Gut metabolite L-lactate supports Campylobacter jejuni population expansion during acute infection.

How the microaerobic pathogen Campylobacter jejuni establishes its niche and expands in the gut lumen during infection is poorly understood. Using 6-wk-old ferrets as a natural disease model, we examined this aspect of C. jejuni pathogenicity. Unlike mice, which require significant genetic or physiological manipulation to become colonized with C. jejuni, ferrets are readily infected without the need to disarm the immune system or alter the gut microbiota. Disease after C. jejuni infection in ferrets reflects closely how human C. jejuni infection proceeds. Rapid growth of C. jejuni and associated intestinal inflammation was observed within 2 to 3 d of infection. We observed pathophysiological changes that were noted by cryptic hyperplasia through the induction of tissue repair systems, accumulation of undifferentiated amplifying cells on the colon surface, and instability of HIF-1α in colonocytes, which indicated increased epithelial oxygenation. Metabolomic analysis demonstrated that lactate levels in colon content were elevated in infected animals. A C. jejuni mutant lacking lctP, which encodes an L-lactate transporter, was significantly decreased for colonization during infection. Lactate also influences adhesion and invasion by C. jejuni to a colon carcinoma cell line (HCT116). The oxygenation required for expression of lactate transporter (lctP) led to identification of a putative thiol-based redox switch regulator (LctR) that may repress lctP transcription under anaerobic conditions. Our work provides better insights into the pathogenicity of C. jejuni.

Transcription of Cystathionine ß-Lyase (MetC) Is Repressed by HeuR in Campylobacter jejuni, and Methionine Biosynthesis Facilitates Colonocyte Invasion.

A previously identified transcriptional regulator in Campylobacter jejuni, termed HeuR, was found to positively regulate heme utilization. Additionally, transcriptomic work demonstrated that the putative operons CJJ81176_1390 to CJJ81176_1394 (CJJ81176_1390-1394) and CJJ81176_1214-1217 were upregulated in a HeuR mutant, suggesting that HeuR negatively regulates expression of these genes. Because genes within these clusters include a cystathionine ß-lyase (metC) and a methionine synthase (metE), it appeared HeuR negatively regulates C. jejuni methionine biosynthesis. To address this, we confirmed mutation of HeuR reproducibly results in metC overexpression under nutrient-replete conditions but did not affect expression of metE, while metC expression in the wild type increased to heuR mutant levels during iron limitation. We subsequently determined that both gene clusters are operonic and demonstrated the direct interaction of HeuR with the predicted promoter regions of these operons. Using DNase footprinting assays, we were able to show that HeuR specifically binds within the predicted -35 region of the CJJ81176_1390-1394 operon. As predicted based on transcriptional results, the HeuR mutant was able to grow and remain viable in a defined medium with and without methionine, but we identified significant impacts on growth and viability in metC and metE mutants. Additionally, we observed decreased adherence, invasion, and persistence of metC and metE mutants when incubated with human colonocytes, while the heuR mutant exhibited increased invasion. Taken together, these results suggest that HeuR regulates methionine biosynthesis in an iron-responsive manner and that the ability to produce methionine is an important factor for adhering to and invading the gastrointestinal tract of a susceptible host. IMPORTANCE As the leading cause of bacterium-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health. Investigating colonization factors that allow C. jejuni to successfully infect a host furthers our understanding of genes and regulatory elements necessary for virulence. In this study, we have begun to characterize the role of the transcriptional regulatory protein, HeuR, on methionine biosynthesis in C. jejuni. When the ability to synthesize methionine is impaired, detrimental impacts on growth and viability are observed during growth in limited media lacking methionine and/or iron. Additionally, mutations in the methionine biosynthetic pathway result in decreased adhesion, invasion, and intracellular survival of C. jejuni when incubated with human colonocytes, indicating the importance of regulating methionine biosynthesis.

The Host Cellular Immune Response to Infection by Campylobacter Spp. and Its Role in Disease.

