Role of E. coli ETT2 in Endothelial Disruption and Sepsis

E. coli has been implicated in a wide range of diseases that affect both animals and humans worldwide. Pathogenic E. coli is not only the major causal agent of enteric/diarrheal disease and urinary tract infections, but also among the most common bacteria that lead to sepsis. E. coli K1 strain is one of the leading causes of neonatal sepsis and meningitis, with around 40% mortality rate. Our overall goal is to elucidate the molecular mechanism of E. coli pathogenesis, especially in septic shock. A common infection strategy exploited by gram-negative pathogens involves the injection of virulence proteins, known as effectors, by the type III secretion system (T3SS). The T3SS contains a molecular syringe assembled by apparatus proteins and a cluster of effector protein cargos that can be transported directly from bacteria to the cytoplasm of the host cell via the syringe. Once translocated, the effectors can manipulate host cells in many ways. While enteropathogenic E. coli (EPEC) relies on the type III secretion system 1 (ETT1) to deliver effector proteins directly into host cells and thus subvert a myriad of host cellular functions, most invasive extraintestinal pathogenic E. coli (ExPEC), such as uropathogenic E. coli, sepsis-associated E. coli, and meningitis associated E. coli, do not depend on ETT1. Instead, they express a different T3SS, the type III secretion system 2, termed ETT2. ETT1 genes are encoded together in the locus of the enterocyte effacement (LEE) pathogenicity island, while ETT2 is homologous to the Salmonella T3SS located on Salmonella pathogenicity island 1 (SPI-1). It remains a mystery how ETT2 affects the virulence of the pathogen. Unlike ETT1, E. coli ETT2 normally contains large deletion and premature stop codons in several genes encoding the ETT2 apparatus proteins, thus is unlikely to assemble into a functional syringe to deliver effector proteins into host cells. Surprisingly, our preliminary data indicate that the survival rate of TLR4 knockout mice infected with a T3SS-deletion mutant (referred to as TTKO) was significantly lower than that in the mice infected with its parent strain EC10. This observation suggests an important role for ETT2 in E. coli virulence. Accordingly, we found that EC10 elicited RhoA activation in human umbilical vein endothelial cells (HUVECs), while EC10 TTKO did not have such effect. In addition, using transendothelial electrical resistance (TER) assay, a measure of junctional integrity, we found that EC10 dose-dependently caused disruption of endothelial barrier function in a LPS-independent manner. Based on our preliminary data, we hypothesize that ExPEC activates endothelial cells and disrupts endothelial barrier function via ETT2 through the RhoA dependent pathway. We will test this hypothesis in the following specific aims: 1. To identify ETT2 proteins responsible for EC10-induced RhoA activation and actin polymerization in endothelial cells. 2. To determine the role of E. coli ETT2 in the disruption of the endothelial barrier in vitro and in vivo, and in septic shock.