Role of NleH from attaching effacing bacteria in host pathogen interactions

Abstract

Enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), and Citrobacter rodentium are diarrhoeal pathogens grouped together on the basis of their ability to intimately adhere to host intestinal epithelia and efface brush border microvilli to form the broader category of attaching effacing (A/E) pathogens. While EPEC and EHEC are human pathogens of global health concern, C. rodentium is a natural murine pathogen which serves as an excellent animal model for in vivo studies. A/E pathogens encode a filamentous type 3 secretion system (T3SS) which is used to deliver virulence factors called effectors directly into the host cell. Both in vivo and in vitro studies have demonstrated that the T3SS is integral to the A/E pathogen virulence strategy. Once translocated to the host cell, effector proteins modulate and disrupt a wide range of host cell signalling pathways and processes including the immune response, cytoskeletal dynamics, GTPase signalling pathways, phagocytosis and apoptosis. As a defence strategy, the host responds by activating an immune response and apoptosis. However, several studies have reported that epithelial cells infected with EPEC do not undergo apoptosis despite the presence of early markers of apoptosis. Recently the NleH effectors were identified in a secretomic and genomic analysis, however their function remains unknown. NleH effectors are present in all sequenced A/E pathogen strains and can be found in duplicate copies (nleH1 and nleH2). We verified that EPEC NleH1 and NleH2 are secreted and translocated in a T3SS dependent manner. In this study we demonstrate that NleH effectors have anti-apoptotic function. During infection of cultured cell lines, we found increased nuclear condensation, membrane blebbing, caspase-3 cleavage and cell death and detachment in an nleH1 and nleH2 double mutant in comparison to wild type EPEC. Furthermore, treatment with a global caspase inhibitor abolished cell loss due to cell death or detachment. Using ectopic expression, we showed that NleH1 alone is sufficient to inhibit caspase-3 cleavage in the presence of the general apoptosis inducers staurosporine and the ER stress apoptosis inducers tunicamycin and brefeldin A. Interestingly we found that NleH effectors are kinases, however their kinase activity is not involved in the inhibition of apoptosis. However, an intact C terminus is essential for NleH’s anti-apoptotic activity. To determine the pathway by which NleH effectors inhibit apoptosis a HeLa human cDNA library was used to screen for potential binding partners using a Y2H assay. The ER antiapoptotic protein Bax inhibitor 1 (BI-1) was identified as a putative binding partner and verified using a direct 2 hybrid assay. Knockdown of BI-1 resulted in loss of NleH’s cytoprotective function. As BI-1 plays a role in calcium homeostasis, the effect of NleH effectors on cytosolic Ca2+ levels was assessed. We found that NleH effectors reduced cytosolic Ca2+ levels in a BI-1 dependent manner. Using the C. rodentium animal model we verified the ability of NleH to inhibit apoptosis in vivo by demonstrating that NleH inhibits caspase-3 activation and nuclear condensation at the site of bacterial attachment. Furthermore, we found a reduction in cell exfoliation in the presence of NleH. Additionally we showed that the NleH effectors play a role in competitiveness and also induce a mild but significant increase in NF-KB activation and TNF-[alpha] expression. Together our results demonstrate that NleH effectors are multi-functional proteins that inhibit apoptosis both in vitro and in vivo, and are kinases with an unknown function. Furthermore, NleH effectors induce a local NF-KB and TNF-[alpha] upregulation which could be linked to its anti-apoptotic activity. NleH effectors may provide a competitive advantage by preventing shedding of infected cells to prolong infection.