Campylobacter spp. are the leading cause of bacterium-derived gastroenteritis worldwide, impacting 96 million individuals annually. Unlike other bacterial pathogens of the gastrointestinal tract, Campylobacter spp. lack many of the classical virulence factors that are often associated with the ability to induce disease in humans, including an array of canonical secretion systems and toxins. Consequently, the clinical manifestations of human campylobacteriosis and its resulting gastrointestinal pathology are believed to be primarily due to the host immune response toward the bacterium. Further, while gastrointestinal infection is usually self-limiting, numerous postinfectious disorders can occur, including the development of Guillain-Barré syndrome, reactive arthritis, and irritable bowel syndrome. Because gastrointestinal disease likely results from the host immune response, the development of these postinfectious disorders may be due to dysregulation or misdirection of the same inflammatory response. As a result, it is becoming increasingly important to the Campylobacter field, and human health, that the cellular immune responses toward Campylobacter be better understood, including which immunological events are critical to the development of disease and the postinfectious disorders mentioned above. In this review, we collectively cover the cellular immune responses across susceptible hosts to Campylobacter jejuni infection, along with the tissue pathology and postinfectious disorders which may develop.

Induction of neutrophil extracellular traps by Campylobacter jejuni.

Campylobacter jejuni is the leading cause of bacterial-derived gastroenteritis worldwide and can lead to several post-infectious inflammatory disorders. Despite the prevalence and health impacts of the bacterium, interactions between the host innate immune system and C. jejuni remain poorly understood. To expand on earlier work demonstrating that neutrophils traffic to the site of infection in an animal model of campylobacteriosis, we identified significant increases in several predominantly neutrophil-derived proteins in the faeces of C. jejuni-infected patients, including lipocalin-2, myeloperoxidase and neutrophil elastase. In addition to demonstrating that these proteins significantly inhibited C. jejuni growth, we determined they are released during formation of C. jejuni-induced neutrophil extracellular traps (NETs). Using quantitative and qualitative methods, we found that purified human neutrophils are activated by C. jejuni and exhibit signatures of NET generation, including presence of protein arginine deiminase-4, histone citrullination, myeloperoxidase, neutrophil elastase release and DNA extrusion. Production of NETs correlated with C. jejuni phagocytosis/endocytosis and invasion of neutrophils suggesting that host- and bacterial-mediated activities are responsible for NET induction. Further, NET-like structures were observed within intestinal tissue of C. jejuni-infected ferrets. Finally, induction of NETs significantly increased human colonocyte cytotoxicity, indicating that NET formation during C. jejuni infection may contribute to observed tissue pathology. These findings provide further understanding of C. jejuni-neutrophil interactions and inflammatory responses during campylobacteriosis.

The Host Antimicrobial Protein Calgranulin C Participates in the Control of Campylobacter jejuni Growth via Zinc Sequestration.

Campylobacter jejuni is a leading cause of bacterially derived gastroenteritis worldwide. Campylobacter is most commonly acquired through the consumption of undercooked poultry meat or through drinking contaminated water. Following ingestion, Campylobacter adheres to the intestinal epithelium and mucus layer, causing toxin-mediated inflammation and inhibition of fluid reabsorption. Currently, the human response to infection is relatively unknown, and animal hosts that model these responses are rare. As such, we examined patient fecal samples for the accumulation of the neutrophil protein calgranulin C during infection with Campylobacter jejuni In response to infection, calgranulin C was significantly increased in the feces of humans. To determine whether calgranulin C accumulation occurs in an animal model, we examined disease in ferrets. Ferrets were effectively infected by C. jejuni, with peak fecal loads observed at day 3 postinfection and full resolution by day 12. Serum levels of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α) significantly increased in response to infection, which resulted in leukocyte trafficking to the colon. As a result, calgranulin C increased in the feces of ferrets at the time when C. jejuni loads decreased. Further, the addition of purified calgranulin C to C. jejuni cultures was found to inhibit growth in a zinc-dependent manner. These results suggest that upon infection with C. jejuni, leukocytes trafficked to the intestine release calgranulin C as a mechanism for inhibiting C. jejuni growth.

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target.

Peptidoglycan LD-carboxypeptidase Pgp2 influences Campylobacter jejuni helical cell shape and pathogenic properties and provides the substrate for the DL-carboxypeptidase Pgp1.

