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After entering the host cells, Salmonella Typhimurium (STM) stays inside a modified membrane-bound compartment called Salmonella containing vacuole (SCV). The biogenesis and stability of SCV are crucial for the intracellular proliferation of Salmonella. Our research has provided a novel mechanistic view on the role of a bacterial porin OmpA in maintaining the stability of SCV. We found that the deletion of OmpA forces the bacteria to escape from the SCV during the immediate early stage of infection. In the absence of OmpA, the bacteria failed to retain the LAMP-1 and came into the host cell’s cytosol. Subsequently, the cytosolic population of STM ΔompA activated the host autophagy machinery after colocalizing with syntaxin 17 and LC3B. The autophagosomes carrying STM ΔompA were targeted to the lysosomes for degradation. Inhibition of autophagy pathway using bafilomycin A1 restored the intracellular proliferation of STM ΔompA. We further showed that the four extracellular loops of OmpA played a crucial role in holding the LAMP-1 pool around the SCV. We have altered the extracellular loop sequences of Salmonella OmpA by site-directed mutagenesis and observed that the bacteria failed to maintain the LAMP-1 pool around the SCV, which finally resulted in their release into the cytosol of the host macrophages. Surprisingly, the cytosolic population of Salmonella having mutations in the extracellular loops of OmpA didn’t activate the lysosomal degradation pathway like STM ΔompA, which helped them to survive within the murine macrophages. In summary, our study revealed an OmpA dependent novel strategy utilized by Salmonella to combat host autophagy by promoting the stability of SCV.

Salmonella continues to be a threat to the human population by taking a toll on the lives of about 20,000 individuals globally per year. Host sirtuins or NAD+-dependent deacetylases are the major players in host immuno-metabolic regulation. However, the role of sirtuins in the modulation of the immune metabolism pertaining to Salmonellosis is largely unknown. Here, we investigated the role of SIRT1 and SIRT3 in mediating immuno-metabolic switch along the progression of Salmonella infection. Gene profiling (Nanostring) data along with flow cytometric analysis indicated the ability of the wildtype Salmonella Typhimurium to skew the polarization state of the macrophages from a pro-inflammatory M1 state toward an immunosuppressive M2 state along its course of infection. Further, using shRNA-mediated knockdown studies and catalytic inhibitor treatment, we showed that SIRT1 and SIRT3 play a crucial role in mediating the immuno-metabolic switch that governs macrophage polarization in Salmonella-infected macrophages. SIRT1 or SIRT3 knockdown or inhibition led to decreased expression of M2 surface markers such as CD206 and showed increased production of pro-inflammatory cytokines and ROS generation which together resulted in attenuated bacterial intracellular proliferation within the infected RAW264.7 macrophages. Alongside immunological functions by modulating p65 NF-kappaB acetylation, SIRT1, and SIRT3 knockdown or inhibition also skews Salmonella-induced host metabolic shift towards increased fatty acid oxidation by regulating acetylation status of HIF-1alpha and PDHA1 which in turn impacts the metabolism of Salmonella within the infected macrophages. In vivo mice-model infection studies highlighted the role of SIRT1 and SIRT3 in controlling bacterial burden and dissemination.

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