Wayne T. Muraoka

Campylobacter jejuni is an enteric pathogen and a major cause of foodborne illness worldwide. It has been shown that C. jejuni primarily utilizes amino acids as its preferred energy source, but its ability to utilize L-fucose can grant a competitive advantage during intestinal colonization. In C. jejuni, fucose utilization is encoded by a variable region named plasticity region 2 (PR2); however, the regulatory mechanism for the region remains unknown and is investigated in this study. Genomic sequence analysis revealed that immediately upstream of the fucose utilization operon is a putative IclR-type transcriptional regulator, cj0480c (named fucR here). To determine whether fucR regulates the expression of the fucose utilization operon, we generated a knock-out mutant of fucR. RT-PCR and microarray analysis found that all the genes in the operon were polycistronic and significantly upregulated in the fucR mutant compared with their expression in the wild-type strain. In the presence of fucose, expression of the fucose utilization genes was induced in the wild-type strain but no longer inducible in the fucR mutant, suggesting that FucR functions as a repressor for the fucose utilization operon. To determine whether FucR directly or indirectly regulates the fucose utilization operon, a 6xHis-tagged full-length FucR was produced in Escherichia coli, and the purified recombinant FucR was used in electrophoretic mobility shift assay, which demonstrated that FucR bound specifically to the promoter region of the fucose utilization operon. Together, these results indicate that the L-fucose utilization operon in C. jejuni is directly regulated by FucR, which functions as a transcriptional repressor and modulates the expression of the operon in response to fucose.

Accumulating evidence suggests that ischemia–reperfusion‐induced injury is associated with the formation of reactive oxygen species (ROS). This study demonstrates the therapeutic effectiveness of novel europium‐doped cerium oxide nanoparticles (Eu‐doped Ceria NPs) as ROS scavengers in a mouse model of intestinal ischemia–reperfusion‐induced injury. An increased production of superoxide radicals is detected in the intestine throughout the ischemia stage and again after initiating reperfusion. These changes in superoxide radical formation are associated with the induction of inflammatory cytokines in the intestine. This study further shows that Eu‐Ceria NPs exhibit superoxide scavenging activity in vitro. Importantly, administration of Eu‐Ceria NPs into the intestinal lumen during the onset of ischemia effectively blocks superoxide accumulation, reduces the expression of IL‐1b, and ameliorates the intestinal pathology. These results suggest that early increased production of ROS during the ischemia–reperfusion promotes intestinal pathology and that mucosal delivery of Eu‐Ceria NPs may be a potential therapeutic approach to block ROS accumulation and ameliorate the severity of intestinal disease.

Mutator strains play an important role in the emergence of antibiotic-resistant bacteria. Campylobacter jejuni is a leading cause of foodborne illnesses worldwide and is increasingly resistant to clinically important antibiotics. The objective of this study was to identify the genetic basis that contributes to a mutator phenotype in Campylobacter and determine the role of this phenotype in the development of antibiotic resistance. A C. jejuni isolate (named CMT) showing a mutator phenotype was subjected to WGS analysis. Comparative genomics, site-specific reversion and mutation, and gene knockout were conducted to prove the mutator effect was caused by a single nucleotide change in the mutY gene of C. jejuni. The C. jejuni CMT isolate showed ∼100-fold higher mutation frequency to ciprofloxacin than the WT strain. Under selection by ciprofloxacin, fluoroquinolone-resistant mutants emerged readily from the CMT isolate. WGS identified a single nucleotide change (G595→T) in the mutY gene of the CMT isolate. Further experiments using defined mutant constructs proved its specific role in elevating mutation frequencies. The mutY point mutation also led to an ∼700-fold increase in the emergence of ampicillin-resistant mutants, indicating its broader impact on antibiotic resistance. Structural modelling suggested the G595→T mutation probably affects the catalytic domain of MutY and consequently abolishes the anti-mutator function of this DNA repair protein. The G595→T mutation in mutY abolishes its anti-mutator function and confers a mutator phenotype in Campylobacter, promoting the emergence of antibiotic-resistant Campylobacter.

Macrolide is the drug of choice to treat human campylobacteriosis, but Campylobacter resistance to this antibiotic is rising. The mechanisms employed by Campylobacter jejuni to adapt to erythromycin treatment remain unknown and are examined in this study. The transcriptomic response of C. jejuni NCTC 11168 to erythromycin (Ery) treatment was determined by competitive microarray hybridizations. Representative genes identified to be differentially expressed were further characterized by constructing mutants and assessing their involvement in antimicrobial susceptibility, oxidative stress tolerance, and chicken colonization. Following the treatment with an inhibitory dose of Ery, 139 genes were up-regulated and 119 were down-regulated. Many genes associated with flagellar biosynthesis and motility was up-regulated, while many genes involved in tricarboxylic acid cycle, electron transport, and ribonucleotide biosynthesis were down-regulated. Exposure to a sub-inhibitory dose of Ery resulted in differential expression of much fewer genes. Interestingly, two putative drug efflux operons (cj0309c-cj0310c and cj1173-cj1174) were up-regulated. Although mutation of the two operons did not alter the susceptibility of C. jejuni to antimicrobials, it reduced Campylobacter growth under high-level oxygen. Another notable finding is the consistent up-regulation of cj1169c-cj1170c, of which cj1170c encodes a known phosphokinase, an important regulatory protein in C. jejuni. Mutation of the cj1169c-cj1170c rendered C. jejuni less tolerant to atmospheric oxygen and reduced Campylobacter colonization and transmission in chickens. These findings indicate that Ery treatment elicits a range of changes in C. jejuni transcriptome and affects the expression of genes important for in vitro and in vivo adaptation. Up-regulation of motility and down-regulation of energy metabolism likely facilitate Campylobacter to survive during Ery treatment. These findings provide new insight into Campylobacter adaptive response to antibiotic treatment and may help to understand the mechanisms underlying antibiotic resistance development.

