' success as a pathogen comes from its ability to evade degradation by macrophages. Normally macrophages clear microorganisms that activate pathogen-recognition receptors (PRRs) through a lysosomal-trafficking pathway called "LC3-associated phagocytosis" (LAP). Although activates numerous PRRs, for reasons that are poorly understood LAP does not substantially contribute to control. LAP depends upon reactive oxygen species (ROS) generated by NADPH oxidase, but fails to generate a robust oxidative response. Here, we show that CpsA, a LytR-CpsA-Psr (LCP) domain-containing protein, is required for to evade killing by NADPH oxidase and LAP. Unlike phagosomes containing wild-type bacilli, phagosomes containing the Δ mutant recruited NADPH oxidase, produced ROS, associated with LC3, and matured into antibacterial lysosomes. Moreover, CpsA was sufficient to impair NADPH oxidase recruitment to fungal particles that are normally cleared by LAP. Intracellular survival of the Δ mutant was largely restored in macrophages missing LAP components (, ,, ,, or ) but not in macrophages defective in a related, canonical autophagy pathway (, , or). The Δ mutant was highly impaired in vivo, and its growth was partially restored in mice deficient in NADPH oxidase, , or, demonstrating that CpsA makes a significant contribution to the resistance of to NADPH oxidase and LC3 trafficking in vivo. Overall, our findings reveal an essential role of CpsA in innate immune evasion and suggest that LCP proteins have functions beyond their previously known role in cell-wall metabolism.

Macrophages are first-line responders against microbes. The success of Mycobacterium tuberculosis (Mtb) rests upon its ability to convert these antimicrobial cells into a permissive cellular niche. This is a remarkable accomplishment, as the antimicrobial arsenal of macrophages is extensive. Normally bacteria are delivered to an acidic, degradative lysosome through one of several trafficking pathways, including LC3-associated phagocytosis (LAP) and autophagy. Once phagocytozed, the bacilli are subjected to reactive oxygen and nitrogen species, and they induce the expression of proinflammatory cytokines, which serve to augment host responses. However, Mtb hijacks these host defense mechanisms, manipulating host cellular trafficking, innate immune responses, and cell death pathways to its benefit. The complex series of measures and countermeasures between host and pathogen ultimately determines the outcome of infection. In this review, we focus on the diverse effectors that Mtb uses in its multipronged effort to subvert the innate immune responses of macrophages. We highlight recent advances in understanding the molecular interface of the Mtb-macrophage interaction.

Mycobacterium tuberculosis causes tuberculosis, which kills more people than any other infection. M. tuberculosis grows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease, M. tuberculosis damages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer of M. tuberculosis can inhibit ESCRT, raising the possibility that M. tuberculosis impairs this host response. Here, we show that ESCRT is recruited to damaged M. tuberculosis phagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.

No abstract

For decades, investigators have studied the interaction of Mycobacterium tuberculosis (Mtb) with macrophages, which serve as a major cellular niche for the bacilli. Because Mtb are prone to aggregation, investigators rely on varied methods to disaggregate the bacteria for these studies. Here, we examined the impact of routinely used preparation methods on bacterial cell envelope integrity, macrophage inflammatory responses, and intracellular Mtb survival. We found that both gentle sonication and filtering damaged the mycobacterial cell envelope and markedly impacted the outcome of infections in mouse bone marrow-derived macrophages. Unexpectedly, sonicated bacilli were hyperinflammatory, eliciting dramatically higher TLR2-dependent gene expression and elevated secretion of IL-1β and TNF-α. Despite evoking enhanced inflammatory responses, sonicated bacilli replicated normally in macrophages. In contrast, Mtb that had been passed through a filter induced little inflammatory response, and they were attenuated in macrophages. Previous work suggests that the mycobacterial cell envelope lipid, phthiocerol dimycocerosate (PDIM), dampens macrophage inflammatory responses to Mtb. However, we found that the impact of PDIM depended on the method used to prepare Mtb. In conclusion, widely used methodologies to disaggregate Mtb may introduce experimental artifacts in Mtb-host interaction studies, including alteration of host inflammatory signaling, intracellular bacterial survival, and interpretation of bacterial mutants.

The pathogenic traits of TlyA proteins of Mycobacterium tuberculosis are not known. Expressions of TlyA in bacteria that do not express endogenous TlyA adhere better to RAW264.7 macrophages and get phagocytosed efficiently. The internalized bacteria avoid acidification to the extent of greater than 65 percent in the case of both TlyA-expressing E. coli and M. smegmatis. Consistent with this observation, we have observed decreased co-localizaton of Lysosomal Membrane Associated Protein-1 (approx. 35 percent), Early Endosomal Antigen-1 (approx. 34 percent), Rab5 (approx. 30 percent) and Rab7 (approx. 35 percent) and enhanced colocalizaton of Rab14 (approx. 80 percent) on both TlyA-expressing bacteria as well as on TlyA-coated latex beads. These results suggest that the mycobacterial TlyA, in general, can modulate phagolysosome maturation pathway immediately after entry into macrophages, while other important molecules may aid the bacterium for long-term, intracellular survival at later point of time.

No abstract

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 the 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.