Despite the importance of Campylobacter jejuni as a pathogen, little is known about the fundamental aspects of its peptidoglycan (PG) structure and factors modulating its helical morphology. A PG dl-carboxypeptidase Pgp1 essential for maintenance of C. jejuni helical shape was recently identified. Bioinformatic analysis revealed the CJJ81176_0915 gene product as co-occurring with Pgp1 in several organisms. Deletion of cjj81176_0915 (renamed pgp2) resulted in straight morphology, representing the second C. jejuni gene affecting cell shape. The PG structure of a Δpgp2 mutant showed an increase in tetrapeptide-containing muropeptides and a complete absence of tripeptides, consistent with ld-carboxypeptidase activity, which was confirmed biochemically. PG analysis of a Δpgp1Δpgp2 double mutant demonstrated that Pgp2 activity is required to generate the tripeptide substrate for Pgp1. Loss of pgp2 affected several pathogenic properties; the deletion strain was defective for motility in semisolid agar, biofilm formation, and fluorescence on calcofluor white. Δpgp2 PG also caused decreased stimulation of the human nucleotide-binding oligomerization domain 1 (Nod1) proinflammatory mediator in comparison with wild type, as expected from the reduction in muropeptide tripeptides (the primary Nod1 agonist) in the mutant; however, these changes did not alter the ability of the Δpgp2 mutant strain to survive within human epithelial cells or to elicit secretion of IL-8 from epithelial cells after infection. The pgp2 mutant also showed significantly reduced fitness in a chick colonization model. Collectively, these analyses enhance our understanding of C. jejuni PG maturation and help to clarify how PG structure and cell shape impact pathogenic attributes.

Narrow-spectrum inhibitors of Campylobacter jejuni flagellar expression and growth.

Campylobacter jejuni is a major cause of food-borne illness due to its ability to reside within the gastrointestinal tracts of chickens. Multiple studies have identified the flagella of C. jejuni as a major determinant of chicken colonization. An inhibitor screen of approximately 147,000 small molecules was performed to identify compounds that are able to inhibit flagellar expression in a reporter strain of C. jejuni. Several compounds that modestly inhibited motility of wild-type C. jejuni in standard assays were identified, as were a number of small molecules that robustly inhibited C. jejuni growth, in vitro. Examination of similar bacterial screens found that many of these small molecules inhibited only the growth of C. jejuni. Follow-up assays demonstrated inhibition of other strains of C. jejuni and Campylobacter coli but no inhibition of the closely related Helicobacter pylori. The compounds were determined to be bacteriostatic and nontoxic to eukaryotic cells. Preliminary results from a day-of-hatch chick model of colonization suggest that at least one of the compounds demonstrates promise for reducing Campylobacter colonization loads in vivo, although further medicinal chemistry may be required to enhance bioavailability.

High-throughput sequencing of Campylobacter jejuni insertion mutant libraries reveals mapA as a fitness factor for chicken colonization.

Campylobacter jejuni is a leading cause of gastrointestinal infections worldwide, due primarily to its ability to asymptomatically colonize the gastrointestinal tracts of agriculturally relevant animals, including chickens. Infection often occurs following consumption of meat that was contaminated by C. jejuni during harvest. Because of this, much interest lies in understanding the mechanisms that allow C. jejuni to colonize the chicken gastrointestinal tract. To address this, we generated a C. jejuni transposon mutant library that is amenable to insertion sequencing and introduced this mutant pool into day-of-hatch chicks. Following deep sequencing of C. jejuni mutants in the cecal outputs, several novel factors required for efficient colonization of the chicken gastrointestinal tract were identified, including the predicted outer membrane protein MapA. A mutant strain lacking mapA was constructed and found to be significantly reduced for chicken colonization in both competitive infections and monoinfections. Further, we found that mapA is required for in vitro competition with wild-type C. jejuni but is dispensable for growth in monoculture.

Campylobacter infection of young children in Colombia and its impact on the gastrointestinal environment.

