagy in gastric epithelial cells. Autophagy is dependent on the unfolded protein response (UPR) which activates the expression of PKR-like ER kinase (PERK). This is accompanied by phosphorylation of eIF2-α and transcriptional activation of ATF4 and CHOP. Knockdown of UPR- related genes inhibits the conversion of LC3I to LC3-II, a marker of autophagy. The autophagy- inducing ability of H. pylori is compromised when cells are infected with an isogenic hp0175 mutant. Autophagy precedes apoptosis. Silencing of BECLIN 1 augments cleavage of caspase 3 as well as apoptosis. Increased apoptosis of gastric epithelial cells is known to be linked to H. pylori- mediated gastric inflammation and carcinogenesis. To the best of our knowledge, this study provides the first demonstration of how HP0175, endowed with moonlighting functions, links UPR- dependent autophagy and apoptosis during H. pylori infection.
No preview · Article · Dec 2014 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis is endowed with the abi-lity to persist within its intracellular niche for years, often tilting the balance of the host-pathogen interac-tion in its favour. The host resists infection by releas-ing damaging free radicals, pushing the pathogen towards lysosomal degradation, releasing an arsenal of cytokines for triggering adaptive immunity and facilitating apoptosis for effective T cell antigen pre-sentation. The bacterium counters these mechanisms and also metabolically reprograms the macrophage to its own benefit. Recent advances in these areas are reviewed here.
[Show abstract][Hide abstract] ABSTRACT: Polyphosphate (Poly P) metabolism regulates the stress response in mycobacteria. Here we describe the regulatory architecture of a signal transduction system involving the two-component system SenX3-RegX3, the extracytoplasmic function sigma factor Sigma E (SigE) and the poly P-synthesizing enzyme polyphosphate kinase 1 (PPK1). The ppk1 promoter of M. tuberculosis is activated under phosphate starvation. This is attenuated upon deletion of an imperfect palindrome likely representing a binding site for the response regulator RegX3, a component of the two-component system (TCS) SenX3-RegX3 which responds to phosphate starvation. Binding of phosphorylated RegX3 to this site was confirmed by electrophoretic mobility shift assay (EMSA). Ppk1 promoter activity was abrogated upon deletion of a putative SigE binding site. Pull-down of SigE from M. tuberculosis lysates of phosphate-starved cells with a biotinylated DNA harbouring the SigE binding site, confirmed the likely binding of SigE to the ppk1 promoter. In vitro transcription corroborated the involvement of SigE in ppk1 transcription. Finally, the overexpression of RseA (anti-SigE) attenuated ppk1 expression under phosphate starvation supporting the role of SigE in ppk1 transcription. The regulatory elements identified in ppk1 transcription in this study, combined with our earlier observations that PPK1 is itself capable of regulating sigE expression via the MprAB TCS, suggest the presence of multiple positive feedback loops in this signaling circuit. In combination with the sequestering effect of RseA, we hypothesize that this architecture could be linked to bistability in the system which, in turn, could be a key element of persistence in M. tuberculosis.
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori is an intriguing bacterium because of its ability to survive at low pH in the stomach and its relationship with gastric inflammation and cancer. Among its armamentarium of virulence factors is HP0175, a peptidylprolyl isomerase (PPIase), which is secreted and is a major antigen in patients with H. pylori-induced pathology. This review summarizes the moonlighting functions of HP0175, revealing that this secreted protein-folding catalyst regulates cell signalling in gastric epithelial cells and in monocytic cells to modulate the inflammatory response and apoptosis during H. pylori infection. In addition, it elicits a Th17 response thereby modulating the adaptive immune response. These activities reveal the importance of protein moonlighting in the generation of H. pylori-induced disease states.
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori infection induces a chronic gastric inflammatory infiltrate. This study was undertaken to evaluate the type of the innate immune responses elicited by the secreted peptidyl-prolyl cis-trans isomerase of H. pylori (HP0175). The cytokine production induced by HP0175 in neutrophils, and monocytes was evaluated. HP0175 was able to induce the expression of IL-23 in neutrophils, and monocytes, and IL-6, IL-1beta and TGF-beta in monocytes. These findings indicate that HP0175 is able to promote the activation of innate cells and the production of a cytokine milieu that may favour the development of Th17 response.
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori infection is characterized by an inflammatory infiltrate, consisting mainly of neutrophils and T cells. This study was undertaken to evaluate the type of gastric T cell response elicited by the secreted peptidyl prolyl cis, trans-isomerase of H. pylori (HP0175) in patients with distal gastric adenocarcinoma. The cytokine profile and the effector functions of gastric tumor-infiltrating lymphocytes (TILs) specific for HP0175 was investigated in 20 patients with distal gastric adenocarcinoma and H. pylori infection. The helper function of HP0175-specific TILs for monocyte MMP-2, MMP-9, and VEGF production was also investigated. TILs cells from H. pylori infected patients with distal gastric adenocarcinoma produced Interleukin (IL)-17 and IL-21 in response to HP0175. HP0175-specific TILs showed poor cytolytic activity while expressing helper activity for monocyte MMP-2, MMP-9 and VEGF production. These findings indicate that HP0175 is able to drive gastric Th17 response. Thus, HP0175, by promoting pro-inflammatory low cytotoxic TIL response, matrix degradation and pro-angiogenic pathways, may provide a link between H. pylori and gastric cancer.
