Investigating how enteropathogenic Escherichia coli (EPEC) subverts AKT signalling

Abstract

The phosphoinositide 3-kinase (PI3K) signalling pathway is activated in macrophages in response to many bacterial pathogens, triggering phagocytic uptake mechanisms and phosphorylation-associated activation of the serine/threonine kinase AKT. Enteropathogenic E. coli (EPEC) inhibits both PI3K mediated phagocytosis and AKT phosphorylation; dependent on a type 3-secretion system (T3SS) critical for delivering up to 24 known effector proteins into target cells. The efficient translocation of most EPEC effectors is dependent on the T3SS effector chaperone CesT. Although the effectors and mechanisms for inhibiting phagocytosis are well described, little is known how EPEC inhibits AKT phosphorylation. AKT activation is a multi-step process involving its recruitment to the cell membrane and phosphorylation of Thr308 by PDK1 and Ser473 by mTORC2. This activation process is supressed by inositol (such as PTEN) and protein (such as PP1 & PP2A) phosphatases. Altered AKT signalling is associated with many cancers, diabetes, cardiovascular and infectious disease, thus identifying how EPEC inhibits AKT activity could provide insight into its complex regulatory process and/or new therapeutic strategies. Screening of bacterial strains, lacking or expressing subsets of EPEC effectors, by western blot analysis suggests that the inhibitory mechanism depends on the CesT chaperone but not the function of the 21 most studied effectors. The EPEC inhibitory mechanism was investigated through the development of a two-wave infection model, examining for T3SS dependent changes in AKT phosphorylation (Thr308 & Ser473), membrane localisation and activity of AKT associated signalling proteins (PDK1 & PTEN). This strategy revealed inhibition of AKT phosphorylation to be stable (up to 3 h) and linked to increased activity of serine/threonine protein phosphatase(s). This finding was supported by phosphatase inhibitor studies, suggesting the involvement of a host activated or bacterial delivered protein phosphatase. Thus, this study provides new insights into the requirement of the EPEC effector repertoire and suggests a novel mechanism by which EPEC inhibits AKT signalling.