Characterisation of the Type III Secretion System EspO effector family in enteric pathogens

Abstract

Many enteric pathogens employ a type III secretion system to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Once inside the cell, effector proteins may interact with host cell proteins to control various cellular processes. This enables enteric pathogens to establish their infective niche within the human host. The T3SS effectors EspO of enterohaemorrhagic Escherichia coli and Citrobacter rodentium, OspE of Shigella flexnerie and SopO of Salmonella enterica Serovar Typhimurium are known to interact with integrin-linked kinase to enhance cellular attachment to the substratum. In this project we demonstrate that the EspO family of effectors also interacts with the anti-apoptotic regulator Hax-1 and the co-chaperone DnaJB6. Employing fluorescence microscopy we found that ectopically expressed EspO resides on large cytosolic vacuoles, which stained positive for the endoplasmic reticulum marker CREB3. Moreover, ectopic expression of EspO changed the cellular distribution of endogenous Hax-1, which co-localized with the EspO vacuoles. We found that the EspO orthologs are anti apoptotic, protecting cells from apoptosis triggered by multiple inducers including staurosporine (a global kinase inhibitor), tunicamycin (induces unfolded protein response) and thapsigargin (interferes with Ca2+ homeostasis). Additionally, EspO protects HeLa cells from the apoptosis induced in EPEC infections. Hax-1 depleted HeLa cells by siRNA showed that the anti apoptotic activity of the EspO orthologs is Hax-1 dependent. Using the C. rodentium in-vivo model of EHEC / EPEC infections, we found that deletion of espO results in decreased levels of hyperplasia similar to the uninfected control. The mutant strain also showed low levels of neutrophils and CD4+T cells, whereas the EspO complemented strain showed a dramatic increase in both inflammatory cells. These results suggest new mechanisms unique to the T3SS EspO family of effectors, which might shed new insights regarding the infection strategies followed by enteric pathogens. These results suggest new mechanisms unique to the T3SS EspO family of effectors, which might shed new insights regarding the infection strategies followed by enteric pathogens.