The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will develop a life-threatening sequela called the hemolytic uremic syndrome is unpredictable. We reasoned that conditions that enhance Stx binding and uptake within the gut after E. coli O157:H7 infection should result in greater disease severity. Because the receptor for Stx, globotriaosylceramide, is up-regulated in the presence of butyrate in vitro, we asked whether a high fiber diet (HFD) that reportedly enhances butyrate production by normal gut flora can influence the outcome of an E. coli O157 infection in mice. To address that question, groups of BALB/c mice were fed high (10%) or low (2%) fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). Mice fed an HFD exhibited a 10-to 100-fold increase in colonization, lost 15% more body weight, exhibited signs of morbidity, and had 25% greater mortality relative to the low fiber diet (LFD)-fed group. Additionally, sections of intestinal tissue from HFD-fed mice bound more Stx1 and expressed more globotriaosylceramide than did such sections from LFD-fed mice. Furthermore, the gut microbiota of HFDfed mice compared with LFD-fed mice contained reduced levels of native Escherichia species, organisms that might protect the gut from colonization by incoming E. coli O157:H7. Taken together, these results suggest that susceptibility to infection and subsequent disease after ingestion of E. coli O157:H7 may depend, at least in part, on individual diet and/or the capacity of the commensal flora to produce butyrate. tubular necrosis | HCT-8 | microbiome

In Helicobacter pylori, iron balance is controlled by the Ferric uptake regulator (Fur), an iron-sensing repressor protein that typically regulates expression of genes implicated in iron transport and storage. Herein, we carried out extensive analysis of Fur-regulated promoters and identified a 7-1-7 motif with dyad symmetry (5’-TAATAATnATTATTA-3’), which functions as the Fur box core sequence of H. pylori. Addition of this sequence to the promoter region of a typically non-Fur regulated gene was sufficient to impose Fur-dependent regulation in vivo. Moreover, mutation of this sequence within Fur-controlled promoters negated regulation. Analysis of the H. pylori chromosome for the occurrence of the Fur box established the existence of well-conserved Fur-boxes in the promoters of numerous known Fur-regulated genes, and revealed novel putative Fur targets. Transcriptional analysis of the new candidate genes demonstrated Fur-dependent repression of HPG27_51, HPG27_52, HPG27_199, HPG27_445, HPG27_825 and HPG27_1063, as well as Fur-mediated activation of the cytotoxin associated gene A, cagA (HPG27_507). Furthermore, electrophoretic mobility shift assays confirmed specific binding of Fur to the promoters of each of these genes. Future experiments will determine whether loss of Fur regulation of any of these particular genes contributes to the defects in colonization exhibited by the H. pylori fur mutant.

SUMMARY Microbial evolution and subsequent species diversification enable bacterial organisms to perform common biological processes by a variety of means. The epsilonproteobacteria are a diverse class of prokaryotes that thrive in diverse habitats. Many of these environmental niches are labeled as extreme, whereas other niches include various sites within human, animal, and insect hosts. Some epsilonproteobacteria, such as Campylobacter jejuni and Helicobacter pylori , are common pathogens of humans that inhabit specific regions of the gastrointestinal tract. As such, the biological processes of pathogenic Campylobacter and Helicobacter spp. are often modeled after those of common enteric pathogens such as Salmonella spp. and Escherichia coli . While many exquisite biological mechanisms involving biochemical processes, genetic regulatory pathways, and pathogenesis of disease have been elucidated from studies of Salmonella spp. and E. coli , these paradigms often do not apply to the same processes in the epsilonproteobacteria. Instead, these bacteria often display extensive variation in common biological mechanisms relative to those of other prototypical bacteria. In this review, five biological processes of commonly studied model bacterial species are compared to those of the epsilonproteobacteria C. jejuni and H. pylori . Distinct differences in the processes of flagellar biosynthesis, DNA uptake and recombination, iron homeostasis, interaction with epithelial cells, and protein glycosylation are highlighted. Collectively, these studies support a broader view of the vast repertoire of biological mechanisms employed by bacteria and suggest that future studies of the epsilonproteobacteria will continue to provide novel and interesting information regarding prokaryotic cellular biology.

