Mycobacterium tuberculosis subverts the TLR-2 - MyD88 pathway to facilitate its translocation into the cytosol

Mycobacterium tuberculosis (M.tb) has evolved mechanisms to evade its destruction in phagolysosomes, where it successfully survives and replicates within phagocytes. Recent studies have shown that virulent strains of M.tb can translocate from the phagosome into the cytosol of dendritic cells (DC). The molecular mechanisms by which virulent M.tb strains can escape the phagosome remain unknown. Here we show that the virulent M.tb strain H37Rv, but not the vaccine strain Bacille Calmette-Guérin (BCG), escapes from the phagolysosome and enters the cytosol by interfering with the TLR-2-MyD88 signaling pathway. Using H37Rv mutants, we further demonstrate that the region of difference-1 (RD-1) locus and ESAT-6, a gene within the RD-1 locus, play an important role in the capacity of M.tb to migrate from the phagosome to the cytosol of macrophages. H37Rv, BCG, H37RvΔRD1, and H37RvΔESAT6 were able to translocate to the cytosol in macrophages derived from TLR-2- and MyD88-deficient animals, whereas only virulent H37Rv was able to enter the cytosol in macrophages from wild type mice. Therefore, signaling through the TLR-2–MyD88 pathway in macrophages plays an important role in confining M.tb within phagolysomes. Virulent strains of M.tb have evolved mechanisms to subvert this pathway, thus facilitating their translocation to the cytosol and to escape the toxic microenvironment of the phagosome or phagolysosome

Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: A non-conventional hemolysin and a ribosomal RNA methyl transferase

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis </it>is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous <it>Mycobacterium tuberculosis </it>genes are being characterized. In this study we report the dual functionality of <it>tlyA </it>gene product of <it>Mycobacterium tuberculosis </it>annotated as Rv1694, a 268 amino acid long basic protein.</p> <p>Results</p> <p>The recombinant purified Rv1694 protein was found to exhibit hemolytic activity <it>in vitro</it>. It showed concentration and time-dependent hemolysis of rabbit and human erythrocytes. Multiple oligomeric forms (dimers to heptamers) of this protein were seen on the membranes of the lysed erythrocytes. Like the oligomers of conventional, well-known, pore-forming toxins, the oligomers of Rv1694 were found to be resistant to heat and SDS, but were susceptible to reducing agents like β-mercaptoethanol as it had abolished the hemolytic activity of Rv1694 indicating the role of disulfide bond(s). The Rv1694 generated <it>de novo </it>by <it>in vitro </it>transcription and translation also exhibited unambiguous hemolysis confirming the self assembly and oligomerization properties of this protein. Limited proteolytic digestion of this protein has revealed that the amino terminus is susceptible while in solution but is protected in presence of membrane. Striking feature of Rv1694 is its presence on the cell wall of <it>E. coli </it>as visualized by confocal microscopy. The surface expression is consistent with the contact dependent haemolytic ability of <it>E. coli </it>expressing this protein. Also, immune serum specific to this protein inhibits the contact dependent hemolysis. Moreover, Rv1694 protein binds to and forms stable oligomers on the macrophage phagosomal membranes. In addition to all these properties, <it>E. coli </it>expressing Rv1694 was found to be susceptible to the antibiotic capreomycin as its growth was significantly slower than mock vector transformed <it>E. coli</it>. The S30 extract of <it>E. coli </it>expressing the Rv1694 had poor translational activity in presence of capreomycin, further confirming its methylation activity. Finally, incorporation of methyl group of [³H]-S-adenosylmethionine in isolated ribosomes also confirmed its methylation activity.</p> <p>Conclusions</p> <p>The Rv1694 has an unusual dual activity. It appears to contain two diverse functions such as haemolytic activity and ribosomal RNA methylation activity. It is possible that the haemolytic activity might be relevant to intra-cellular compartments such as phagosomes rather than cell lysis of erythrocytes and the self-assembly trait may have a potential role after successful entry into macrophages by <it>Mycobacterium tuberculosis</it>.</p

Membrane Bound Monomer of Staphylococcal α-Hemolysin Induces Caspase Activation and Apoptotic Cell Death despite Initiation of Membrane Repair Pathway

