\u3ci\u3eCampylobacter\u3c/i\u3e Pathogenesis and Subunit Vaccine Development

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis in the United States. Increasing resistance of Campylobacter to clinical antibiotics raises an urgent need for novel strategies to prevent and control infections in humans and animal reservoirs, which necessitates a better understanding of Campylobacter pathogenesis. We hypothesize that multidrug efflux pump CmeABC and ferric enterobactin (FeEnt) iron acquisition systems, which play a critical role in Campylobacter pathogenesis, are novel targets for developing effective measures against Campylobacter. To test this, the molecular, antigenic, functional, and protective characteristics of two outer membrane proteins, CmeC (an essential component of CmeABC drug efflux pump) and CfrA (a FeEnt receptor), were examined. Both CmeC and CfrA are highly conserved and widely produced in C. jejuni strains. Anti-CmeC and Anti-CfrA antibodies inhibited the function of CmeABC efflux pump and CfrA, resulting enhanced susceptibility to bile salts and reduced utilization of FeEnt of C. jejuni, respectively. Immunoblotting analysis also indicated that CfrA is expressed and immunogenic in vivo. Amino acid substitution mutagenesis demonstrated that a highly conserved basic amino acid R327 in CfrA plays a critical role in FeEnt acquisition. The purified recombinant CmeC and a Salmonella live vaccine expressing the protective epitope of CfrA were evaluated as subunit vaccines against Campylobacter infection in the chicken model. CmeC vaccination elicited immune response but failed to reduce C. jejuni colonization in the intestine. However, Salmonella-vectored vaccine conferred significant protection against C. jejuni challenge. To further elucidate the role of iron acquisition in the pathogenesis of Campylobacter, whole genome sequence of a unique C. jejuni strain was determined using a 454 GS FLX sequencer with Titanium series reagents. Comparative genomics analysis led to the identification of a novel Campylobacter Enterobactin Esterase (Cee) that is essential in the CfrB-dependent FeEnt utilization pathway. Extensive genetic manipulation revealed molecular pathways and mechanistic features of the two orchestrated FeEnt acquisition systems in Campylobacter. This project provides critical information about the feasibility of targeting CmeC and CfrA for immune protection against Campylobacter colonization in the intestine, and increases our understanding of the critical role of FeEnt acquisition in the pathophysiology of Campylobacter

A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency in Campylobacter jejuni

Conjugation is an important mechanism for horizontal gene transfer in Campylobacter jejuni, the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency in Campylobacter spp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC) C. jejuni strain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFC C. jejuni strain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFC C. jejuni strain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme (aka CjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (\u3e5,000-fold). Chromosomal complementation of three diverse HFC C. jejuni strains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest the Escherichia coli-derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency in C. jejuni. Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni

Campylobacter jejuni genotypes are associated with post-infection irritable bowel syndrome in humans

Campylobacter enterocolitis may lead to post-infection irritable bowel syndrome (PI-IBS) and while some C. jejuni strains are more likely than others to cause human disease, genomic and virulence characteristics promoting PI-IBS development remain uncharacterized. We combined pangenome-wide association studies and phenotypic assays to compare C. jejuni isolates from patients who developed PI-IBS with those who did not. We show that variation in bacterial stress response (Cj0145_phoX), adhesion protein (Cj0628_CapA), and core biosynthetic pathway genes (biotin: Cj0308_bioD; purine: Cj0514_purQ; isoprenoid: Cj0894c_ispH) were associated with PI-IBS development. In vitro assays demonstrated greater adhesion, invasion, IL-8 and TNFα secretion on colonocytes with PI-IBS compared to PI-no-IBS strains. A risk-score for PI-IBS development was generated using 22 genomic markers, four of which were from Cj1631c, a putative heme oxidase gene linked to virulence. Our finding that specific Campylobacter genotypes confer greater in vitro virulence and increased risk of PI-IBS has potential to improve understanding of the complex host-pathogen interactions underlying this condition

Molecular, Antigenic, and Functional Characteristics of Ferric Enterobactin Receptor CfrA in Campylobacter jejuni ▿

