Investigations on how the enteropathogenic Escherichia coli (EPEC) EspF effector inhibits PI-3 kinase-dependent phagocytosis

Abstract

Enteropathogenic E. coli inhibits phosphoinositide 3 (PI-3) kinase dependent phagocytosis via a Type Three Secretion System (T3SS) that delivers up to twenty effector proteins into target cells. The T3SS components are encoded on the Locus of Enterocyte Effacement (LEE) pathogenicity island alongside genes for a surface protein, Intimin, and seven effectors (Tir, Map, EspF, EspG, EspZ, EspH and EspB; latter also needed to deliver effectors). Inhibiting phagocytosis is linked to EspB, EspH, EspG and EspF activities with inhibitory mechanisms described for all except EspF. The aim of this study was to determine if EspF alone could inhibit phagocytosis and define the inhibitory mechanism. Initial anti-phagocytosis studies, with J774A.1 macrophages, not only confirmed EspF and T3SS-dependent inhibition but suggested a T3SS-independent contribution. Moreover, studies with effectors-deficient EPEC and non-pathogenic E. coli carrying LEE on a plasmid argued for LEE sufficiency. Surprisingly, delivering EspF into macrophages without most (EPEC multi-mutant) or all (via T3SS of another pathogen, Yersinia) other EPEC effectors argued against EspF sufficiency. Interestingly, the data also argued against EspG driving the anti-phagocytosis process but suggested that EspF’s contribution required Map, EspH, Tir, and/or the Intimin activities. Surprisingly, screening EspF/Map/EspH/Tir/Intimin single, double, triple, quadruple and quintuple mutants failed to confirm the critical roles for EspF or EspH that are linked to phenotypic instability and/or indirect contributions. Preliminary investigations on EspF’s involvement revealed possible features and domains required for its efficient expression, secretion and/or inhibiting phagocytosis. Crucially, the screening data provided many hypotheses including Tir and Intimin being able to drive T3SS-dependent anti-phagocytosis, an idea supported by complementation studies. Collectively, this study provides important new insights on EPEC’s ability to inhibit its uptake by J774A.1 macrophages and reveals unknown levels of complexity.