Molecular mechanisms of Mycoplasma hyopneumoniae adherence to swine respiratory epithelial cells

A microtiter plate adherence assay for Mycoplasma hyopneumoniae was established by using purified swine tracheal cilia which are the natural targets for the mycoplasma. M. hyopneumoniae bound specifically to solubilized cilia immobilized onto microtiter plates. Dextran sulfate, heparin, chondroitin sulfate, laminin, mucin, and fucoidan significantly inhibited binding of the mycoplasmas to cilia. Heparin, mucin, fucoidan, and chondroitin sulfate interacted with the adhesins on the surface of mycoplasmas, whereas laminin blocked the receptors in cilia. Treatment of cilia with neuraminidase appeared to promote adherence of the mycoplasmas; whereas, treatment of cilia with sodium metaperiodate decreased the binding. In the second study, the natural glycolipid receptors in cilia for Mycoplasma hyopneumoniae adherence were analyzed by using a thin-layer chromatography (TLC) overlay assay. M. hyopneumoniae bound specifically to sulfatide, globoside, ganglioside GM3, and three glycolipids (La, Lb, and Lc) extracted from swine tracheal cilia. La, Lb, and Lc were determined to be sulfated glycolipids. Sensitive and dose-dependent binding of M. hyopneumoniae was also detected to La, Lb, and Lc immobilized onto microtiter plates. In the third study, the adhesins of M. hyopneumoniae were identified and characterized. A monoclonal antibody (Mab F2G5) predominantly recognized a 97 KDa (P97) protein of M. hyopneumoniae and inhibited the adherence of the mycoplasma to cilia. Immunolabelling demonstrated that the proteins recognized by Mab F2G5 were located at the surface of the mycoplasmas, predominantly on a surface fuzzy layer. Antibody affinity chromatography-purified P97 was able to bind to cilia and blocked the adherence of intact M. hyopneumoniae cells to cilia. Surface proteolysis of M. hyopneumoniae with trypsin selectively digested P97 and decreased the adherence activity of the mycoplasma. The predominant proteins detected by Mab F2G5 were different in size among various strains, and multiple proteins were detected with Mab F2G5 in a single strain, indicating that the antigens bearing the epitope for Mab F2G5 undergo intraspecies and intrastrain size variations. In conclusion, adherence of M. hyopneumoniae to cilia was mainly mediated by ligand-receptor interactions. Three sulfated glycolipids (La, Lb, and Lc) in cilia were the major native receptors for M. hyopneumoniae. A major adhesin of M. hyopneumoniae was determined to be P97; evidence was obtained that M. hyopneumoniae adhesins undergo size variations, which may be a pathogenic mechanism utilized by M. hyopneumoniae to evade the porcine immune system

Cj0011c, a Periplasmic Single- and Double-Stranded DNA-Binding Protein, Contributes to Natural Transformation in Campylobacter jejuni

Campylobacter jejuni is an important bacterial pathogen causing gastroenteritis in humans. C. jejuni is capable of natural transformation, which is considered a major mechanism mediating horizontal gene transfer and generating genetic diversity. Despite recent efforts to elucidate the transformation mechanisms of C. jejuni, the process of DNA binding and uptake in this organism is still not well understood. In this study, we report a previously unrecognized DNA-binding protein (Cj0011c) in C. jejuni that contributes to natural transformation. Cj0011c is a small protein (79 amino acids) with a partial sequence homology to the C-terminal region of ComEA in Bacillus subtilis. Cj0011c bound to both single- and double-stranded DNA. The DNA-binding activity of Cj0011c was demonstrated with a variety of DNAs prepared from C. jejuni or Escherichia coli, suggesting that the DNA binding of Cj0011c is not sequence dependent. Deletion of the cj0011c gene from C. jejuni resulted in 10- to 50-fold reductions in the natural transformation frequency. Different from the B. subtilis ComEA, which is an integral membrane protein, Cj0011c is localized in the periplasmic space of C. jejuni. These results indicate that Cj0011c functions as a periplasmic DNA receptor contributing to the natural transformation of C. jejuni