Campylobacter infections are a leading cause of bacterial-derived gastroenteritis worldwide with particularly profound impacts on pediatric patients in low-and-middle income countries. It remains unclear how Campylobacter impacts these hosts, though it is becoming increasingly evident that it is a multifactorial process that depends on the host immune response, the gastrointestinal microbiota, various bacterial factors, and host nutritional status. Since these factors likely vary between adult and pediatric patients in different regions of the world, it is important that studies define these attributes in well characterized clinical cohorts in diverse settings. In this study, we analyzed the fecal microbiota and the metabolomic and micronutrient profiles of asymptomatic and symptomatic pediatric patients in Colombia that were either infected or uninfected with Campylobacter during a case-controlled study on acute diarrheal disease. Here, we report that the microbiome of Campylobacter- infected children only changed in their abundance of Campylobacter spp. despite the inclusion of children with or without diarrhea. In addition to increased Campylobacter, computational models were used to identify fecal metabolites that were associated with Campylobacter infection and found that glucose-6-phosphate and homovanillic acid were the strongest predictors of infection in these pediatric patients, which suggest that colonocyte metabolism are impacted during infection. Despite changes to the fecal metabolome, the concentrations of intestinal minerals and trace elements were not significantly impacted by Campylobacter infection, but were elevated in uninfected children with diarrhea. Importance: Gastrointestinal infection with pathogenic Campylobacter species has long been recognized as a significant cause of human morbidity. Recently, it has been observed that pediatric populations in low-and-middle income countries are uniquely impacted by these organisms in that infected children can be persistently colonized, develop enteric dysfunction, and exhibit reduced development and growth. While the association of Campylobacter species with these long-term effects continues to emerge, the impact of infection on the gastrointestinal environment of these children remains uncharacterized. To address this knowledge gap, our group leveraged clinical samples collected during a previous study on gastrointestinal infections in pediatric patients to examine the fecal microbiota, metabolome, and micronutrient profiles of those infected with Campylobacter species, and found that the metabolome was impacted in a way that suggests gastrointestinal cell metabolism is affected during infection, which is some of the first data indicating how gastrointestinal health in these patients may be affected.

Crystal structure of the MrkD1P receptor binding domain of Klebsiella pneumoniae and identification of the human collagen V binding interface.

Klebsiella species are members of the family enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. Among other virulence factors in Klebsiella, type 3 pili exhibit a unique binding pattern in the human kidney via interaction of two MrkD adhesion variants 1C1 and 1P to type IV and/or V collagen. However, very little is known about the nature of this recognition. Here we present the crystal structure of the plasmid born MrkD1P receptor domain (MrkDrd). The structure reveals a jelly-roll ß-barrel fold comprising 17 ß-strands very similar to the receptor domain of GafD, the tip adhesin from the F17 pilus that recognizes n-acetyl-d-glucosamine (GlcNAc). Analysis of collagen V binding of different MrkD1P mutants revealed that two regions were responsible for its binding: a pocket, that aligns approximately with the GlcNAc binding pocket of GafD involving residues R105 and Y155, and a transversally oriented patch that spans strands ß2a, ß9b and ß6 including residues V49, T52, V91, R102 and I136. Taken together, these data provide structural and functional insights on MrkD1P recognition of host cells, providing a tool for future development of rationally designed drugs with the prospect of blocking Klebsiella adhesion to collagen V.

A secreted sirtuin from Campylobacter jejuni contributes to neutrophil activation and intestinal inflammation during infection.

Histone modifications control numerous processes in eukaryotes, including inflammation. Some bacterial pathogens alter the activity or expression of host-derived factors, including sirtuins, to modify histones and induce responses that promote infection. In this study, we identified a deacetylase encoded by Campylobacter jejuni which has sirtuin activities and contributes to activation of human neutrophils by the pathogen. This sirtuin is secreted from the bacterium into neutrophils, where it associates with and deacetylates host histones to promote neutrophil activation and extracellular trap production. Using the murine model of campylobacteriosis, we found that a mutant of this bacterial sirtuin efficiently colonized the gastrointestinal tract but was unable to induce cytokine production, gastrointestinal inflammation, and tissue pathology. In conclusion, these results suggest that secreted bacterial sirtuins represent a previously unreported class of bacterial effector and that bacterial-mediated modification of host histones is responsible for the inflammation and pathology that occurs during campylobacteriosis.

Gut metabolite L-lactate supports Campylobacter jejuni population expansion during acute infection.