BACKGROUND: Wild type Staphylococcal alpha-hemolysin (alpha-HL) assembly on target mammalian cells usually results in necrotic form of cell death; however, caspase activation also occurs. The pathways of caspase activation due to binding/partial assembly by alpha-HL are unknown till date. RESULTS: Cells treated with H35N (a mutant of alpha-HL that remains as membrane bound monomer), have been shown to accumulate hypodiploid nuclei, activate caspases and induce intrinsic mitochondrial apoptotic pathway. We have earlier shown that the binding and assembly of alpha-HL requires functional form of Caveolin-1 which is an integral part of caveolae. In this report, we show that the caveolae of mammalian cells, which undergo a continuous cycle of 'kiss and run' dynamics with the plasma membrane, have become immobile upon the binding of the monomer. The cells treated with H35N were unable to recover despite activation of membrane repair mechanism involving caspase-1 dependent activation of sterol regulatory element binding protein-1. CONCLUSIONS: This is for the first time we show the range of cellular changes and responses that take place immediately after the binding of the monomeric form of staphylococcal alpha-hemolysin

Mycobacterium tuberculosis TlyA protein negatively regulates T helper (Th) 1 and Th17 differentiation and promotes tuberculosis pathogenesis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence

Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence

The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as 'hemolysin' which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms

Mycobacterium Tuberculosis Arrests Host Cycle At The G1/S Transition To Establish Long Term Infection

Signals modulating the production of Mycobacterium tuberculosis (Mtb) virulence factors essential for establishing long-term persistent infection are unknown. The WhiB3 redox regulator is known to regulate the production of Mtb virulence factors, however the mechanisms of this modulation are unknown. To advance our understanding of the mechanisms involved in WhiB3 regulation, we performed Mtb in vitro, intraphagosomal and infected host expression analyses. Our Mtb expression analyses in conjunction with extracellular flux analyses demonstrated that WhiB3 maintains bioenergetic homeostasis in response to available carbon sources found in vivo to establish Mtb infection. Our infected host expression analysis indicated that WhiB3 is involved in regulation of the host cell cycle. Detailed cell-cycle analysis revealed that Mtb infection inhibited the macrophage G1/S transition, and polyketides under WhiB3 control arrested the macrophages in the G0-G1phase. Notably, infection with the Mtb whiB3 mutant or polyketide mutants had little effect on the macrophage cell cycle and emulated the uninfected cells. This suggests that polyketides regulated by Mtb WhiB3 are responsible for the cell cycle arrest observed in macrophages infected with the wild type Mtb. Thus, our findings demonstrate that Mtb WhiB3 maintains bioenergetic homeostasis to produce polyketide and lipid cyclomodulins that target the host cell cycle. This is a new mechanism whereby Mtb modulates the immune system by altering the host cell cycle to promote long-term persistence. This new knowledge could serve as the foundation for new host-directed therapeutic discovery efforts that target the host cell cycle

TLR-2-deficient macrophages fail to restrict <i>M. bovis</i> BCG to phagolysosomes.

<p>Bacteria were labelled green with FITC and used to infect peritoneal macrophages from TLR-2^−/− or wild type C57BL/6 mice at an MOI of 5∶1. The average number of bacteria present per cell after 5 hr of infection was the same. Representative confocal images show data at the 72 hr time point after infection in macrophages. In cells from TLR-2^−/− mice, a population of H37Rv and <i>M. bovis</i> BCG organisms are not co-localized (green after merging pictures) with either (<b><i>A</i></b>) LAMP-1 (red) or (<b><i>B</i></b>) Rab5 (red). (<b><i>C</i></b>) Infected cells were permeabilized with digitonin and stained with rabbit anti-Mtb antibody followed by anti-rabbit IgG-Alexa 594 (red). The <i>M.tb</i> organisms in the cytosol accessible to these antibodies stained red and yellow after merging pictures. Bacteria that were green in merged pictures were localized to phagolysosomes. The nucleus of the cells was stained with DAPI (blue). The upper row of each section shows H37Rv and the lower row shows BCG. (<b><i>D</i></b>) The kinetics of bacterial translocation to the cytosol of macrophages from 0 to 96 hr after infection. This was calculated from confocal studies of digitonin-permeabilized cells (♦ H37Rv- and ▪ BCG-infected macrophages from TLR-2^−/− mice; ▴ H37Rv- and • BCG-infected macrophage from C57BL/6 mice), as in panel (<b><i>C</i></b>). Bacteria in the cytoplasm of macrophages were counted from an average of 50 infected cells that were accessible to the anti-Mtb antibody (red) and turned yellow after merging pictures. Experiments were run in triplicates and repeated three times. Representative data are shown.</p