Advances in Campylobacter biology and implications for biotechnological applications

Campylobacter jejuni is a major foodborne pathogen of animal origin and a leading cause of bacterial gastroenteritis in humans. During the past decade, especially since the publication of the first C. jejuni genome sequence, major advances have been made in understanding the pathobiology and physiology of this organism. It is apparent that C. jejuni utilizes sophisticated mechanisms for effective colonization of the intestinal tracts in various animal species. Although Campylobacter is fragile in the environment and requires fastidious growth conditions, it exhibits great flexibility in the adaptation to various habitats including the gastrointestinal tract. This high adaptability is attributable to its genetically, metabolically and phenotypically diverse population structure and its ability to change in response to various challenges. Unlike other enteric pathogens, such as Escherichia coli and Salmonella, Campylobacter is unable to utilize exogenous glucose and mainly depends on the catabolism of amino acids as a carbon source. Campylobacter proves highly mutable in response to antibiotic treatments and possesses eukaryote-like dual protein glycosylation systems, which modify flagella and other surface proteins with specific sugar structures. In this review we will summarize the distinct biological traits of Campylobacter and discuss the potential biotechnological approaches that can be developed to control this enteric pathogen

Anti-Campylobacter Activities and Resistance Mechanisms of Natural Phenolic Compounds in Campylobacter

Background Campylobacter is a major foodborne pathogen and alternative antimicrobials are needed to prevent or decrease Campylobacter contamination in foods or food producing animals. The objectives of this study are to define the anti-Campylobacter activities of natural phenolic compounds of plant origin and to determine the roles of bacterial drug efflux systems in the resistance to these natural phenolics in Campylobacter jejuni. Methodology/Principal Findings Anti-Campylobacter activities were evaluated by an MIC assay using microdilution coupled with ATP measurement. Mutants of the cmeB and cmeF efflux genes and the cmeR transcriptional repressor gene were compared with the wild-type strain for their susceptibilities to phenolics in the absence and presence of efflux-pump inhibitors (EPIs). The phenolic compounds produced significant, but variable activities against both antibiotic-susceptible and antibiotic resistant Campylobacter. The highest anti-Campylobacter activity was seen with carnosic and rosmarinic acids in their pure forms or in enriched plant extracts. Inactivation of cmeB rendered C. jejuni significantly more susceptible to the phenolic compounds, while mutation of cmeF or cmeR only produced a moderate effect on the MICs. Consistent with the results from the efflux pump mutants, EPIs, especially phenylalanine-arginine β-naphthylamide and NMP, significantly reduced the MICs of the tested phenolic compounds. Further reduction of MICs by the EPIs was also observed in the cmeB and cmeF mutants, suggesting that other efflux systems are also involved in Campylobacter resistance to phenolic compounds. Conclusion/Significance Natural phenolic compounds of plant origin have good anti-Campylobacter activities and can be further developed for potential use in controlling Campylobacter. The drug efflux systems in Campylobacter contribute significantly to its resistance to the phenolics and EPIs potentiate the anti-Campylobacter activities of plant phenolic compounds

Genomic Insights into Campylobacter jejuni Virulence and Population Genetics

Campylobacter jejuni has long been recognized as a main food-borne pathogen in many parts of the world. Natural reservoirs include a wide variety of domestic and wild birds and mammals, whose intestines offer a suitable biological niche for the survival and dissemination of the organism. Understanding the genetic basis of the biology and pathogenicity of C. jejuni is vital to prevent and control Campylobacter-associated infections. The recent progress in sequencing techniques has allowed for a rapid increase in our knowledge of the molecular biology and the genetic structures of Campylobacter. Single-molecule realtime (SMRT) sequencing, which goes beyond four-base sequencing, revealed the role of DNA methylation in modulating the biology and virulence of C. jejuni at the level of epigenetics. In this review, we will provide an up-to-date review on recent advances in understanding C. jejuni genomics, including structural features of genomes, genetic traits of virulence, population genetics, and epigenetics

