Analysis of the Function of EnteropathogenicEscherichia coliEspB by Random Mutagenesis

Abstract

Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrhea, especially in developing countries. EspB, a key virulence factor of EPEC, is required for the attaching and effacing effect characteristic of EPEC and enterohemorrhagic E. coli and has been posited to play several functions in the process of infection. Attaching and effacing activity is associated with the accumulation of filamentous actin beneath adherent bacteria as measured in the fluorescence actin staining (FAS) test. To determine whether different domains of EspB are responsible for different functions, 42 plasmids carrying mutated espB were introduced into an espB deletion mutant. Two major groups of espB mutants were identified. One group of 17 mutants exhibited positive FAS results and normal levels of hemolytic activity. Another group of 22 mutants exhibited negative FAS results and low levels of hemolytic activity. Three mutants were exceptional. One mutant was FAS positive but had significantly reduced hemolytic activity. Conversely, a second mutant was FAS negative but had full hemolytic activity. A third mutant had a significantly reduced FAS level compared to the wild type but full hemolytic activity. The results of EspF and Tir translocation assays confirmed that FAS-negative insertions disrupt effector translocation and mutants with FAS-positive insertions retain protein translocation activity. These results suggest that EspB has distinct domain functions involved in effector translocation that can be distinguished from its role as a component of the translocation pore.Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. The hallmark of EPEC infection is the ability of the pathogen to attach intimately to the host cell membrane, destroy microvilli, and induce the formation of cup-like pedestals composed of cytoskeletal proteins directly underneath the adherent bacteria. This phenomenon, known as the attaching and effacing (A/E) effect (38), has been observed in vitro in cell culture and in duodenal or rectal biopsy specimens from infants with EPEC infection (45, 58). A 35,624-bp genetic element known as the locus of enterocyte effacement (LEE) is necessary and sufficient for the A/E effect (35, 36). The LEE has 41 open reading frames whose products can be divided into five categories: a type III secretion system (

Interactions and Predicted Host Membrane Topology of the Enteropathogenic Escherichia coli Translocator Protein EspB

“…For example, an S. flexneri ipaB mutant is nonhemolytic, while an ipaC mutant retains detectable, albeit with reduced hemolytic activity (2). Similar results were reported for YopB and YopD and for EspD and EspB (28,35,41). Thus, YopB homologues, including PopB, IpaB, and EspD, seem to play a primary role in pore formation.…”

“…While several YopD homologues, such as EspB, have one predicted transmembrane domain beginning approximately 100 residues from the amino terminus, no direct evidence of host cell membrane topology has been reported previously. In this study, we exploited knowledge of permissive sites of EspB that could accept amino acid insertions without compromising function (28) to test the hypothesis that EspB adopts a specific host cell membrane topology and to identify domains of EspB required for interactions with EspD.…”

SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

“…4A). While it is not predicted to form any obvious secondary structures, this region does overlap a weak coiled-coil domain predicted by Luo and Donnenberg (46). Interestingly, this EspB domain is particularly sensitive to insertion mutagenesis of a five-codon linker, and insertions in this region often interfere with the proper translocator function of EspB (46,47), indicating that this EspB domain is essential for EspB function.…”

Secretion of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase by the LEE-encoded type III secretion system in enteropathogenic Escherichia coli

“…It is well known that mutations in escN, the gene encoding the T3SS ATPase, abolish secretion of both translocator and effector proteins through this system. In contrast, mutations in espB, the gene encoding a hydrophobic translocon protein that is thought to form with EspD a translocation pore through the host membrane, disrupt translocation of effector proteins into host cells during infection but do not impair secretion of these proteins into the extracellular medium (Deng et al, 2004, Luo and Donnenberg, 2006, Luo and Donenberg, 2011.…”

“…Translocators are needed for translocation of the effectors into host cells (Luo and Donnenberg, 2006). To date multiple type III effectors have been identified in EPEC, some of them are encoded within the LEE whereas others are encoded in distinct pathogenicity islands through the chromosome (reviewed by Dean and Kenny, 2009).…”