Chengru Zhu

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 α-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.

Complete Nucleotide Sequence and Analysis of the Locus of Enterocyte Effacement from Rabbit Diarrheagenic Escherichia coli RDEC-1

ABSTRACT The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species. To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic Escherichia coli strain RDEC-1 (O15:H−) and compared it with those from human enteropathogenicE. coli (EPEC, O127:H6) and enterohemorrhagic E. coli (EHEC, O157:H7) strains. Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at selC and is flanked by an IS2 element and the lifA toxin gene. The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC. orf3 and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE. The predicted promoters of LEE1, LEE2, LEE3, tir, andLEE4 operons are highly conserved among the LEEs, although the upstream regions varied considerably fortir and the crucial LEE1 promoter, suggesting differences in regulation. Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator. In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps). A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed. Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity. Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.

Protection against Hemorrhagic Colitis in an Animal Model by Oral Immunization with Isogeneic Rabbit Enteropathogenic Escherichia coli Attenuated by Truncating Intimin

ABSTRACT 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-1Δeae860-939) was well tolerated, as observed by the absence of clinical signs of disease or evidence of intestinal A/E lesions. The efficacy of RDEC-1Δeae860-939 as a vaccine was evaluated by orogastric inoculation of rabbits with RDEC-1Δeae860-939 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-1Δeae860-939-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-1Δeae860-939, 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.

Effect of Zinc in Enteropathogenic Escherichia coli Infection

ABSTRACT Enteropathogenic Escherichia coli (EPEC) infection triggers the release of ATP from host intestinal cells, and the ATP is broken down to ADP, AMP, and adenosine in the lumen of the intestine. Ecto-5′-nucleotidase (CD73) is the main enzyme responsible for the conversion of 5′-AMP to adenosine, which triggers fluid secretion from host intestinal cells and also has growth-promoting effects on EPEC bacteria. In a recent study, we examined the role of the host enzyme CD73 in EPEC infection by testing the effect of ecto-5′-nucleotidase inhibitors. Zinc was a less potent inhibitor of ecto-5′-nucleotidase in vitro than the nucleotide analog α,β-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than α,β-methylene-ADP. This discrepancy between the in vitro and in vivo potencies of the two inhibitors prompted us to search for potential targets of zinc other than ecto-5′-nucleotidase. Zinc, at concentrations that produced little or no inhibition of EPEC growth, caused a decrease in the expression of EPEC protein virulence factors, such as bundle-forming pilus (BFP), EPEC secreted protein A, and other EPEC secreted proteins, and reduced EPEC adherence to cells in tissue culture. The effects of zinc were not mimicked by other transition metals, such as manganese, iron, copper, or nickel, and the effects were not reversed by an excess of iron. Quantitative real-time PCR showed that zinc reduced the abundance of the RNAs encoded by the bfp gene, by the plasmid-encoded regulator (per) gene, by the locus for the enterocyte effacement (LEE)-encoded regulator (ler) gene, and by several of the esp genes. In vivo, zinc reduced EPEC-induced fluid secretion into ligated rabbit ileal loops, decreased the adherence of EPEC to rabbit ileum, and reduced histopathological damage such as villus blunting. Some of the beneficial effects of zinc on EPEC infection appear to be due to the action of the metal on EPEC bacteria as well as on the host.

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.