Insights into the impact of flhF inactivation on Campylobacter jejuni colonization of chick and mice gut
BMC Microbiology volume 18, Article number: 149 (2018)
Campylobacter 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.
The 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.
Inactivation 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.
Campylobacter jejuni is the most common cause of foodborne gastroenteritis in humans worldwide, and infections caused by this microbe often leads to diarrhea and sometimes to a severe inflammatory response with clinical symptoms of fever, abdominal cramping, bloody stools and other symptoms [1, 2]. C. jejuni is prevalent in the environment and can infect humans in a variety of ways [3, 4]. Although the incidence of C. jejuni infection is very high at present, causing it to receive increased attention , a complete understanding of the mechanisms of C. jejuni-associated disease is still an ongoing effort . An analysis of the genome sequence of C. jejuni revealed that the cytolethal distending toxin (CDT) is the only toxin produced by this bacterium. Many other classical virulence factors that are common to other pathogens are missing in the C. jejuni genome . It has now been shown that colonization in the host intestinal tract and persistence to a sufficient load is crucial for C. jejuni pathogenesis [3, 7, 8].
Intestinal colonization by C. jejuni is influenced by multiple factors, including the capsule, glycosylation system, catabolism of L-serine, cytochrome c peroxidase, transport systems, putative adhesins, chemotaxis and flagellar motility [9,10,11,12,13,14,15,16]. The flagellum is particularly important among these factors . C. jejuni produces a single flagellum at one or both poles, which not only provides chemotactic motility for migration to replicative niches but also helps the bacteria penetrate the mucus that covers the epithelial cells. In addition, the flagellum is involved in interactions with and invasion of the host epithelium, the secretion of invasion antigens, and the evasion of the innate immune system, is the essential virulence and colonization factor of C. jejuni [6, 15, 18,19,20]. The flagellum is a multicomponent organelle composed of a basal body, motor, rod, hook and flagellin filament. It appears that the C. jejuni flagellar substructures, flagellin glycosylation, motility and secreted invasion antigens are all required for host colonization but are not the only determinants [9, 16, 19, 21,22,23]. There are over 50 flagellum-related genes in the C. jejuni genome, and their corresponding contributions to colonization are also diverse [15, 16, 18, 22,23,24,25]. Considering the multifunctional role played by flagella during C. jejuni pathogenesis , a comprehensive understanding of their virulence potential is urgently needed.
The importance of FlhF for flagellar biosynthesis has been observed for several pathogens (primarily for the polarly flagellated bacteria) in recent years [27,28,29,30,31,32,33]. In C. jejuni, the inactivation of flhF has been observed to inhibit the synthesis of flagella and motility in previous studies [34, 35], but its contribution to C. jejuni colonization has not yet been characterized. In this study, the influence of flhF on C. jejuni colonization was systematically evaluated both in the reference strain and in an isolate. The results showed the significant effect this gene has in the host colonization process. The possible factors that may contribute to the colonization defect were assayed, including the flagella-associated abilities such as adherence and invasion to the host cells, autoagglutination (AAG) and biofilm formation. The mechanism of by which FlhF influences host colonization was also explored at the gene expression level. We observed that flhF is continuously upregulated during the cell infection process, and in addition to its major role in the flagellar system, some other infection-related genes were observed to be downregulated in the flhF mutant strain. The results of this study provide a reference for the further analysis of this virulence factor and investigations of the mechanism of flagella mediated co-regulation of pathogenesis in this bacterium.
Bacteria and cell culture
C. jejuni strains were grown on Campylobacter blood-free selective agar containing charcoal cefoperazone deoxycholate (CCDA) (Oxoid, Basingstoke, UK) or in Mueller-Hinton (MH) broth (BD Biosciences, Sparks, MD, USA) in a microaerophilic environment (5% O2, 10% CO2, and 85% N2) and incubated at 42 °C. The C. jejuni isolate used in this study was isolated from the anal swabs of a monkey with diarrhea and was further identified using multiplex PCR (polymerase chain reaction) (Additional file 1: Figure S1) . Appropriate antibiotics (50 μg/ml kanamycin, 20 μg/ml chloramphenicol, 10 μg/ml rifampicin, 6 μg/ml polymyxin B, 10 μg/ml trimethoprim, 100 μg/ml cycloheximide, 40 μg/ml vancomycin, 20 μg/ml amphotericin B or 60 μg/ml cefoperazone) were added to the medium as required. Inactivation of flhF and complementation of the mutant in C. jejuni NCTC 11168 and in the isolate were performed as previously described . Human colon cancer cell line (Caco-2) was acquired from American Type Cell Culture (ATCC) and routinely cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained in a humidified 37 °C atmosphere containing 5% CO2.