A mutator phenotype promoting the emergence of spontaneous oxidative stress-resistant mutants in Campylobacter jejuni

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. As a microaerophilic organism, C. jejuni must be able to defend against oxidative stress encountered both in the host and in the environment. How Campylobacter utilizes a mutation-based mechanism for adaptation to oxidative stress is still unknown. Here we present a previously undescribed phenotypic and genetic mechanism that promotes the emergence of oxidative stress resistant mutants. Specifically, we showed that a naturally occurring mutator phenotype, resulting from a loss of function mutation in the DNA repair enzyme MutY, increased oxidative stress resistance (OXR) in C. jejuni. We further demonstrated that MutY malfunction didn\u27t directly contribute to the OXR phenotype, but increased the spontaneous mutation rate in the peroxide regulator gene perR, which functions as a repressor for multiple genes involved in oxidative stress resistance. Mutations in PerR resulted in loss of its DNA binding function and derepression of PerR-controlled oxidative stress defense genes, thereby conferring an OXR phenotype and facilitating Campylobacter survival under oxidative stress. These findings reveal a new mechanism that promotes the emergence of spontaneous OXR mutants in bacterial organisms

Fitness of antimicrobial-resistant Campylobacter and Salmonella

Campylobacter and Salmonella are the most commonly reported bacterial causes of human foodborne infections, and increasing proportions of these pathogens become resistant to medically important antimicrobial agents, imposing a burden on public health. Acquisition of resistance to antibiotics affects the adaptation and evolution of Salmonella and Campylobacter in various environments. Many resistance-conferring mutations entail a biological fitness cost, while others (e.g. fluoroquinolone resistance in Campylobacter) have no cost or even enhanced fitness. In Salmonella, the fitness disadvantage due to antimicrobial resistance can be restored by acquired compensatory mutations, which occur both in vitro and in vivo. The compensated or even enhanced fitness associated with antibiotic resistance may facilitate the spread and persistence of antimicrobial-resistant Salmonella and Campylobacter in the absence of selection pressure, creating a significant barrier for controlling antibiotic-resistant foodborne pathogens

Lack of Evidence for erm(B) Infiltration Into Erythromycin-Resistant Campylobacter coli and Campylobacter jejuni from Commercial Turkey Production in Eastern North Carolina: A Major Turkey-Growing Region in the United States

In Campylobacter spp., resistance to erythromycin and other macrolides has typically implicated ribosomal mutations, especially substitutions in the 23S rRNA genes. However, in 2014, the macrolide resistance gene erm(B) was reported for the first time in Campylobacter and shown to be harbored by a multidrug resistance island in the chromosome of the swine-derived strain Campylobacter coli ZC113. erm(B)-positive C. coli and Campylobacter jejuni strains from the food supply have been mostly reported from China. However, erm(B)-positive C. coli isolates were also detected recently in fecal samples from turkeys in Spain. To determine whether erm(B) may be harbored by erythromycin-resistant Campylobacter from commercial turkey production in eastern North Carolina, a major turkey-growing region in the United States, we investigated a panel of 178 erythromycin-resistant isolates (174 C. coli, 4 C. jejuni) using PCR with erm(B)-specific primers. None of the isolates were PCR-positive for erm(B) and sequence analysis of a subset of these erythromycin-resistant isolates revealed that all harbored A2075G substitutions in the 23S rRNA genes. Data fail to provide evidence for infiltration of erm(B) into erythromycin-resistant Campylobacter from commercial turkey production in this region and suggest the need for continuing surveillance

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

Constitutive and Inducible Expression of the rRNA Methylase Gene erm(B) in Campylobacter

Macrolides are the antimicrobials of choice for treating human campylobacteriosis. The recent emergence of erm(B) in Campylobacter bacteria threatens the utility of this class of antibiotics. Here we report the constitutive and inducible expression of erm(B) in Campylobacter isolates derived from diarrheal patients and food-producing animals. Constitutive expression of erm(B) was associated with insertion and deletion in the regulatory region of the gene, providing the first documentation of the differential expression of erm(B) in Campylobacter bacteria