Full-text · Article · Oct 2012 · Internal and Emergency Medicine
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis (M.tb.) replicates in host macrophages to cause tuberculosis. We have investigated the role of miRNAs in M.tb.-infected murine RAW264.7 cells and bone marrow-derived macrophages (BMDMs), focusing on miR-155, the most highly upregulated miRNA. We observed that miR-155 upregulation is directly linked to the attenuation of expression of BTB and CNC homology 1 (Bach1) and SH2-containing inositol 5'-phosphatase (SHIP1). Bach1 is a transcriptional repressor of haem oxygenase-1 (HO-1), whereas SHIP1 inhibits the activation of the serine/threonine kinase AKT. We hypothesize that M.tb.-induced miR-155 induction leads to repression of Bach1, which augments the expression of HO-1, a documented activator of the M.tb. dormancyregulon. SHIP1 repression facilitates AKT activation, which is required for M.tb. survival. In addition, M.tb.-induced miR-155 inhibits expression of cyclooxygenase-2 (Cox-2) and interleukin-6 (Il-6), two modulators of the innate immune response. Importantly, we observed that the virulence-associated secreted protein ESAT-6 plays a key role in miR-155 induction and its subsequent effects on Bach1 and SHIP1 repression. Inhibition of miR-155 hindered survival of M.tb. in RAW264.7 and in murine BMDMs. Thus, our results offer new insights into the role of miRNAs in modulation of the host innate immune response by M.tb. for its own benefit.
No preview · Article · Jun 2012 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: A prerequisite for successful establishment of Mycobacterium tuberculosis in the host is its ability to survive after internalization in alveolar macrophages that they encounter after inhalation. The innate immune response protects some individuals to the extent that they remain uninfected. In others, the innate immune system is not sufficient and an adaptive immune response is generated. This is usually protective, but not sterilizing, and individuals remain latently infected. In susceptible individuals, M. tuberculosis successfully escapes immune surveillance. The interplay between the host innate immune response and the bacterial mechanisms in play to offset this response, is of considerable importance in dictating the course of the disease. In order to gain an understanding of this interplay it is of importance to analyze how M. tuberculosis interacts with innate immune receptors and makes its entry into macrophages, how it subverts the bactericidal effects of macrophages, and dampens processes required for protective immunity, including cytokine and chemokine induction. This review will focus on some of the Indian efforts in these areas, concentrating mainly on the interaction of M. tuberculosis with macrophages and dendritic cells (DCs). The role of the PE/PPE family of proteins in regulating the immune response, will not be discussed in this chapter. The genome-wide approaches of analyzing host-M. tuberculosis interactions will also be discussed elsewhere.
No preview · Article · May 2011 · Tuberculosis (Edinburgh, Scotland)
[Show abstract][Hide abstract] ABSTRACT: A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity.
In the present study, we characterise quantitatively the single cell promoter activity of the three key genes, namely, mprA, sigE and rel, in the stringent response pathway with gfp as the reporter. The origin of bimodality in the GFP distribution lies in two stable expression states, i.e., bistability. We develop a theoretical model to study the dynamics of the stringent response pathway. The model incorporates a recently proposed mechanism of bistability based on positive feedback and cell growth retardation due to protein synthesis. Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time. This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise.
The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.
Full-text · Article · Jan 2011 · BMC Systems Biology
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppA(MTB).
OppA(MTB) was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppA(MTB) confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1beta, IL-6 and TNF-alpha was also compromised after inactivation of oppD.
Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis.
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus-1 (HIV-1) impairs tumor necrosis factor-alpha (TNF-alpha)-mediated macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb). HIV Nef protein plays an important role in the pathogenesis of AIDS. We have tested the hypothesis that exogenous Nef is a factor that inhibits TNF-alpha production/apoptosis in macrophages infected with Mtb. We demonstrate that Mtb and Nef individually trigger TNF-alpha production in macrophages. However, TNF-alpha production is dampened when the two are present simultaneously, probably through cross-regulation of the individual signaling pathways leading to activation of the TNF-alpha promoter. Mtb-induced TNF-alpha production is abrogated upon mutation of the Ets, Egr, Sp1, CRE, or AP1 binding sites on the TNF-alpha promoter, whereas Nef-mediated promoter activation depends only on the CRE and AP1 binding sites, pointing to differences in the mechanisms of activation of the promoter. Mtb-dependent promoter activation depends on the mitogen-activated kinase (MAPK) kinase kinase ASK1 and on MEK/ERK signaling. Nef inhibits ASK1/p38 MAPK-dependent Mtb-induced TNF-alpha production probably by inhibiting binding of ATF2 to the TNF-alpha promoter. It also inhibits MEK/ERK-dependent Mtb-induced binding of FosB to the promoter. Nef-driven TNF-alpha production occurs in an ASK1-independent, Rac1/PAK1/p38 MAPK-dependent, and MEK/ERK-independent manner. The signaling pathways used by Mtb and Nef to trigger TNF-alpha production are therefore distinctly different. In addition to attenuating Mtb-dependent TNF-alpha promoter activation, Nef also reduces Mtb-dependent TNF-alpha mRNA stability probably through its ability to inhibit ASK1/p38 MAPK signaling. These results provide new insight into how HIV Nef probably exacerbates tuberculosis infection by virtue of its ability to dampen Mtb-induced TNF-alpha production.
[Show abstract][Hide abstract] ABSTRACT: The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, namely PknA (encoded by Rv0014c) and FtsZ-interacting protein A, FipA (encoded by Rv0019c) and the products of the division cell wall (dcw) cluster, namely FtsZ and FtsQ. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A) showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress.