The disordered arrangement of flagella biosynthetic genes, combined with a simplified regulatory mechanism, has made elucidating the process of Campylobacter jejuni flagellation difficult. FlhF is a recently identified element that controls the assembly of the flagella, although its function mechanism and regulatory preference are not well defined at present. In this study, we found that inactivation of FlhF caused the transcription of most flagella genes down-regulated. The importance of FlhF was systematically evaluated by analyzing changes in the transcription profiles between wild-type and flhF mutant strains, which showed that FlhF affects late flagella genes obviously. FlhF is constitutively expressed during C. jejuni growth, demonstrating that it is a class I flagella element that participates in early flagella assembly. In addition, the early flagella component FlhB was not localized to the cell pole in the flhF mutant. Thus, flagella assembly was impeded at the initial stage. We propose a model in which FlhF helps target the early flagella components to the cell pole, functioning prior to the formation of the flagella export apparatus, and thus places FlhF at the top of the flagella regulatory cascade hierarchy. Inactivation of FlhF impeded flagella assembly at the initial stage and decreased transcription of flagella genes through a feed-back control mechanism, leading to FlhF having a significant influence on the expression of late flagella components and resulting in the aflagellate C. jejuni phenotype. Our present study has uncovered how FlhF influences C. jejuni flagella biosynthesis, which will be helpful in understanding the C. jejuni flagella biosynthetic pathway and bacterial flagellation in general.

Campylobacter is one of the most important foodborne pathogens worldwide, and poultry is regarded as the main reservoir of Campylobacter. The contamination of Campylobacter in broiler chickens at the farm level is closely related to the transmission of Campylobacter in the poultry production chain. This study identified 464 Campylobacter isolates from 1,534 samples from broiler rearing period and slaughtering process including 233 Campylobacter jejuni isolates and 231 Campylobacter coli isolates. We have observed a dynamic distribution of Campylobacter during broiler chicken production, that 66.3% of Campylobacter isolates were C. jejuni during broiler rearing period, while C. coli occupied 60.4% of Campylobacter isolates during the broiler slaughtering process. A tag-label method allowed us to track the dynamic of Campylobacter in each broiler chicken from 31-day age at rearing to the partition step in the slaughterhouse. At the 31-day during rearing, 150 broiler chicken were labeled, and was tracked for Campylobacter positive from rearing period to slaughtering process. Among the labeled broiler, 11 of the tracking broiler samples were able to detect Campylobacter from rearing period to slaughtering. All Campylobacter isolates from the 11 tracking samples were sequenced and analyzed. C. jejuni isolates were divided into four STs and C. coli isolates were divided into six STs. Isolates with identical core genome were observed from the same tag-labeled samples at different stages indicating a vertical transmission of Campylobacter in the early broiler meat production. Meanwhile, the core genome analysis elucidated the cross-contamination of Campylobacter during the rearing period and the slaughtering process. The virulotyping analysis revealed that all C. jejuni isolates shared the same virulotypes, while C. coli isolates were divided into three different virulotypes. The antimicrobial resistance gene analysis demonstrated that all Campylobacter isolates contained at least two antibiotic resistance genes (ARGs), Tang et al. Campylobacter From Broiler in China and the ARG profiles were well-corresponding to each ST type. Our study observed a high prevalence of Campylobacter during the early chicken meat production, and further studies will be needed to investigate the diversity and transmission of Campylobacter in the poultry production chain.

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.

FlhF is a key protein required for complete flagellar synthesis, and its deletion results in the complete absence of a flagella and thus motility in Campylobacter jejuni. However, the specific mechanism still remains unknown. In this study, RNA-Seq, EMSAs, ChIP-qPCR and β-Galactosidase assays were performed to elucidate the novel interactions between FlhF and genes involved in flagellar synthesis. Results showed that FlhF has an overall influence on the transcription of flagellar genes with an flhF mutant displaying down-regulation of most flagellar related genes. FlhF can directly bind to the flgI promoter to regulate its expression, which has significant expression change in an flhF mutant. The possible binding site of FlhF to the flgI promoter was explored by continuously narrowing the flgI promoter region and performing further point mutations. Meanwhile, FlhF can directly bind to the promoters of rpoD, flgS, and fliA encoding early flagellin regulators, thereby directly or indirectly regulating the synthesis of class I, II, and III flagellar genes, respectively. Collectively, this study demonstrates that FlhF may directly regulate the transcription of flagellar genes by binding to their promoters as a transcriptional regulator, which will be helpful in understanding the mechanism of FlhF in flagellar biosynthetic and bacterial flagellation in general.

