DECIPHERING THE MECHANISM OF ACTION OF MOONLIGHTING VIRULENCE FACTOR LISTERIA ADHESION PROTEIN: FROM BACTERIOLOGY TO HOST PATHOLOGY

Some cytosolic proteins like housekeeping enzymes are relic virulence factors from more ancient prokaryotes and eukaryotes like mycoplasma and amoeba (protozoa). In certain extreme circumstances, these cytosolic proteins have the potential to temporarily transform into virulence determinants, promoting a variety of functions such as host colonization, immunomodulation, immune evasion, and disruption of host nutritional immunity. These cytosolic proteins must be secreted and reassembled on the pathogens’ surface to execute these functions. However, the mechanism of action of these moonlighting pathogenic proteins is elusive, highly variable, and largely depends on each protein. 

Listeria adhesion protein (LAP), a bifunctional alcohol acetaldehyde dehydrogenase (AdhE, lmo1634) is one of the moonlight virulence families found only in pathogenic Listeria such as L. monocytogenes and L. ivanovii. LAP serves as an adhesive factor by anchoring on the bacterial cell surface. By co-immunoprecipitation and mass-spectrum analysis, we identified that Internalin B (InlB), another Lm surface invasive protein, is the major surface receptor of LAP. Secreted and cell wall-associated LAP is reduced in ∆inlB and naturally InlB-deficient strains but restored in ∆inlB inlB+ complemented strains. Protein structure analysis of the LAP-InlB complex suggests the C-terminal pocket region is responsible for docking onto the Gly-Trp (GW) module of InlB.  In-silico simulation further indicates electrostatic forces primarily drive the interaction, which was confirmed with LAP variants containing mutations in negatively charged (Glu621Ser, Glu523Ser) amino acids, showing altered binding conformations and weaker affinity for InlB.Cell surface anchoring of LAP is critical for Lm host adhesion and translocation, which is validated in both cell culture and murine models. In summary, these data shed light on a working surface anchoring mechanism of a new type of non-traditional virulence factor in Lm. 

Furthermore, we also studied Lm-induced gastroenteritis in mice when inoculated with high loads of Lm (~1 x 1010 CFU/mouse) resembling human cases. Lm-induced gastroenteritis is an overlooked field with great diagnostic potential. We confirmed that the Lm-induced gastroenteritis is not isolate-dependent because both Lm F4244 (neuroinvasive strain) and H7014 (gastroenteritis strain) cause diarrhea and intestinal inflammation. Interestingly, diarrhea does not depend on the severity of the Lm invasion and barrier breach. We observed minor signs of diarrhea in the small intestine of CD11b-/- mice over wild-type mice during the Lm oral route of infection, even though Lm tissue burden is generally higher in CD11b-/- mice. The data suggest that Lm-induced diarrhea could be a specific host-initiated defense mechanism mediated by immune cells for pathogen clearance. Overall, these studies offer fresh perspectives on the surface anchoring mechanism of moonlight protein, LAP, on the Lm cell surface and the host intestinal defense system during Lm-induced gastroenteritis.