UC San Diego

The clearance of enteric bacteria by phagocytes is an essential role of the host response against infection. Previously, we have reported that BAI1 (Brain Angiogenesis Inhibitor 1) binds bacterial lipopolysaccharide (LPS) of gram-negative bacteria and mediates its engulfment by activating ELMO1 (Engulfment and cell motility protein 1).

While both pathogenic and commensal-Gram-negative bacteria express lipopolysaccharide (LPS), intestinal phagocytes are able to discriminate commensals from enteric pathogens. Using Salmonella as a model organism, we showed that ELMO1 interacts with Salmonella effector protein called SifA (Salmonella induced filaments A) which involves in the survival of bacteria inside macrophages. SifA shares the WxxxE signature motif that present only in enteric pathogens and absent in commensals. The signature motif is important for interaction with ELMO1. Therefore, we hypothesized that the ELMO1-SifA interaction could play a role in the bacterial survival by interfering with host cellular pathways. Pulldown assays with GST-SifA on murine macrophage lysate revealed that SifA interacts with late endosomal protein Rab9. Rab 9 is a host GTPase involved in the retrograde trafficking of CI-MPRs (Cation-independent mannose 6 phosphate receptors) from the late endosome to the trans-Golgi network. Further analysis using GST-Rab9 pulldowns with direct recombinant proteins in solution revealed that increasing concentrations of SifA displaces the strong ELMO1 interaction with Rab9. To elucidate the functional relevance of ELMO1-SifA interaction, immunofluorescence microscopy was performed to monitor the integrity of Salmonella-containing vacuole and the localization of CI-M6PR. Our data shows an accumulation of CI-MPR at the surface plasma membrane in the presence of SifA. Bacterial clearance assays using mutant strains of Salmonella showed that clearance was delayed in ELMO1-depleted murine macrophages when compared to control macrophages. Interestingly, macrophages infected with Salmonella mutant strains lacking SifA had significantly higher intracellular bacteria. Our findings provide new insight on how Salmonella modulates host pathways through its interaction with ELMO1 to create its own replicative niche in the form of Salmonella containing vacuoles within host cells.