Acinetobacter baumannii is an important emerging pathogen that is capable of causing many types of severe infection, especially in immunocompromised hosts. Since A. baumannii can rapidly acquire antibiotic resistance genes, many infections are on the verge of being untreatable, and novel therapies are desperately needed. To investigate the potential utility of copper-based antibacterial strategies against Acinetobacter infections, we characterized copper resistance in a panel of recent clinical A. baumannii isolates. Exposure to increasing concentrations of copper in liquid culture and on solid surfaces resulted in dose-dependent and strain-dependent effects; levels of copper resistance varied broadly across isolates, possibly resulting from identified genotypic variation among strains. Examination of the growth-phase-dependent effect of copper on A. baumannii revealed that resistance to copper increased dramatically in stationary phase. Moreover, A. baumannii biofilms were more resistant to copper than planktonic cells but were still susceptible to copper toxicity. Exposure of bacteria to subinhibitory concentrations of copper allowed them to better adapt to and grow in high concentrations of copper; this copper tolerance response is likely achieved via increased expression of copper resistance mechanisms. Indeed, genomic analysis revealed numerous putative copper resistance proteins that share amino acid homology to known proteins in Escherichia coli and Pseudomonas aeruginosa. Transcriptional analysis revealed significant upregulation of these putative copper resistance genes following brief copper exposure. Future characterization of copper resistance mechanisms may aid in the search for novel antibiotics against Acinetobacter and other highly antibiotic-resistant pathogens. IMPORTANCEAcinetobacter baumannii causes many types of severe nosocomial infections; unfortunately, some isolates have acquired resistance to almost every available antibiotic, and treatment options are incredibly limited. Copper is an essential nutrient but becomes toxic at high concentrations. The inherent antimicrobial properties of copper give it potential for use in novel therapeutics against drug-resistant pathogens. We show that A. baumannii clinical isolates are sensitive to copper in vitro, both in liquid and on solid metal surfaces. Since bacterial resistance to copper is mediated though mechanisms of efflux and detoxification, we identified genes encoding putative copper-related proteins in A. baumannii and showed that expression of some of these genes is regulated by the copper concentration. We propose that the antimicrobial effects of copper may be beneficial in the development of future therapeutics that target multidrug-resistant bacteria.

Helicobacter pylori must be able to rapidly respond to fluctuating conditions within the stomach. Despite this need for constant adaptation, H. pylori encodes few regulatory proteins. Of the identified regulators, the ferric uptake regulator (Fur), the nickel response regulator (NikR), and the two-component acid response system (ArsRS) are each paramount to the success of this pathogen. While numerous studies have individually examined these regulatory proteins, little is known about their combined effect. Therefore, we constructed a series of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS. A growth curve analysis revealed minor variation in growth kinetics across the strains; these were most pronounced in the triple mutant and in strains lacking ArsS. Visual analysis showed that strains lacking ArsS formed large aggregates and a biofilm-like matrix at the air-liquid interface. Biofilm quantification using crystal violet assays and visualization via scanning electron microscopy (SEM) showed that all strains lacking ArsS or containing a nonphosphorylatable form of ArsR (ArsR-D52N mutant) formed significantly more biofilm than the wild-type strain. Molecular characterization of biofilm formation showed that strains containing mutations in the ArsRS pathway displayed increased levels of cell aggregation and adherence, both of which are key to biofilm development. Furthermore, SEM analysis revealed prevalent coccoid cells and extracellular matrix formation in the ArsR-D52N, ⌬nikR ⌬arsS, and ⌬fur ⌬nikR ⌬arsS mutant strains, suggesting that these strains may have an exacerbated stress response that further contributes to biofilm formation. Thus, H. pylori ArsRS has a previously unrecognized role in biofilm formation. IMPORTANCEDespite a paucity of regulatory proteins, adaptation is key to the survival of H. pylori within the stomach. While prior studies have focused on individual regulatory proteins, such as Fur, NikR, and ArsRS, few studies have examined the combined effect of these factors. Analysis of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS revealed a previously unrecognized role for the acid-responsive two-component system ArsRS in biofilm formation.