TccP2 of O157:H7 and Non-O157 Enterohemorrhagic Escherichia coli (EHEC): Challenging the Dogma of EHEC-Induced Actin Polymerization

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir EHEC O157 is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF U , which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, ␤-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 ⌬tccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.Enterohemorrhagic Escherichia coli (EHEC) is a subgroup of verocytotoxin (VT)-producing E. coli (VTEC) that can cause bloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (reviewed in references 31 and 32). There are two major types of VT (VT1 and VT2), and VT2 can be subdivided into at least five subtypes (26). E. coli O157:H7 is the most common and virulent EHEC serotype, and it is implicated worldwide in human disease (31). Typical strains of EHEC O157:H7 do not ferment sorbitol and lack ␤-D-glucuronidase activity. However, ␤-D-glucuronidase-positive and sorbitol-fermenting EHEC O157:HϪ strains have been implicated in diarrhea and hemolytic-uremic syndrome in Germany and in other European countries (22). Recent epidemiological studies show that there is a steady increase in the isolation of non-O157 EHEC from humans (1, 35) and animals (21, 27), particularly of serogroups O26, O111, and O103. A case control study of risk factors associated with EHEC infection in Argentina revealed that while EHEC O157 was responsible for 60% of cases, EHEC O145 and O121 were found in 14.5% and 1% of the cases, respectively (29).The hallmark of infections with EHEC and enteropathogenic E. coli (EPEC), a major cause of infantile diarrhea in developing countries (8), is the ability of the bacteria to modulate, while remaining extracellular, the cytoskeleton of eukaryotic cells (reviewed in reference 6), which respond to infection by producing elongated actin-rich pedestal-like structures under...

Enteropathogenic Escherichia coli O125:H6 Triggers Attaching and Effacing Lesions on Human Intestinal Biopsy Specimens Independently of Nck and TccP/TccP2

“…However, while typical EHEC O157:H7 strains use the effector protein TccP (5,11), EPEC strains use the host adaptor protein Nck, which binds tyrosinephosphorylated Tir (2,12). Interestingly, non-O157 EHEC (29) and EPEC strains belonging to lineage 2 (34) can use both the Nck and TccP2 actin polymerization pathways, while atypical sorbitol-fermenting EHEC O157 expresses both TccP and TccP2 (29). The fact that EPEC and EHEC express what seem to be redundant mechanisms to efficiently trigger actin polymerization is suggestive of an essential role and of selective pressure to maintain this capability.…”

Virulence Genes and Molecular Typing of Different Groups of Escherichia coli O157 Strains in Cattle

“…EAF1/EAF2 PCR primers were used for eaf (17) and BFP1/BFP2 were used for bfp (22) according to the published protocols. The eae genes were typed as described by Oswald et al (45), and tir genes were typed as described by Ogura et al (42). Ehly1/Ehly5 primers were used for ehxA (57).…”

Prevalence and Sequence Diversity of a Factor Required for Actin-Based Motility in Natural Populations of Burkholderia Species

“…Natural diversity exists in the prevalence and sequences of bacterial factors required for actin-dependent movement, including Listeria ActA (10,17), Shigella and enteroinvasive Escherichia coli IcsA (4), Rickettsia sp. RickA (9,14), and the Tir and TccP proteins of attaching and effacing E. coli (7,20,22). Such factors are not ubiquitous in all species of these genera, and the impact of polymorphisms on actin assembly, cell-to-cell spread, and pathogenesis is ill defined.…”