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.

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.

Although campylobacteriosis is a zoonotic foodborne illness, high-risk isolates from animal sources are rarely characterized, and the pathogenic potential of zoonotic strains remains an obstacle to effective intervention against human infection. HS19 has been acknowledged as a maker serotype represented by Campylobacter jejuni (C. jejuni) isolates from patients with post-infection Guillain-Barré syndrome (GBS), which is circulation in developed countries. However, a previous serotype epidemiological study of C. jejuni isolates in an animal population revealed that HS19 was also prevalent in isolates from cattle in China. In this study, to investigate the hazardous potential of zoonotic strains, 14 HS19 isolates from cattle were systematically characterized both by genotype and phenotype. The results showed that all of these cattle isolates belonged to the ST-22 complex, a high-risk lineage represented by 77.2% HS19 clinical isolates from patients worldwide in the PubMLST database, indicating that the ST-22 complex is the prominent clonal complex of HS19 isolates, as well as the possibility of clonal spread of HS19 isolates across different regions and hosts. Nevertheless, these cattle strains clustered closely with the HS19 isolates from patients, suggesting a remarkable phylogenetic relatedness and genomic similarity. Importantly, both tetracycline genes tet(O) and gyrA (T86I) reached a higher proportional representation among the cattle isolates than among the human clinical isolates. A worrying level of multidrug resistance (MDR) was observed in all the cattle isolates, and two MDR profiles of the cattle isolates also existed in human clinical isolates. Notably, although shared with the same serotype HS19 and sequence type ST-22, 35.7% of cattle isolates induced severe gastrointestinal pathology in the IL-10–/– C57BL/6 mice model, indicating that some bacteria could change due to host adaptation to induce a disease epidemic, thus the associated genetic elements deserve further investigation. In this study, HS19 isolates from cattle were first characterized by a systematic evaluation of bacterial genomics and in vitro virulence, which improved our understanding of the potential zoonotic hazard from food animal isolates with high-risk serotypes, and provided critical information for the development of targeted C. jejuni mitigation strategies.

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This study supplied malic acid-supplemented drinking water to flocks that were naturally Campylobacter-positive and assessed the effect of feeding malic acid to chickens on Campylobacter reduction and poultry production. In Experiment 1, chickens were provided with malic acid-supplemented drinking water for three weeks. The contamination loads of Campylobacter were decreased by 0.91–0.98 log after the first week of use (p < 0.05). However, this effect did not persist over time and significant decontamination could not be found in the second and third weeks of application. Thus, in Experiment 2 malic acid-supplemented drinking water was given to chickens for a period of five days at slaughter age. The Campylobacter carriage was found to be effectively decreased by 1.05–1.55 log (p < 0.05). Malic acid had no adverse effects on chicken body weight, weight gain, intestinal indices, or the microbiota. In addition, it could change the composition of chicken meat since the moisture content was increased by 5.12–5.92% (p < 0.05) and the fat content was decreased by 1.60% (p < 0.05). Our study provides an effective means for reducing the contamination of Campylobacter during the chicken rearing period and this method can be applied to promote the safe development of poultry farming and its products.