Campylobacter jejuni remains among the leading causes of bacterial food-borne illness. The current understanding of Campylobacter physiology suggests that it is asaccharolytic and is unable to catabolize exogenous carbohydrates. Contrary to this paradigm, we provide evidence for l -fucose utilization by C. jejuni . The fucose phenotype, shown in chemically defined medium, is strain specific and linked to an 11-open reading frame (ORF) plasticity region of the bacterial chromosome. By constructing a mutation in fucP (encoding a putative fucose permease), one of the genes in the plasticity region, we found that this locus is required for fucose utilization. Consistent with their function in fucose utilization, transcription of the genes in the locus is highly inducible by fucose. PCR screening revealed a broad distribution of this genetic locus in strains derived from various host species, and the presence of this locus was consistently associated with fucose utilization. Birds inoculated with the fucP mutant strain alone were colonized at a level comparable to that by the wild-type strain; however, in cocolonization experiments, the mutant was significantly outcompeted by the wild-type strain when birds were inoculated with a low dose (10 ⁵ CFU per bird). This advantage was not observed when birds were inoculated at a higher inoculum dose (10 ⁸ CFU per bird). These results demonstrated a previously undescribed substrate that supports growth of C. jejuni and identified the genetic locus associated with the utilization of this substrate. These findings substantially enhance our understanding of the metabolic repertoire of C. jejuni and the role of metabolic diversity in Campylobacter pathobiology.

Summary: 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.

This study is aimed to determine the role of capsular polysaccharide (CPS) and lipooligosaccharide (LOS) in modulating antimicrobial resistance and natural transformation of Campylobacter jejuni, an important food-borne human pathogen. A series of C. jejuni mutants, which are defective in either CPS or LOS or both, were constructed. The antimicrobial susceptibility, bacterial surface hydrophobicity, natural transformation frequency and DNA binding and uptake were measured and compared between the mutants and the wild-type strain. Truncation of LOS greatly reduced (8-fold) the intrinsic resistance of C. jejuni to erythromycin, a key antibiotic used for treating human campylobacteriosis, while the loss of CPS did not result in significant changes in the susceptibility to antimicrobial agents. Notably, mutation of LOS also significantly increased (>16-fold) the susceptibility to erythromycin in C. jejuni mutants carrying the A2074G mutation in 23S rRNA. The increased susceptibility to erythromycin in the LOS mutant was probably due to enhanced permeability to this antibiotic, because the LOS mutation rendered the surface of C. jejuni more hydrophobic. Loss of CPS and truncation of LOS increased the transformation frequency by 4- and 25-fold, respectively, and mutation of both CPS and LOS resulted in a 97-fold increase in the transformation frequency. Consistent with the increased transformation frequencies, the CPS and LOS mutants showed enhanced rates of DNA uptake. These results demonstrate that the surface polysaccharides in C. jejuni contribute to the resistance to erythromycin, a clinically important antibiotic, but restrict natural transformation.

Campylobacter jejuni, an important food-borne human pathogen, is increasingly resistant to antimicrobials. Natural transformation is considered to be a main mechanism for mediating the transfer of genetic materials encoding antibiotic resistance determinants in C. jejuni, but direct evidence for this notion is still lacking. In this study, we determined the role of Cj1211 in natural transformation and in the development of antibiotic resistance in C. jejuni. Insertional mutagenesis of Cj1211, a Helicobacter pylori ComH3 homolog, abolished natural transformation in C. jejuni. In vitro coculture of C. jejuni strains carrying either kanamycin or tetracycline resistance markers demonstrated the development of progenies that were resistant to both antibiotics, indicating that the horizontal transfer of antibiotic resistance determinants actively occurs in mixed Campylobacter populations. A mutation of Cj1211 or the addition of DNase I in culture media completely inhibited the formation of progenies that were resistant to both antibiotics, indicating that the horizontal transfer of the resistance determinants is mediated by natural transformation. Interestingly, the mutation of Cj1211 also reduced the frequency of emergence of spontaneous mutants that were resistant to fluoroquinolone (FQ) and streptomycin but did not affect the outcome of FQ resistance development under FQ treatment, suggesting that natural transformation does not play a major role in the emergence of FQ-resistant Campylobacter strains during treatment with FQ antimicrobials. These results define Cj1211 as a competence factor in Campylobacter, prove the role of natural transformation in the horizontal transfer of antibiotic resistance determinants in Campylobacter, and provide new insights into the mechanism underlying the development of FQ-resistant Campylobacter strains.