How the microaerobic pathogen Campylobacter jejuni establishes its niche and expands in the gut lumen during infection is poorly understood. Using six-week-old ferrets as a natural disease model, we examined this aspect of C. jejuni pathogenicity. Unlike mice, which require significant genetic or physiological manipulation to become colonized with C. jejuni , ferrets are readily infected without the need to disarm the immune system or alter the gut microbiota. Disease after C. jejuni infection in ferrets reflects closely how human C. jejuni infection proceeds. Rapid growth of C. jejuni and associated intestinal inflammation was observed within two-three days of infection. We observed pathophysiological changes that were noted by cryptic hyperplasia through the induction of tissue repair systems, accumulation of undifferentiated amplifying cells on the colon surface, and instability of HIF-1α in colonocytes, which indicated increased epithelial oxygenation. Metabolomic analysis demonstrated that lactate levels in colon content were elevated in infected animals. A C. jejuni mutant lacking lctP , which encodes an L-lactate transporter, was significantly decreased for colonization during infection. Lactate also influences adhesion and invasion by C. jejuni to a colon carcinoma cell line (HCT116). The oxygenation required for expression of lactate transporter ( lctP ) led to discovery of a putative thiol based redox switch regulator (LctR) that may repress lctP transcription under anaerobic conditions. Our work provides new insights into the pathogenicity of C. jejuni . Significance: There is a gap in knowledge about the mechanisms by which C. jejuni populations expand during infection. Using an animal model which accurately reflects human infection without the need to alter the host microbiome or the immune system prior to infection, we explored pathophysiological alterations of the gut after C. jejuni infection. Our study identified the gut metabolite L-lactate as playing an important role as a growth substrate for C. jejuni during acute infection. We identified a DNA binding protein, LctR, that binds to the lctP promoter and may repress lctP expression, resulting in decreased lactate transport under low oxygen levels. This work provides new insights about C. jejuni pathogenicity.

Campylobacter jejuni induces differentiation of human neutrophils to the CD16hi /CD62Llo subtype.

The discovery of neutrophil subtypes has expanded what is known about neutrophil functions, yet there is still much to learn about the role of these subtypes during bacterial infection. We investigated whether Campylobacter jejuni induced differentiation of human neutrophils into the hypersegmented, CD16hi /CD62Llo subtype. In addition, we investigated whether C. jejuni-dependent differentiation of this neutrophil subtype induced cancer-promoting activities of human T cells and colonocytes, which were observed in other studies of hypersegmented, CD16hi /CD62Llo neutrophils. We found that C. jejuni causes a significant shift in human neutrophil populations to the hypersegmented, CD16hi /CD62Llo subtype and that those populations exhibit delayed apoptosis, elevated arginase-1 expression, and increased reactive oxygen species production. Furthermore, incubation of C. jejuni-infected neutrophils with human T cells resulted in decreased expression of the ζ-chain of the TCR, which was restored upon supplementation with exogenous l-arginine. In addition, incubation of C. jejuni-infected neutrophils with human colonocytes resulted in increased HIF-1α stabilization and NF-κB activation in those colonocytes, which may result in the up-regulation of protumorigenic genes.

Type 3 fimbriae and biofilm formation are regulated by the transcriptional regulators MrkHI in Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production. One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate type 3 fimbrial expression.

Transposon-Based Identification of Factors That Promote Campylobacter jejuni Nuclease Activity.

Nucleases are ubiquitous in pathogens and allow bacteria to acquire nucleotide nutrients, take up foreign DNA, induce tissue damage, degrade neutrophil extracellular traps, and modulate the host inflammatory response. Furthermore, nucleases can modulate numerous bacterial virulence factors, promoting bacterial growth and disease. To understand how bacteria can produce nucleases, an unbiased approach is needed to identify these systems. Campylobacter jejuni is the leading cause of bacterial-derived gastroenteritis and utilizes numerous systems to damage host DNA. Therefore, it is imperative to identify C. jejuni nucleases to understand the molecular mechanism of both infection and pathology. Detailed protocols for a transposon insertion sequencing-based DNase agar screen, a quantitative PCR nuclease screen, and PCR transposon insertion confirmation are included in this article. © 2021 Wiley Periodicals LLC. Basic Protocol 1: DNase agar colony screen of Campylobacter jejuni transposon insertion sequencing library isolates Basic Protocol 2: Quantitative PCR nuclease screen of transposon insertion sequencing library isolates Basic Protocol 3: PCR transposon insertion confirmation.

Characterization of Campylobacter jejuni-Neutrophil Interactions.

