Haemolysin E- and enterohaemolysin-derived haemolytic activity of O55/O157 strains and other Escherichia coli lineages

Abstract

Among three haemolysins identified thus far in Escherichia coli, alpha-haemolysin (HlyA) is encoded on the pathogenicity islands of extraintestinal pathogenic strains, while enterohaemolysin (EhxA) is encoded on the virulence plasmids of enterohaemorrhagic E. coli (EHEC) strains. In contrast, the gene for haemolysin E (HlyE) is located on the E. coli chromosome backbone and is therefore widely distributed among E. coli strains. However, because hlyE gene expression is repressed by the H-NS protein and because the gene has been disrupted in many strains, its haemolytic activity cannot be detected in wild-type strains by routine screening on blood agar plates. In this study, we found that the HlyE-derived haemolytic activity of enteropathogenic E. coli (EPEC) O55 : H7 can be detected after anaerobic cultivation on a washed blood agar plate (EHX plate) that is used to detect the production of EhxA. We also found that the haemolytic activity of EHEC O157 : H7 observed on EHX plates under aerobic and anaerobic growth conditions is derived from EhxA and HlyE, respectively; this differential expression of the two haemolysins occurs at the transcriptional level. Our analysis of 60 E. coli strains of various pathotypes and phylogenies for their repertoires of haemolysin genes, haemolytic phenotypes and hlyE gene sequences revealed that HlyE activity can generally be detected on EHX plates under anaerobic growth conditions if the gene is intact. Furthermore, our results indicate that hlyE gene inactivation occurred in three of the five E. coli lineages (phylogroups A, B1 and B2), which demonstrates phylogroup-specific gene disruption patterns.

Prevalence of Hemolysin Genes and Comparison of ehxA Subtype Patterns in Shiga Toxin-Producing Escherichia coli (STEC) and Non-STEC Strains from Clinical, Food, and Animal Sources

“…The majority of the sheA-positive strains carried other hemolysin genes, mainly ehxA; however, 63 hemolytic strains carried only sheA, suggesting that sheA might be responsible for hemolytic expression. In fact, previous studies have revealed that sheA is not totally silent in some E. coli strains, and construction of sheA deletion mutants uncovered a complete loss of hemolytic activity, indicating a sheA-dependent hemolytic phenotype (22,40,48,74). The addition of mitomycin C into the SHIBAM agar used in this study (56) may have further contributed to an increased release of sheA, presumably resulting in detectable levels of hemolytic activity (40).…”

“…The addition of mitomycin C into the SHIBAM agar used in this study (56) may have further contributed to an increased release of sheA, presumably resulting in detectable levels of hemolytic activity (40). Additionally, anaerobic conditions during incubation of blood agar plates resulted in increased hemolytic expression due to sheA, and strains that were nonhemolytic under aerobic conditions expressed hemolytic activity when incubated anaerobically (22). These findings were not confirmed in our study; most of the strains either did not grow anaerobically, or hemolytic activity was decreased or completely absent compared to aerobic conditions (unpublished data).…”

“…Shiga toxin (stx 1 and stx 2 ), its variants, and the protein intimin (encoded by the eae allele), which is involved in attachment of the organisms to and effacing of gut mucosal cells, are only a few examples of established virulence factors (16)(17)(18)(19)(20)(21). Also, several different types of hemolysin genes have been identified in various bacterial species and are often regarded as major virulence factors (22,23). Four types of hemolysin have been identified in E. coli: alpha-hemolysin (hlyA), plasmid-and phage-carried enterohemolysin (ehxA, e-hlyA), and silent hemolysin (sheA).…”

An Environmental Shiga Toxin-Producing Escherichia coli O145 Clonal Population Exhibits High-Level Phenotypic Variation That Includes Virulence Traits

“…All strains were tested for hemolytic activity on blood agar plates as described previously (48). Briefly, the washed blood agar plates were made with blood agar base (BBL) supplemented with 10 mM CaCl 2 and 5% defibrinated sheep blood that was washed three times in phosphate-buffered saline (pH 7.4).…”

Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

“…[34][35][36][37][38][39] In E. coli, hlyE transcription is activated from a complex FNR-dependent class II promoter and HlyE activity is detected under anaerobic growth conditions. [40][41][42][43][44][45] For these enteric bacteria, oxygen starvation could signal entry into a host and prompt expression of the HlyE cytolysin. In Salmonella Typhi, the causative agent of typhoid fever, hlyE mutants exhibited impaired invasion of human epithelial (HEp-2) cells and heterologous hlyE expression in Salmonella Typhimurium enhanced colonization of the spleen and liver in a mouse model of infection.…”