The Escherichia coli TolC efflux pump protein is immunogenic and elicits protective antibodies

Antimicrobial resistance is an increasing worldwide public health burden that threatens to make the existent antimicrobials obsolete. Among the mechanisms of antimicrobial resistance is the overexpression of efflux pumps, such as the AcrA–AcrB–TolC which extrude diverse compounds, reducing the intracellular concentration of antimicrobials. TolC is the outer membrane protein of this pump and has recently gained attention as a therapeutic target. However, little is known about the immune response generated against the TolC protein. Here we evaluated the cellular and humoral immune response against the TolC from Escherichia coli . An in silico epitope prediction of the E. coli TolC showed several residues could bind to human antibodies, and we showed that human plasma presented anti-TolC IgG and IgA antibodies. Gram-negative infected patients presented a slight increase in anti-TolC IgM amounts, compared to controls. Recombinant E. coli TolC protein stimulated macrophages in vitro to produce nitric oxide, as well as IL-6 and TNF-α, assessed by Griess assay and ELISA, respectively. Immunization of mice with TolC intraperitoneally and an in vitro re-stimulation of lymph node cells led to increased percentage of T cell proliferation and IFNγ production, evaluated by flow cytometry and ELISA, respectively. We observed that TolC mouse immunization stimulated anti-TolC IgM and IgG production, with a higher level of IgG1 and IgG2, amongst the IgG subclasses. Finally, TolC IgG from mouse immune serum could bind to live E. coli, increase bacterial uptake by macrophages in vitro . TolC immunized mice had a survival rate increased in 60% post infection with E. coli . Our results showed that TolC is immunogenic, activating macrophages, T and B cells, leading to the production of protective antibodies against E. coli . ### Competing Interest Statement The authors have declared no competing interest.