Campylobacter jejuni is the major cause of human bacterial diarrhea worldwide. Its pathogenic mechanism remains poorly understood. cj0371 is a novel gene that was uncovered using immunoscreening. There have been no previous reports regarding its function. In this study, we constructed an insertion mutant and complement of this gene in C. jejuni and examined changes in virulence. We observed that the cj0371 mutant showed significantly increased invasion and colonization ability. We also investigated the role of cj0371 in motility, chemotaxis, and growth kinetics to further study its function. We found that the cj0371 mutant displays hypermotility, enhanced chemotaxis, and enhanced growth kinetics. In addition, we localized the Cj0371 protein at the poles of C. jejuni by fluorescence microscopy. We present data that collectively significantly proves our hypothesis that cj0371 is a new virulence-associated gene and through the influence of chemotaxis plays a negative role in C. jejuni pathogenicity.

A lack of relevant disease models for Campylobacter jejuni has long been an obstacle to research into this common enteric pathogen. Here we used an infant rabbit to study C. jejuni infection, which enables us to define several previously unknown but key features of the organism. C. jejuni is capable of systemic invasion in the rabbit, and developed a diarrhea symptom that mimicked that observed in many human campylobacteriosis. The large intestine was the most consistently colonized site and produced intestinal inflammation, where specific cytokines were induced. Genes preferentially expressed during C. jejuni infection were screened, and acs, cj1385, cj0259 seem to be responsible for C. jejuni invasion. Our results demonstrates that the infant rabbit can be used as an alternative experimental model for the study of diarrheagenic Campylobacter species and will be useful in exploring the pathogenesis of other related pathogens.

Campylobacter jejuni is a prevalent foodborne pathogen worldwide. Human infection by C. jejuni primarily arises from contaminated poultry meats. Genes expressed in vivo may play an important role in the pathogenicity of C. jejuni. We applied an immunoscreening method, in vivo-induced antigen technology (IVIAT), to identify in vivo-induced genes during human infection by C. jejuni. An inducible expression library of genomic proteins was constructed from sequenced C. jejuni NCTC 11168 and was then screened using adsorbed, pooled human sera obtained from clinical patients. We successfully identified 24 unique genes expressed in vivo. These genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, and other processes. We selected six genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results showed that the selected six genes were significantly upregulated in vivo but not in vitro. In short, these identified in vivo-induced genes may contribute to human infection of C. jejuni, some of which may be meaningful vaccine candidate antigens or diagnosis serologic markers for campylobacteriosis. IVIAT may present a significant and efficient method for understanding the pathogenicity mechanism of Campylobacter and for finding targets for its prevention and control.

BackgroundCampylobacter jejuni (C. jejuni) is a leading cause of foodborne gastroenteritis worldwide. This bacterium lacks many of the classical virulence factors, and flagellum-associated persistent colonization has been shown to be crucial for its pathogenesis. The flagellum plays a multifunctional role in C. jejuni pathogenesis, and different flagellar elements make diverse contributions. The flhF gene encodes the flagellar biosynthesis regulator, which is important for flagellar biosynthesis. In this study, the influence of flhF on C. jejuni colonization was systematically studied, and the possible mechanisms were also analyzed.ResultsThe flhF gene has a significant influence on C. jejuni colonization, and its inactivation resulted in severe defects in the commensal colonization of chicks, with approximately 104- to 107-fold reductions (for NCTC 11168 and a C. jejuni isolate respectively) observed in the bacterial caecal loads. Similar effects were observed in mice where the flhF mutant strain completely lost the ability to continuously colonize mice, which cleared the isolate at 7 days post inoculation. Characterization of the phenotypic properties of C. jejuni that influence colonization showed that the adhesion and invasion abilities of the C. jejuni flhF mutant were reduced to approximately 52 and 27% of that of the wild-type strain, respectively. The autoagglutination and biofilm-formation abilities of the flhF mutant strain were also significantly decreased. Further genetic investigation revealed that flhF is continuously upregulated during the infection process, which indicates a close association of this gene with C. jejuni pathogenesis. The transcription of some other infection-related genes that are not directly involved in flagellar assembly were also influenced by its inactivation, with the flagellar coexpressed determinants (Feds) being apparently affected.ConclusionsInactivation of flhF has a significant influence on C. jejuni colonization in both birds and mammals. This defect may be caused by the decreased adhesion, invasion, autoagglutination and biofilm-formation abilities of the flhF mutant strain, as well as the influence on the transcription of other infection related genes, which provides insights into this virulence factor and the flagellum mediated co-regulation of C. jejuni pathogenesis.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1318-1) contains supplementary material, which is available to authorized users.