Chick and mice colonization assays were largely performed as described in previous studies [16, 24, 37]. Briefly, C. jejuni strains were harvested in phosphate buffered saline (PBS), and 1 × 108 colony-forming units (cfu) of bacteria were subsequently orally inoculated into 1-day-old Campylobacter-free Leghorn chicks obtained from the Jiangsu Institute of Poultry Science. Chicks were randomly allocated into three groups and sacrificed by CO2 asphyxiation at 1 and 7 days post inoculation. For each chick, the intestinal tract was collected, separated following gentle extrusion of the lumenal contents, weighed, homogenized, serially diluted and plated on selective CCDA agar (containing rifampicin, polymyxin B, trimethoprim, cycloheximide, vancomycin, amphotericin B and cefoperazone) to count the bacteria load. Six-week-old C57/BL6 mice were acquired from the Comparative Medicine Center of Yangzhou University. The mice initially received 200 μl of a sodium bicarbonate solution (50 mg/ml), after which 1 × 108 cfu of bacteria were used to infect mice by orogastric inoculation 15–30 min later. The mice were sacrificed by cervical dislocation at the designated time point, and the colonization levels were evaluated using selective media containing amphotericin B and cycloheximide as described above.
Growth and biochemical tests
Overnight cultures of C. jejuni were harvested and diluted in MH broth to a starting optical density at 600 nm (OD600) of 0.08. Two-milliliter aliquots of the suspension were transferred into the wells of a 12-well plate and incubated at 42 °C and 120 rpm under a microaerophilic atmosphere. Samples were collected at indicated intervals and the OD600 values were determined to compare the growth of the different strains. Biochemical testing was performed as described by Valenza et al. . Bacteria were harvested in a 0.45% NaCl solution and the turbidities were adjusted to an equivalent of a McFarland standard of 3. The suspensions were inoculated into a Neisseria-Haemophilus (NH) identification card, which was automatically analyzed by a VITEK 2 microbiology system (bioMérieux, France).
Cell adhesion and invasion assay
Adhesion and invasion assays were performed as described previously . Caco-2 cells were first seeded into a 24-well plate at a density of 1 × 105. After the cells incubated for 18 h, C. jejuni that had grown for 20 h were harvested and resuspended in DMEM, and 1 ml suspensions containing 1 × 107 cfu were then inoculated into each well. The plates were centrifuged at 600×g for 5 min to enhance the contact between bacteria and eukaryotic cells. For bacterial adhesion assays, cells were incubated for 2 h, washed three times with PBS to remove the unattached bacteria, and then were lysed in 1 ml of PBS containing 0.1% TritonX-100, with serial dilutions of the suspensions subsequently spread onto CCDA plates to quantify the number of bacteria. To assess the number of invaded bacteria, the inoculated cells were first incubated for 2 h, washed once with DMEM and then were incubated in DMEM containing gentamicin (100 μg/ml) for another 1 h. The extracellular bacteria were killed during this period, and the number of invaded bacteria was determined as described above.
The autoagglutination assay was performed as previously described . C. jejuni strains were grown for 20 h prior to use, after which each strain was suspended in PBS and adjusted to an OD600 of 1.0. Five-milliliter aliquots of the suspension were transferred into a series of sterile glass tubes (13 × 100 mm), which were incubated under microaerophilic conditions for a predetermined time. After incubation, 200 μl of the bacterial suspension was carefully removed from the top of each tube, and the OD600 was measured to evaluate the degree of AAG. Bacterial cells that strongly agglutinate will fall to the bottom of the tube, leading to a decreased OD600 value.
The ability of C. jejuni strains to form biofilms was measured by crystal violet staining, using a method described by Naito et al. with some modifications . Briefly, bacteria cultured for 20 h were harvested and diluted in MH broth to an OD600 of 0.1, after which 200 μl of each suspension was inoculated into 96-well polypropylene plates and incubated at 42 °C under microaerobic conditions. After incubating for 48, 60, 72, 84 and 90 h, the plates were washed with PBS and dried at 60 °C for 30 min. Next, 200 μl of a 1% crystal violet solution was added to each well to stain the biofilms that had formed. After staining at room temperature for 30 min, the plates were rinsed thoroughly with PBS three times to remove the unbound crystal violet. The plates were subsequently dried at 60 °C for 15 min, and the biofilms were quantified by determining the OD570 values after the remaining crystal violet was solubilized in a solution composed of 30% methanol and 10% acetic acid.