Campylobacter jejuni is the leading cause of bacterial-derived gastroenteritis worldwide, infecting 96 million individuals annually. During infection, inflammation and tissue pathology occur in the lower gastrointestinal tract, including the recruitment of leukocytes. Neutrophils are the most abundant leukocyte in humans, and recruitment is associated with bacterial infections and the development of various inflammatory disorders, including inflammatory bowel disease. Neutrophils possess three main antibacterial functions: phagocytosis and degradation of microbes, degranulation to release antimicrobial proteins, and extrusion of neutrophil extracellular traps (NETs). Because neutrophils are recruited to the site of C. jejuni infection and they are associated with damaging inflammation in other diseases, it is imperative to understand the immunopathology that occurs during C. jejuni infection and thoroughly study the neutrophil response to the pathogen. Detailed protocols for human and ferret neutrophil isolations, neutrophil gentamicin protection assay, neutrophil activation flow cytometry assay, NET induction and quantification, and neutrophil western blot analysis are included in this article. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human and ferret neutrophils Basic Protocol 2: Neutrophil gentamicin protection assay Basic Protocol 3: Neutrophil activation flow cytometry analyses Basic Protocol 4: Neutrophil extracellular trap induction and quantification Basic Protocol 5: Western blot detection of neutrophil-derived antimicrobial proteins.

Metal homeostasis in pathogenic Epsilonproteobacteria: mechanisms of acquisition, efflux, and regulation.

Epsilonproteobacteria are a diverse class of eubacteria within the Proteobacteria phylum that includes environmental sulfur-reducing bacteria and the human pathogens, Campylobacter jejuni and Helicobacter pylori. These pathogens infect and proliferate within the gastrointestinal tracts of multiple animal hosts, including humans, and cause a variety of disease outcomes. While infection of these hosts provides nutrients for the pathogenic Epsilonproteobacteria, many hosts have evolved a variety of strategies to either sequester metals from the invading pathogen or exploit the toxicity of metals and drive their accumulation as an antimicrobial strategy. As a result, C. jejuni and H. pylori have developed mechanisms to sense changes in metal availability and regulate their physiology in order to respond to either metal limitation or accumulation. In this review, we will discuss the challenges of metal availability at the host-pathogen interface during infection with C. jejuni and H. pylori and describe what is currently known about how these organisms alter their gene expression and/or deploy bacterial virulence factors in response to these environments.

Role of MrkJ, a phosphodiesterase, in type 3 fimbrial expression and biofilm formation in Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen that has been shown to adhere to human extracellular matrices using the type 3 fimbriae. Introduction of plasmids carrying genes known to alter intracellular cyclic-di-GMP pools in Vibrio parahaemolyticus revealed that these genes also altered type 3 fimbrial surface expression in K. pneumoniae. Immediately adjacent to the type 3 fimbrial gene cluster is a gene, mrkJ, that is related to a family of bacterial genes encoding phosphodiesterases. We identify here a role for MrkJ, a functional phosphodiesterase exhibiting homology to EAL domain-containing proteins, in controlling type 3 fimbria production and biofilm formation in K. pneumoniae. Deletion of mrkJ resulted in an increase in type 3 fimbria production and biofilm formation as a result of the accumulation of intracellular cyclic-di-GMP. This gene was shown to encode a functional phosphodiesterase via restoration of motility in a V. parahaemolyticus strain previously shown to accumulate cyclic-di-GMP and in vitro using phosphodiesterase activity assays. The effect of the mrkJ mutation on type 3 fimbrial expression was shown to be at the level of mrkA gene transcription by using quantitative reverse transcription-PCR. These results reveal a previously unknown role for cyclic-di-GMP in type 3 fimbrial production.

Whole-Genome Sequencing and Bioinformatic Analysis of Environmental, Agricultural, and Human Campylobacter jejuni Isolates From East Tennessee.

As a leading cause of bacterial-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health in both the developed and developing worlds. Despite its prevalence as a human pathogen, the source of these infections remains poorly understood due to the mutation frequency of the organism and past limitations of whole genome analysis. Recent advances in both whole genome sequencing and computational methods have allowed for the high-resolution analysis of intraspecies diversity, leading multiple groups to postulate that these approaches may be used to identify the sources of Campylobacter jejuni infection. To address this hypothesis, our group conducted a regionally and temporally restricted sampling of agricultural and environmental Campylobacter sources and compared isolated C. jejuni genomes to those that caused human infections in the same region during the same time period. Through a network analysis comparing genomes from various sources, we found that human C. jejuni isolates clustered with those isolated from cattle and chickens, indicating these as potential sources of human infection in the region.