Gut microbes, the innate immune system and inflammatory bowel disease: location, location, location.
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Clostridioides difficile↗
Biomedical subjects
Edgar C Boedeker
Publications and source records attributed to Edgar C Boedeker.
The QseC sensor kinase: a bacterial adrenergic receptor.
Quorum sensing is a cell-to-cell signaling mechanism in which bacteria respond to hormone-like molecules called autoinducers (AIs). The AI-3 quorum-sensing system is also involved in interkingdom signaling with the eukaryotic hormones epinephrine/norepinephrine. This signaling activates transcription of virulence genes in enterohemorrhagic Escherichia coli O157:H7. However, this signaling system has never been shown to be involved in virulence in vivo, and the bacterial receptor for these signals had not been identified. Here, we show that the QseC sensor kinase is a bacterial receptor for the host epinephrine/norepinephrine and the AI-3 produced by the gastrointestinal microbial flora. We also found that an alpha-adrenergic antagonist can specifically block the QseC response to these signals. Furthermore, we demonstrated that a qseC mutant is attenuated for virulence in a rabbit animal model, underscoring the importance of this signaling system in virulence in vivo. Finally, an in silico search found that the periplasmic sensing domain of QseC is conserved among several bacterial species. Thus, QseC is a bacterial adrenergic receptor that activates virulence genes in response to interkingdom cross-signaling. We anticipate that these studies will be a starting point in understanding bacterial-host hormone signaling at the biochemical level. Given the role that this system plays in bacterial virulence, further characterization of this unique signaling mechanism may be important for developing novel classes of antimicrobials.
Animals↗
Towards a vaccine for attaching/effacing Escherichia coli: a LEE encoded regulator (ler) mutant of rabbit enteropathogenic Escherichia coli is attenuated, immunogenic, and protects rabbits from lethal challenge with the wild-type virulent strain.
The ler (LEE encoded regulator) gene product is a central regulator for the genes encoded on the locus of enterocyte effacement (LEE) pathogenicity island of attaching/effacing (A/E) pathogens, including human enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) as well as animal isolates. Although an in vivo role for Ler in bacterial virulence has not been documented, we hypothesized that a Ler deletion mutant should be attenuated for virulence but might retain immunogenicity. The goals of this study were to genetically characterize ler of a rabbit EPEC (rEPEC) strain (O103:H2), to examine the effect of ler on in vivo virulence, and to determine if intragastric inoculation of an attenuated rEPEC ler mutant was immunogenic and could protect rabbits against subsequent challenge with the wild-type virulent parent strain. The predicted ler gene product of rEPEC strain O103:H2 shares high homology (over 95% amino acid identity) with the Lers of another rEPEC strain RDEC-1 (O15:H-) and human EPEC and EHEC. A defined internal ler deletion mutant of rEPEC O103:H2 showed reduced production of secreted proteins. Although orogastric inoculation of rabbits with the virulent parent O103:H2 strain induced severe diarrhea, significant weight loss and early mortality with adherent mucosal bacteria found at sacrifice, the isogeneic ler mutant strain was well tolerated. Animals gained weight and showed no clinical signs of disease. Examination of histological sections of intestinal segments revealed the absence of mucosal bacterial adherence. This result demonstrates an essential role for Ler in in vivo pathogenicity of A/E E. coli. Single dose orogastric immunization with the rEPEC ler mutant induced serum IgG antibody to whole bacteria (but not to intimin). Immunized animals were protected against enteric infection with the WT virulent parent strain exhibiting normal weight gain, absence of diarrhea and absence of mucosally adherent bacteria at sacrifice. Such attenuated ler mutant strains may have potential for use as oral vaccines, or as vaccine vectors for delivery of foreign antigens. It remains to be determined whether such regulatory mutants can protect against infection with A/E bacteria of differing serotypes affecting different hosts.
Adhesins, Bacterial↗
Delivery of heterologous protein antigens via hemolysin or autotransporter systems by an attenuated ler mutant of rabbit enteropathogenic Escherichia coli.
