Jeremy J. Gilbreath

Change Is Good: Variations in Common Biological Mechanisms in the Epsilonproteobacterial Genera Campylobacter and Helicobacter

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.

Dietary choice affects Shiga toxin-producing Escherichia coli (STEC) O157:H7 colonization and disease

Significance We demonstrated that dietary fiber content affects susceptibility to Shiga toxin (Stx)-producing Escherichia coli (STEC) infection in mice. We showed that high fiber diet (HFD)-fed mice had elevated levels of butyrate, a beneficial gut metabolite that paradoxically enhances the cell-killing capacity of Stx. We also found that the amount of gut bacteria in HFD-fed mice increased whereas the percent of commensal Escherichia species (spp) decreased compared with animals fed a low fiber diet (LFD). These changes led to higher E. coli O157:H7 colonization levels, more weight loss, and greater rates of death in HFD-fed than in LFD-fed STEC-infected animals. 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 HFD-fed 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.

Polymorphism in the interleukin-17A promoter contributes to gastric cancer.

AIM To evaluate the contribution of the G-197A polymorphism in the interleukin-17 (IL-17) promoter region to gastric cancer risk in an Iranian population. METHODS We performed a case control study using samples from 161 individuals with gastric cancer and 171 healthy controls. For each individual, the G-197A genotype was determined by restriction fragment length polymorphism analysis of polymerase chain reaction-amplified fragments. Statistical analyses were performed to determine whether any demographic or behavioral factors, infection with Helicobacter pylori (H. pylori), or a particular G-197A genotype was associated with gastric cancer risk. RESULTS We found that the G-197A genotype was significantly associated with increased gastric cancer risk (P = 0.001). Patients who were homozygous (AA) at position -197 were 2.9 times more likely to develop disease (95%CI: 1.56-5.4; P = 0.001). Furthermore, logistic regression analysis revealed that the presence of a single A allele increased the risk of gastric cancer up to 1.7-fold (95%CI: 1.26-2.369; P = 0.001). This association was observed for early stage gastric adenocarcinomas only, and was not linked to H. pylori infection. CONCLUSION These results suggest that carrying one or more G-197A polymorphisms at position -197 in the IL-17 promoter region significantly increases gastric cancer risk in this patient population.

Detailed analysis of Helicobacter pylori Fur-regulated promoters reveals a Fur box core sequence and novel Fur-regulated genes

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.

Fur Activates Expression of the 2-Oxoglutarate Oxidoreductase Genes (oorDABC) in Helicobacter pylori

Helicobacter pylori is a highly successful pathogen that colonizes the gastric mucosa of ∼50% of the worlds population. Within this colonization niche, the bacteria encounter large fluctuations in nutrient availability. As such, it is critical that this organism regulate expression of key metabolic enzymes so that they are present when environmental conditions are optimal for growth. One such enzyme is the 2-oxoglutarate (α-ketoglutarate) oxidoreductase (OOR), which catalyzes the conversion of α-ketoglutarate to succinyl coenzyme A (succinyl-CoA) and CO(2). Previous studies from our group suggested that the genes that encode the OOR are activated by iron-bound Fur (Fe-Fur); microarray analysis showed that expression of oorD, oorA, and oorC was altered in a fur mutant strain of H. pylori. The goal of the present work was to more thoroughly characterize expression of the oorDABC genes in H. pylori as well as to define the role of Fe-Fur in this process. Here we show that these four genes are cotranscribed as an operon and that expression of the operon is decreased in a fur mutant strain. Transcriptional start site mapping and promoter analysis revealed the presence of a canonical extended -10 element but a poorly conserved -35 element upstream of the +1. Additionally, we identified a conserved Fur binding sequence ∼130 bp upstream of the transcriptional start site. Transcriptional analysis using promoter fusions revealed that this binding sequence was required for Fe-Fur-mediated activation. Finally, fluorescence anisotropy assays indicate that Fe-Fur specifically bound this Fur box with a relatively high affinity (dissociation constant [K(d)] = 200 nM). These findings provide novel insight into the genetic regulation of a key metabolic enzyme and add to our understanding of the diverse roles Fur plays in gene regulation in H. pylori.