The ferric enterobactin receptor CfrA not only is responsible for high-affinity iron acquisition in Campylobacter jejuni but also is essential for C. jejuni colonization in animal intestines. In this study, we determined the feasibility of targeting the iron-regulated outer membrane protein CfrA for immune protection against Campylobacter colonization. Alignment of complete CfrA sequences from 15 Campylobacter isolates showed that the levels of amino acid identity for CfrA range from 89% to 98%. Immunoblotting analysis using CfrA-specific antibodies demonstrated that CfrA was dramatically induced under iron-restricted conditions and was widespread and produced in 32 Campylobacter primary strains from various sources and from geographically diverse areas. The immunoblotting survey results were highly correlated with the results of an enterobactin growth promotion assay and a PCR analysis using cfrA-specific primers. Inactivation of the cfrA gene also impaired norepinephrine-mediated growth promotion, suggesting that CfrA is required for C. jejuni to sense intestinal stress hormones during colonization. Complementation of the cfrA mutant with a wild-type cfrA allele in trans fully restored the production and function of CfrA. A growth assay using purified anti-CfrA immunoglobulin G demonstrated that specific CfrA antibodies could block the function of CfrA, which diminished ferric enterobactin-mediated growth promotion under iron-restricted conditions. The inhibitory effect of CfrA antibodies was dose dependent. Immunoblotting analysis also indicated that CfrA was expressed and immunogenic in chickens experimentally infected with C. jejuni. Amino acid substitution mutagenesis demonstrated that R327, a basic amino acid that is highly conserved in CfrA, plays a critical role in ferric enterobactin acquisition in C. jejuni. Together, these findings strongly suggest that CfrA is a promising vaccine candidate for preventing and controlling Campylobacter infection in humans and animal reservoirs

Discovery of bile salt hydrolase inhibitors using an efficient high-throughput screening system.

The global trend of restricting the use of antibiotic growth promoters (AGP) in animal production necessitates the need to develop valid alternatives to maintain productivity and sustainability of food animals. Previous studies suggest inhibition of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization, is a promising approach to promote animal growth performance. To achieve the long term goal of developing novel alternatives to AGPs, in this study, a rapid and convenient high-throughput screening (HTS) system was developed and successfully used for identification of BSH inhibitors. With the aid of a high-purity BSH from a chicken Lactobacillus salivarius strain, we optimized various screening conditions (e.g. BSH concentration, reaction buffer pH, incubation temperature and length, substrate type and concentration) and establish a precipitation-based screening approach to identify BSH inhibitors using 96-well or 384-well microplates. A pilot HTS was performed using a small compound library comprised of 2,240 biologically active and structurally diverse compounds. Among the 107 hits, several promising and potent BSH inhibitors (e.g. riboflavin and phenethyl caffeate) were selected and validated by standard BSH activity assay. Interestingly, the HTS also identified a panel of antibiotics as BSH inhibitor; in particular, various tetracycline antibiotics and roxarsone, the widely used AGP, have been demonstrated to display potent inhibitory effect on BSH. Together, this study developed an efficient HTS system and identified several BSH inhibitors with potential as alternatives to AGP. In addition, the findings from this study also suggest a new mode of action of AGP for promoting animal growth

Effect of Bile Salt Hydrolase Inhibitors on a Bile Salt Hydrolase from Lactobacillus acidophilus

Bile salt hydrolase (BSH), a widely distributed function of the gut microbiota, has a profound impact on host lipid metabolism and energy harvest. Recent studies suggest that BSH inhibitors are promising alternatives to antibiotic growth promoters (AGP) for enhanced animal growth performance and food safety. Using a high-purity BSH from Lactobacillus salivarius strain, we have identified a panel of BSH inhibitors. However, it is still unknown if these inhibitors also effectively inhibit the function of the BSH enzymes from other bacterial species with different sequence and substrate spectrum. In this study, we performed bioinformatics analysis and determined the inhibitory effect of identified BSH inhibitors on a BSH from L. acidophilus. Although the L. acidophilus BSH is phylogenetically distant from the L. salivarius BSH, sequence analysis and structure modeling indicated the two BSH enzymes contain conserved, catalytically important amino residues and domain. His-tagged recombinant BSH from L. acidophilus was further purified and used to determine inhibitory effect of specific compounds. Previously identified BSH inhibitors also exhibited potent inhibitory effects on the L. acidophilus BSH. In conclusion, this study demonstrated that the BSH from L. salivarius is an ideal candidate for screening BSH inhibitors, the promising alternatives to AGP for enhanced feed efficiency, growth performance and profitability of food animals

Dosing effects of selected BSH inhibitors on BSH activity.

<p>(A) Inhibition of BSH activity by caffeic acid phenethyl ester (CAPE). (B) Inhibition of BSH activity by riboflavin. All assays were performed in triplicate. The procedure is detailed in Materials and Methods.</p

Effect of different classes of antibiotics on BSH activity.

<p>The antibiotic hits by HTS.</p

A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency in Campylobacter jejuni

Conjugation is an important mechanism for horizontal gene transfer in Campylobacter jejuni, the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency in Campylobacter spp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC) C. jejuni strain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFC C. jejuni strain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFC C. jejuni strain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme (aka CjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (>5,000-fold). Chromosomal complementation of three diverse HFC C. jejuni strains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest the Escherichia coli-derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency in C. jejuni. Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni.This article is published as Zeng, Ximin, Zuowei Wu, Qijing Zhang, and Jun Lin. "A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency in Campylobacter jejuni." Applied and Environmental Microbiology 84, no. 23 (2018): e02004-18. DOI: 10.1128/AEM.02004-18. Posted with permission.</p