Correlating Levels of Type III Secretion and Secreted Proteins with Fecal Shedding of Escherichia coli O157:H7 in Cattle

Abstract

The locus of enterocyte effacement (LEE) of Escherichia coli O157:H7 (O157) encodes a type III secretion system (T3SS) for secreting LEE-encoded and non-LEE-encoded virulence proteins that promote the adherence of O157 to intestinal epithelial cells and the persistence of this food-borne human pathogen in bovine intestines. In this study, we compared hha sepB and hha mutants of O157 for LEE transcription, T3SS activity, adherence to HEp-2 cells, persistence in bovine intestines, and the ability to induce changes in the expression of proinflammatory cytokines. LEE transcription was upregulated in the hha sepB and hha mutant strains compared to that in the wild-type strain, but the secretion of virulence proteins in the hha sepB mutant was severely compromised. This reduced secretion resulted in reduced adherence of the hha sepB mutant to Hep-2 cells, correlating with a significantly shorter duration and lower magnitude of fecal shedding in feces of weaned (n ‫؍‬ 4 per group) calves inoculated with this mutant strain. The levels of LEE transcription, T3SS activity, and adherence to HEp-2 cells were much lower in the wild-type strain than in the hha mutant, but no significant differences were observed in the duration or the magnitude of fecal shedding in calves inoculated with these strains. Examination of the rectoanal junction (RAJ) tissues from three groups of calves showed no adherent O157 bacteria and similar proinflammatory cytokine gene expression, irrespective of the inoculated strain, with the exception that interleukin-1␤ was upregulated in calves inoculated with the hha sepB mutant. These results indicate that the T3SS is essential for intestinal colonization and prolonged shedding, but increased secretion of virulence proteins did not enhance the duration and magnitude of fecal shedding of O157 in cattle or have any significant impact on the cytokine gene expression in RAJ tissue compared with that in small intestinal tissue from the same calves. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 (referred to here as O157) causes a broad spectrum of diarrheal illnesses, including uncomplicated diarrhea, hemorrhagic colitis (HC), and hemolytic-uremic syndrome (HUS) (34). O157 infections are the major cause of acute renal failure in young children in the United States (34). Shiga toxins encoded by the stx 1 and stx 2 genes (52) are the major virulence factors responsible for the development of HC and HUS. Among ruminants, cattle are considered the major reservoir for O157 and animals colonized with these bacteria serve as major direct and indirect sources of this pathogen (28). O157 colonizes the large intestines of cattle, especially the cecum and tissues of the rectoanal junction (RAJ) (7, 37). Colonization requires intimate adherence of O157 bacteria to intestinal epithelial cells, a process that culminates in the formation of characteristic histopathological lesions called attaching and effacing (A/E) lesions. A/E histopathology results from the rearrangements of cytoskeletal elements of mucosal epithe...

Escherichia coli O157:H7 Lacking the qseBC -Encoded Quorum-Sensing System Outcompetes the Parental Strain in Colonization of Cattle Intestines

“…Since QseBC-mediated quorum-sensing signaling in EHEC O157:H7 enhances flagellar gene expression and motility (13,15), which have indirectly been shown to be important for colonization (36,37), we compared the motility of the qseBC deletion mutant to those of the parental and the qseBCcomplemented mutant strains in the presence or absence of the QseBC-activating signaling molecule norepinephrine (NE). The phenotypic analysis of motility on soft-agar plates revealed that a deletion of the qseBC genes did not compromise bacterial motility.…”

“…In a recent study, we demonstrated the importance of motility and LEE for increased colonization of bovine intestines by EHEC O157:H7, since mutants expressing LEE at very high levels but compromised in bacterial motility were unable to colonize and persist in cattle intestines (37). Thus, regulatory pathways that allow increased expression of LEE and other adhesins, such as curli, without causing significant reductions in bacterial motility might explain the increased colonization of cattle intestines by the qseBC mutant of EHEC O157:H7.…”

“…In addition to a postulated role of motility in colonization of bovine intestines by EHEC O157:H7 (36,37), the type III secretion system-mediated secretion of LEE-as well as non-LEE-encoded effectors are critical for EHEC O157:H7 adherence to and colonization of cattle intestines (29,37,38,41). Thus, significant but low levels of increases in the transcriptional levels of LEEcarried ler and espA, observed in the qseBC mutant irrespective of the presence or lack thereof of NE, could be instrumental in imparting a competitive edge to the mutant relative to the parent strain in intestinal colonization of calves.…”

Evaluation of Animal Genetic and Physiological Factors That Affect the Prevalence of Escherichia coli O157 in Cattle

“…E. coli O157 primarily colonizes the mucosal epithelium at the RAJ [17], although it can be isolated from the gall bladder and along the gastrointestinal tract [18], [19]. More than 100 genes are involved in the colonization of the bovine intestine identified by genetic and biochemical analyses [20], including the E. coli O157 type III secretion system that enables the translocation of effector proteins into host cells and is required for colonization of cattle [21], [22], [23], [24], [25]. Besides the bacterial factors, environmental factors are believed to contribute to the prevalence of E. coli O157 in cattle.…”

Determining the Relative Contribution and Hierarchy of hha and qseBC in the Regulation of Flagellar Motility of Escherichia coli O157:H7

“…That motility and/or flagella might not be required for human virulence is suggested by the increased isolation of sorbitol-fermenting non-motile EHEC O157:NM strains from HUS patients in Germany that adhered at significantly higher levels to human colonic epithelial cells and expressed increased amounts of curli [16]. However, in a recent study we have demonstrated that a hha deletion mutant of EHEC O157:H7 expressing LEE at very high levels but showing reduced motility due to the reduced expression of the flagellar gene fliC failed to establish increased colonization of cattle intestines compared to the wild-type strain [17]. Despite unsettled role of motility in EHEC O157:H7 colonization of bovine intestines, there is increasing evidence that flagella might promote adherence of EHEC O157:H7 to the target sites in the large intestine.…”