In this report, we describe the use of an attenuated regulatory mutant of a rabbit enteropathogenic Escherichia coli (rEPEC) as a live vaccine vector to deliver heterologous protein antigens using two dedicated transport systems, a Salmonella autotransporter and the E. coli hemolysin apparatus. We previously reported that an isogeneic ler (LEE encoded regulator) mutant of rEPEC O103:H2 is attenuated and immunogenic in rabbits. We first evaluated the Salmonella autotransporter MisL containing the immunodominant B-cell epitope of the circumsporozoite protein from Plasmodium falciparum, (NANP)8, fused to the C-terminal translocator domain under the control of the constitutive Tac17 promoter. The rEPEC ler mutant was able to express and to translocate the (NANP)8 passenger peptide to the bacterial surface. We next investigated the delivery of Shiga toxin B subunit (Stx1B) from human enterohemorrhagic E. coli by the rEPEC ler mutant via the MisL autotransporter or the E. coli hemolysin secretion apparatus. The autotransporter and hemolysin plasmids expressed similar levels of Stx1B (30-40 ng/ml/OD600). Only 6% of Stx1B was found in the autotransporter supernatants; the rest was cell-associated, with a small fraction of the Stx1B surface-exposed as determined by immunofluorescence. In contrast, 88% of Stx1B was secreted into culture supernatants by the hemolysin secretion system. In an in vivo study, no significant protection was observed in rabbits inoculated with the ler mutant harboring the Stx1B-autotransporter plasmid following experimental challenge with RDEC-H19A, the prototype rEPEC containing an Stx-converting phage. In contrast, rabbits inoculated with the rEPEC ler mutant containing the Stx1B-hemolysin fusion were partially protected from RDEC-H19A infection as demonstrated by decreased weight loss (p<0.008) when compared to rabbits inoculated with the parent ler mutant. Our results suggest that attenuated rEPEC are capable of serving as vaccine vectors to express heterologous protein antigens from different cellular locations and deliver these antigens to the intestinal mucosa. With this system, secreted proteins may be more effective than cell-associated antigens in generating protection.
Animals↗
Protection against hemorrhagic colitis in an animal model by oral immunization with isogeneic rabbit enteropathogenic Escherichia coli attenuated by truncating intimin.
Strains of Shiga toxin (Stx)-producing Escherichia coli, also called enterohemorrhagic E. coli (EHEC), are important food-borne pathogens for humans. Most EHEC strains intimately adhere to the intestinal mucosa in a characteristic attaching and effacing (A/E) pattern, which is mediated by the bacterial adhesin intimin. Subsequent release of Stx1 and/or Stx2 leads to the frequent development of hemorrhagic colitis and, less commonly, to hemolytic-uremic syndrome. The aim of the present study was to develop an attenuated A/E E. coli strain for use as a vaccine against EHEC infection encoding a truncated intimin lacking adhesive capacity, but which would still express somatic antigens, other products of the locus of enterocyte effacement pathogenicity island, and an immunogenic remnant of the intimin molecule. A single-nucleotide deletion was generated in the eae gene in the prototype rabbit A/E E. coli strain RDEC-1 (O15:H-), which resulted in truncation of intimin by 81 C-terminal residues (860 to 939 amino acids) containing a disulfide loop. Inoculation of rabbits with large doses of the truncated intimin mutant (RDEC-1Deltaeae(860-939)) was well tolerated, as observed by the absence of clinical signs of disease or evidence of intestinal A/E lesions. The efficacy of RDEC-1Deltaeae(860-939) as a vaccine was evaluated by orogastric inoculation of rabbits with RDEC-1Deltaeae(860-939) followed by challenge with the virulent strain RDEC-H19A, an Stx1-producing derivative of wild-type RDEC-1 capable of inducing hemorrhagic colitis in rabbits. Following RDEC-H19A challenge, nonimmunized control rabbits exhibited characteristic weight loss with watery to bloody diarrhea and demonstrated intimate bacterial attachment, effacement of microvilli, submucosal edema, mucosal heterophile infiltrates, and Shiga toxin-induced vascular lesions. In contrast, the RDEC-1Deltaeae(860-939)-immunized rabbits showed no clinical signs of disease, maintained normal weight gain, had reduced fecal shedding of challenge organisms, and showed an absence of gross or microscopic lesions in the intestinal mucosa. Serum antibodies specific to intimin were detected among rabbits immunized with RDEC-1Deltaeae(860-939), indicating that truncation of the intimin functional domain not only attenuated bacterial virulence, but also retained at least some of the immunogenicity of native intimin. Although it is not possible to gauge the exact contribution of residual intimin immunity to protection, this attenuation strategy for A/E E. coli strains shows promise for the development of effective vaccines to prevent EHEC infection in humans and animals.
Adhesins, Bacterial↗
Vaccines for enterotoxigenic Escherichia coli: current status.
PURPOSE OF REVIEW: Enterotoxigenic Escherichia coli (ETEC) remain major causes of infantile diarrhea in the developing world and of travelers' diarrhea in visitors to these areas from industrialized countries, although the relative importance of these pathogens in these settings may be decreasing. The pathogenesis of ETEC infections has been well understood for almost two decades, and there is good evidence for acquired immunity after infection. Nevertheless, currently there is no effective ETEC vaccine. This review evaluates the current status of ETEC vaccine development. RECENT FINDINGS: ETEC organisms express fimbrial (or fibrillar) colonization factor antigens that function as adhesins to promote their attachment to the small intestinal epithelium. They secrete either (or both) of two major protein enterotoxins that induce fluid and electrolyte secretion. Vaccine development during the last decade has targeted the components of the three major colonization factor antigens as well as the immunogenic heat-labile enterotoxin. This strategy was expected to cover 90% of infecting strains, leaving unprotected only those strains without major colonization factor antigens and those that express only the poorly immunogenic heat-stable enterotoxin. Recent experiences have questioned the validity of the current vaccine strategy since new reports indicate that (1) the number of recognized colonization factor antigens of ETEC has increased to more than 21, (2) epidemiologic field studies of children in endemic areas suggest that infection with ETEC of a given colonization factor antigen/toxin phenotype may not confer protection on reinfection with other strains of the same colonization factor antigen/toxin phenotype, and (3) a major field trial of a heat-killed ETEC vaccine expressing colonization factor antigens and containing the B subunit of cholera toxin as a surrogate for E. coli heat-labile enterotoxin was ineffective against ETEC infections that should have been "vaccine preventable." New vaccine strategies have been developed to deliver ETEC antigens to the mucosal immune system. SUMMARY: Although new vaccines are being developed to improve immunogenicity over that of the heat-killed vaccine, the current strategy for antigen inclusion has been challenged and new, common antigens may have to be defined to achieve the goal of an effective vaccine against ETEC.