Identification and Characterization of Novel Helicobacter pylori apo-Fur-Regulated Target Genes

In Helicobacter pylori, the ferric uptake regulator (Fur) has evolved additional regulatory functions not seen in other bacteria; it can repress and activate different groups of genes in both its iron-bound and apo forms. Because little is understood about the process of apo-Fur repression and because only two apo-Fur-repressed genes (pfr and sodB) have previously been identified, we sought to expand our understanding of this type of regulation. Utilizing published genomic studies, we selected three potential new apo-Fur-regulated gene targets: serB, hydA, and the cytochrome c553 gene. Transcriptional analyses confirmed Fur-dependent repression of these genes in the absence of iron, as well as derepression in the absence of Fur. Binding studies showed that apo-Fur directly interacted with the suspected hydA and cytochrome c553 promoters but not that of serB, which was subsequently shown to be cotranscribed with pfr; apo-Fur-dependent regulation occurred at the pfr promoter. Alignments of apo-regulated promoter regions revealed a conserved, 6-bp consensus sequence (AAATGA). DNase I footprinting showed that this sequence lies within the protected regions of the pfr and hydA promoters. Moreover, mutation of the sequence in the pfr promoter abrogated Fur binding and DNase protection. Likewise, fluorescence anisotropy studies and binding studies with mutated consensus sequences showed that the sequence was important for apo-Fur binding to the pfr promoter. Together these studies expand the known apo-Fur regulon in H. pylori and characterize the first reported apo-Fur box sequence.

Nontraditional therapies to treat Helicobacter pylori infection.

The Gram-negative pathogen Helicobacter pylori is increasingly more resistant to the three major antibiotics (metronidazole, clarithromycin and amoxicillin) that are most commonly used to treat infection. As a result, there is an increased rate of treatment failure; this translates into an overall higher cost of treatment due to the need for increased length of treatment and/or the requirement for combination or sequential therapy. Given the rise in antibiotic resistance, the complicated treatment regime, and issues related to patient compliance that stem from the duration and complexity of treatment, there is clearly a pressing need for the development of novel therapeutic strategies to combat H. pylori infection. As such, researchers are actively investigating the utility of antimicrobial peptides, small molecule inhibitors and naturopathic therapies. Herein we review and discuss each of these novel approaches as a means to target this important gastric pathogen.

Enterobacterial Common Antigen Mutants of Salmonella enterica Serovar Typhimurium Establish a Persistent Infection and Provide Protection against Subsequent Lethal Challenge

ABSTRACT Infection with Salmonella spp. is a significant source of disease globally. A substantial proportion of these infections are caused by Salmonella enterica serovar Typhimurium. Here, we characterize the role of the enterobacterial common antigen (ECA), a surface glycolipid ubiquitous among enteric bacteria, in S. Typhimurium pathogenesis. Construction of a defined mutation in the UDP-N-acetylglucosamine-1-phosphate transferase gene, wecA, in two clinically relevant strains of S. Typhimurium, TML and SL1344, resulted in strains that were unable to produce ECA. Loss of ECA did not affect the gross cell surface ultrastructure, production of lipopolysaccharide (LPS), flagella, or motility. However, the wecA mutant strains were attenuated in both oral and intraperitoneal mouse models of infection (P < 0.001 for both routes of infection; log rank test), and virulence could be restored by complementation of the wecA gene in trans. Despite the avirulence of the ECA-deficient strains, the wecA mutant strains were able to persistently colonize systemic sites (spleen and liver) at moderate levels for up to 70 days postinfection. Moreover, immunization with the wecA mutant strains provided protection against a subsequent lethal oral or intraperitoneal challenge with wild-type S. Typhimurium. Thus, wecA mutant (ECA-negative) strains of Salmonella may be useful as live attenuated vaccine strains or as vehicles for heterologous antigen expression.

Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100‐bp insertion in helix 6

Two different versions of the 16S rRNA gene, one of which contained an unusual 100-bp insertion in helix 6, were detected in isolate UFO1 acquired from the Oak Ridge Integrated Field-Research Challenge (ORIFRC) site in Tennessee. rRNA was extracted from UFO1 and analyzed by reverse transcriptase-quantitative PCR with insert- and non-insert-specific primers; only the noninsert 16S rRNA gene sequence was detected. Similarly, PCR-based screening of a cDNA library (190 clones) constructed from reverse-transcribed rRNA from UFO1 did not detect any clones containing the 100-bp insert. Examination of cDNA with primers specific to the insert-bearing 16S rRNA gene, but downstream of the insert, suggests that the insert was excised from rRNA. Inspection of other 16S rRNA genes in the GenBank database revealed that a homologous insert sequence, also found in helix 6, has been reported in other environmental clones, including those acquired from ORIFRC enrichments. These findings demonstrate the existence of widely divergent copies of the 16S rRNA gene within the same organism, which may confound 16S rRNA gene-based methods of estimating microbial diversity in environmental samples.

Shiga Toxins, and the Genes Encoding Them, in Fecal Samples from Native Idaho Ungulates

ABSTRACT Cattle are a known reservoir of Shiga toxin-producing Escherichia coli. The prevalence and stability of Shiga toxin and/or Shiga toxin genes among native wild ungulates in Idaho were investigated. The frequency of both Shiga genes and toxin was similar to that reported for Idaho cattle (∼19%).