<i>Mycobacterium tuberculosis</i> H37Rv, but not <i>M. bovis</i> BCG, translocates into the cytosol of murine peritoneal macrophages.

<p>Bacteria were labelled green with FITC and used to infect peritoneal macrophages from C57BL/6 mice at an MOI of 5∶1. <b>(</b><b><i>A</i></b><b>)</b> Average number of bacteria present per cell after 5 hr of infection. <b>(</b><b><i>B</i></b><b>)</b> Percentage of bacteria that did not co-localize with LAMP-1 and/or Rab5 and, therefore considered to be in the cytosol, among the total number of bacteria at different time points after infection. Representative confocal images shown for the 72 hr time point after infection indicate that <i>M. bovis</i> BCG is completely co-localized with <b>(</b><b><i>C</i></b><b>)</b> LAMP-1 (red) or <b>(</b><b><i>D</i></b><b>)</b> Rab5 (red) but that some of the H37Rv organisms do not. <b>(</b><b><i>E</i></b><b>)</b> Infected cells were permeabilized with digitonin and stained with rabbit anti-Mtb antibody followed by anti-rabbit IgG-Alexa 594 (red). The <i>M.tb</i> organisms in the cytosol accessible to these antibodies stained red and yellow after merge, respectively. Bacteria that stained green in merged pictures were localized in the phagosome or phagolysosome. The nucleus of the cells was stained with DAPI (blue). The upper row of each section shows macrophages infected with H37Rv and the lower row shows macrophages infected with <i>M. bovis</i> BCG. <b>(</b><b><i>F</i></b><b>)</b> Kinetics of bacterial translocation to the cytosol of infected macrophages from 0 to 96 hr after infection. This was calculated from confocal studies with digitonin-permeabilized cells, as in panel <b>(</b><b><i>E</i></b><b>)</b>. Bacteria in the cytoplasm of macrophages were counted from an average of 50 infected cells, which were accessible to the anti-Mtb antibody (red) and turned yellow after merging pictures. Experiments were run in triplicates and repeated three times. Representative data are shown.</p

<i>Mycobacterium tuberculosis</i> H37Rv down-regulates the expression of key genes involved in the TLR-2 - MyD88 signaling pathway.

<p>Macrophages form wild type (C57BL/6) mice were infected with the indicated mycobacterial strains and 48 hr later cells were harvested for either analysis of relative mRNA expression of the indicated genes by quantitative RT-PCR or protein expression using immune-blotting. The gene expression of <b>(</b><b><i>A</i></b><b>)</b> TLR-2, <b>(</b><b><i>B</i></b><b>)</b> MyD88, <b>(</b><b><i>C</i></b><b>)</b> IRAK4, <b>(</b><b><i>D</i></b><b>)</b> TRAF6 and <b>(</b><b><i>E</i></b><b>)</b> TIRAP was down-regulated in macrophages infected with <i>M.tb</i> H37Rv. In contrast, no changes were observed in the expression of these genes in macrophages infected with BCG or the H37RvΔRD-1 and H37RvΔESAT6 mutants of H37Rv. The mRNA expression profiles were normalized with respect to expression of the GAPDH gene for each sample. The relative expression of genes in infected to uninfected macrophages is shown in each panel. Fold increase in the expression of each gene was calculated with respect to uninfected control at the same time point using the 2^−ΔΔCt method. Data shown here are representative of three independent experiments and qPCR assays were set up in triplicate for each target and the GAPDH gene.</p