Temperature- and medium-dependent secretion of proteins by Shiga toxin-producing Escherichia coli

Abstract

Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium-and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37؇C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium-and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes.

Cloning and characterization of the esp region from a dog attaching and effacing Escherichia coli strain 4221 and detection of EspB protein-binding to HEp-2 cells

“…4B). Previous reports have shown that the expression and secretion of EPEC EspB into culture supernatant is activated in DMEM in the absence of bovine serum and that the secretion is not dependent on the presence of cultured eucaryotic cells [25]. However, our results indicated that the secretion of the DEPEC EspB protein is dependent on one or more components found in serum.…”

Effect of culture conditions on Escherichia coli O157:H7-mediated attaching–effacing lesions in a bovine large intestinal mucosal explant model

“…In Study 2, the e¡ects of bacterial culture media and explant maintenance media on A/E lesion development were tested. The hypotheses that both would have an effect were based on previous observations of the e¡ects of culture conditions on transcriptional regulation of the secreted proteins that mediate A/E lesion development [3,4]. Among explants incubated in 95% O 2 , those maintained in RPMI 1640 had a greater % positive tissue sections than those maintained in DMEM (log odds = 30.47 þ 0.25 and 31.44 þ 0.32, respectively ; P = 0.016).…”

“…We hypothesized that culture conditions that enhance expression of secreted proteins involved in the pathogenesis, e.g. EspB [3] and Tir [4], would increase development of A/E lesions in the model. The objective of this study was to optimize the culture conditions for E. coli O157:H7-induced A/E lesions as assessed by di¡erent morphometric methods.…”

The expression of Longus type 4 pilus of enterotoxigenic Escherichia coli is regulated by LngR and LngS and by H‐NS, CpxR and CRP global regulators

“…Host and environmental stimuli trigger or repress the production of bacterial virulence factors such as toxins and pili (Ferraris et al, 1990;Mahan et al, 1993). Some of the signaling factors involved in induction of pilus expression have been studied for some pathogenic E. coli pili (Olson, 1993;Ebel et al, 1996). For example, the environmental signals and molecular mechanisms involved in regulation of transcription of the TCP of V. cholerae, BFP of EPEC and 987P pili of porcine ETEC have been previously studied (Ebel et al, 1996;Edwards and Schifferli, 1997;Skorupski and Taylor, 1997a,b;Schuhmacher and Klose, 1999).…”

“…Some of the signaling factors involved in induction of pilus expression have been studied for some pathogenic E. coli pili (Olson, 1993;Ebel et al, 1996). For example, the environmental signals and molecular mechanisms involved in regulation of transcription of the TCP of V. cholerae, BFP of EPEC and 987P pili of porcine ETEC have been previously studied (Ebel et al, 1996;Edwards and Schifferli, 1997;Skorupski and Taylor, 1997a,b;Schuhmacher and Klose, 1999). In many instances, in vitro growth conditions constitute signals that may typify or mimic certain biologic niches.…”