Bacterial Toxins↗
Gastrointestinal infections: an overview.
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Comment↗
Use of a continuous-flow anaerobic culture to characterize enteric virulence gene expression.
We developed an in vitro culture method to characterize the expression of bacterial genes under conditions mimicking the colonic environment. Our culture system (the intestinal simulator) comprised a continuous-flow anaerobic culture which was inoculated with fecal samples from healthy volunteers. As a test organism, we employed enteroaggregative Escherichia coli (EAEC), an emerging diarrheal pathogen that is thought to cause infection in both the small and large intestines. After the simulator culture achieved equilibrium conditions, we inoculated the system with prototype EAEC strain 042 and assessed the expression of three EAEC virulence-related genes. We focused particularly on expression of aggR, which encodes a global transcriptional regulator of EAEC virulence factors, and two AggR-regulated genes. By using real-time quantitative reverse transcription-PCR, we showed that aggR expression in the simulator is increased 3- to 10-fold when 042 is grown under low-pH (5.5 to 6.0) conditions, compared with results with neutral pH (7.0). Interestingly, however, this effect was seen only when the strain was grown in the presence of commensal bacteria. We also found that expression of aggR is 10- to 20-fold higher at low NaCl concentrations, and this effect was also observed only in the presence of commensal bacteria. Using coculture and conditioned-media experiments, we identified specific strains of Enterococcus and Clostridium that upregulated aggR expression; in contrast, strains of Lactobacillus and Veillonella downregulated aggR expression. Our data provide new insights into regulation of virulence genes in EAEC and suggest the utility of intestinal simulation cultures in characterizing enteric gene regulation.
Anaerobiosis↗
Foodborne enteric infections.
Foodborne infections are estimated to affect one in four Americans each year. Most these (67%) are caused by the Norwalk-like viruses, but Campylobacter and nontyphoidal Salmonellae together account for about one fourth of cases of illness in which a pathogen can be detected. Less common bacterial infections, such as with Listeria monocytogenes and the Shiga toxin-producing Escherichia coli, cause fewer infections but are important because of their severe complications or high mortality rate, or both. This review describes the recent development of a national surveillance system for foodborne illness, newer methods for molecular characterization of organisms for epidemiologic studies, and individual etiologic agents in the order of frequency of occurrence. Methods for decreasing the disease burden are discussed, including education of health care professionals and the public, modification of food-handling behaviors, the use of food irradiation, and the application of probiotics to foods.
Journal Article↗
Helicobacter pylori infection: treatment.
Current standard treatment regimens for Helicobacter pylori infection provide eradication rates 80 to 90%. These rates have been achieved with a variety of 1-week triple therapies using two antibiotics and an acid suppressant. Antibiotic resistance, which may develop during failed treatment, is becoming increasingly common and has led to studies of new regimens for primary therapy, and new strategies for salvage of failed therapy. Other regimens have been designed and tested with the aim of decreasing the cost of initial therapy or to improve compliance, but abbreviated regimens have high incidence of failure and may add to the problem of resistance. Increasing attention has been paid to the need for, and timing of, the determination of antibiotic resistance of H. pylori isolates either at the time of initial diagnosis or after treatment failure. New, simpler, and noninvasive methods are offered for follow-up to determine if eradication has been successful. Treatment regimens should be chosen based on local drug susceptibility patterns and the availability of approved therapeutic agents in each country. Established indications for testing for H. pylori and administering therapy include active or inactive peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma, as well as hyperplastic polyps, hyperplastic gastropathy, postendoscopic resection for gastric malignancy, and acute H. pylori gastritis. It is now largely accepted that noninvestigated dyspepsia is an indication for testing for and treating H. pylori, but that dyspeptic symptoms shown not to be associated with ulcer (nonulcer dyspepsia) do not now provide an indication for testing. Controversial or unresolved indications for testing and treating include planned use of chronic antisecretory therapy, planned use of nonsteroidal anti-inflammatory drugs, and use as a general approach to the prevention of gastric cancer.
Journal Article↗