Deciphering the mechanisms employed by Salmonella to mediate modulation of host endo-lysosomal machinery
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Salmonella genus encompasses Gram-negative, rod-shaped, facultative anaerobic, non-sporulating, and predominantly motile enteric bacteria. It causes pathogenesis in a wide array of hosts, from cold-blooded animals to humans and diseases ranging from occasionally fatal systemic fever (Typhoid) to self-limiting diarrhea (gastroenteritis) in humans. Regardless of current medical advancements, Salmonella remains a significant cause of morbidity and mortality in developing countries. Salmonella remains enclosed in a vacuole inside the host cells, and these modified vacuoles are called Salmonella-containing vacuoles (SCV). In general, intracellular pathogens are subjected to the various host defense mechanisms, among which avoiding fusion with lysosomes is a prime challenge. Several intracellular bacteria have employed strategies to escape lysosomal degradation, such as Listeria spp., Shigella spp., E. coli K12, and Mycobacterium tuberculosis. Similarly, during the intracellular life of Salmonella, the SCVs are also targets for the lysosomes. Hence, Salmonella substantially alters host endo-lysosomal pathways to thrive inside the host cell. In our study, we have tried to decipher a novel molecular mechanism employed by Salmonella to modulate the host endo-lysosomal machinery to establish its replicative niche in otherwise hostile host cells. We found that Salmonella pathogenicity island 1 (SPI-1) effector SopB subverts host xenophagy by altering the phosphatidylinositol phosphate dynamics on the SCV membrane. It also mediates the downregulation of host lysosomal number, which is one of the prime strategies for establishing a replicative niche inside the host cell. Several of these fusion events of endo-lysosomal pathways are with the help of host syntaxin proteins. We further identified a crucial interaction of host Syntaxin 3 (STX3) with SCVs and observed that this interaction is SPI-2 dependent. Furthermore, this interaction with STX3 facilitates the division of the bacterium along with SCV and maintains a single bacterium per vacuole status, thus, facilitating a hospitable niche inside the host cells. Part I: To unravel the mechanisms employed by Salmonella SopB to subvert host cell xenophagy in macrophages. Salmonella survives and utilizes macrophages for effective dissemination throughout the host, causing systemic infection. One of the central host defense mechanisms in macrophages is xenophagy, or macro-autophagy where invading pathogens are targeted for degradation by fusion with lysosomes. The process of autophagy depends on the dynamics of phosphatidyl-inositol phosphates (PIPs) species present on the vesicular membranes. Salmonella, when inside intact SCV, escapes autophagy. However, the exact mechanism remains unknown. Interestingly, we observed that Salmonella SPI-1 effector SopB, which plays a crucial role in inducing uptake of bacteria inside non-phagocytic cells, inhibits the recruitment of various autophagic adaptor proteins such as Syntaxin 17 (STX17), microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B or LC3B), p62/SQSTM (sequestosome 1) onto the SCV membrane. Notably, we also found that SopB downregulates the transcript levels of these adaptor proteins and the overall autophagy flux inside host cells. We further demonstrate that SopB alters the PIP dynamics (PI3P and PI4P) of the SCV membrane. This activity of SopB helps the bacterium escape autophagy by inhibiting the fusion of SCVs with both lysosomes and autophagosomes. Part II: To decipher the mechanism behind the reduction in lysosomal number in Salmonella-infected cells. Previous work from our laboratory has shown that Salmonella infection causes a reduction in the overall lysosomal number inside the cell. This downregulation of lysosomal number facilitates survival of Salmonella inside host cells, as there are not enough lysosomes for the SCVs to fuse with and mediate clearance of the pathogen. While performing infection experiments with SopB mutants, we observed that the levels of LAMP1 and the number of lysosomes were significantly higher in SopB mutant infected cells than in STM WT. SopB has also been shown to activate Akt by phosphorylating Ser437 residue. In an independent study, it has been reported that Akt can further phosphorylate transcription factor EB (TFEB), which is a master regulator of the CLEAR (Coordinated Lysosomal Expression and Regulation) gene network. The phosphorylated TFEB is inactive and cannot translocate into the nucleus and hence cannot initiate the transcription of genes responsible for lysosomal biogenesis and autophagy. Therefore, we hypothesized that Salmonella SopB might also mediate overall downregulation through the Akt-TFEB axis in infected cells. We observed that SopB indeed reduced the number of acidic lysosomes inside the infected cells via the Akt-TFEB axis and thus facilitates the survival of Salmonella in host macrophages. These results were also validated in the mice model of Salmonella infection. Part III: To elucidate the role of host Syntaxins in Salmonella pathogenesis and its virulence. Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in the endosomal trafficking pathways. Recent reports shed light on intracellular bacteria modulating host syntaxin to establish infection successfully. Salmonella actively modulates its vacuole to escape lysosomal fusion. One of the critical SNAREs in macrophages responsible for phagosome maturation is Syntaxin 3 and Syntaxin 4. Also, there is a report which suggests that SCV harbors Syntaxin 12. Therefore, in the third part of the study, based on the literature available, we have screened a few Syntaxins in SCV biogenesis and found that STX3 could play a role in SCV biogenesis. Upon knockdown of STX 3, we have observed that the bacterial proliferation is hindered and is restored upon the overexpression of STX3. We observed using live-cell imaging that during infection, SCV interacts with STX3 and thus might help in fusion and fission events of SCV with other vesicles to acquire membrane for facilitating the division of SCV. We also found this interaction abrogated when we infected with SPI-2 encoded T3SS apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS (STM ∆invC). Together, these results indicated that the effector molecule secreted through SPI-2 encoded T3SS is involved in interaction with host STX3, which is essential to maintain Salmonella division along with SCV and maintenance of a single bacterium per vacuole. Significant Findings and Conclusions: This study concludes that Salmonella modulates host endo-lysosomal machinery to establish replicative niches inside host cells. Mainly, SopB plays a dual role in subverting the host cell xenophagy in macrophages; (1) by modulating the phosphatidylinositol phosphates dynamics on the SCV membrane to inhibit fusion of SCV with autophagosomes or lysosomes and (2) it downregulates the overall lysosomal biogenesis through Akt-TFEB axis inside infected host macrophages. This study also elucidates one of the critical interactions with host STX3, which helps the bacteria inside the host cell to divide along with SCV and is essential for maintaining a single bacterium per vacuole. Overall, we have deciphered that through the involvement of various effector molecules, Salmonella substantially modulates the host endo-lysosomal machinery to cause pathogenesis in the host.