RNA isolation and quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was performed as described previously . C. jejuni was cultured for designated time, after which the RNA was extracted using an RNeasy plus mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The isolated RNA was then used for cDNA synthesis using an RT reagent Kit (TaKaRa, Dalian, China). qRT-PCR was performed with a ABI PRISM 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the FastStart Universal SYBR Green Master (ROX) (Roche Diagnostics, GmbH, Germany). The primers used for qRT-PCR are listed in the Additional file 1: Table S1. The cj0402 gene (glyA) was used as an endogenous control to evaluate the relative expression of the target genes.
Gene collection and microarray analysis
The genes required for C. jejuni adhesion/invasion and colonization that were not directly involved in flagellar assembly were identified and are listed in Additional file 1: Table S2, and this information was primarily obtained from previously published reports. The relative mRNA expression of the selected genes in the C. jejuni flhF mutant strain versus the wild-type strain were acquired and analyzed using our previous whole transcriptome microarray data , which has been submitted to the NCBI Gene Expression Omnibus (GEO) database with the accession number GSE 87852. The fold change in gene expression was graphically presented in a heatmap that was generated using the Hemi software, with different colors representing the degree of variation in expression .
The data are presented as the means ± standard deviation (SD) from three independent experiments. Student’s t-test was used to assess the significance of the observed differences (P < 0.05 *, and P < 0.01 **).
Inactivation of flhF in C. jejuni dramatically influences its colonization in both chicks and mice
The importance of flhF on C. jejuni commensal colonization in chicks was systematically analyzed for both the reference strain NCTC 11168 and for a C. jejuni isolate. For NCTC 11168 (Fig. 1a), the C. jejuni bacterial load in the intestinal tract was relatively low 1 day after inoculation. However, we could not isolate the flhF mutant strain at this time point and a significant difference in colonization was observed compared to the wild-type strain in the large intestine (caeca and colon) (P < 0.05). On day 7, the flhF mutant strain still could not colonize the small intestines of chicks (duodenum, jejunum and ileum), and although it was present in the large intestines of some chicks, the bacterial load was significantly lower than that observed for NCTC 11168-infected group (P < 0.01).
A hyper-colonization phenotype was observed for the C. jejuni isolate, as the bacterial loads in the intestines of chicks infected with the same dose were notably higher than in those of NCTC 11168-infected chicks, thus the effect of flhF was further determined in this C. jejuni isolate. As shown in Fig. 1b, the flhF gene was observed to be important in the initial establishment of colonization, since at 1 day post infection, the number of recovered isogenetic flhF mutant was significantly lower than that observed for chicks infected with the wild-type strain for both the small intestine (P < 0.01) and the caeca (P < 0.05). On day 7, the flhF mutant strain was almost completely cleared from the chicks and could be found only in the caeca of one chick, while the wild-type C. jejuni isolate consistently colonized the large intestines of chicks with a high load (approximately 109 cfu/g in the caeca). Complementation of flhF recovered this deficiency somewhat, both in C. jejuni NCTC 11168 and in the isolate, indicating that flhF significantly influences the commensal colonization of chicks.
The mice were less susceptible to C. jejuni colonization than the chicks. Although the C. jejuni isolate was observed to stably colonize both animals, the reference strain NCTC 11168 failed to colonize the mice in our study. For the C. jejuni isolate (Fig. 2), the bacteria recovered from the intestinal tract of mice were notably less abundant than those recovered from the chicks. However, the results showed that flhF does not greatly influence the initial colonization of mice. Although the average amount of the isolated flhF mutant was less than that of the wild-type strain, no significant differences were observed after 1 day post inoculation. On day 7, the flhF mutant strain could not be recovered from any regions of the intestines, while the wild-type strain continuously colonized the mice, with significant differences observed in the jejunum, ileum, caecum and colon (P < 0.01). These results suggested that inactivation of flhF resulted in the eventual elimination of the bacteria in mice.
flhF does not influence the growth or primary biochemical characteristics of C. jejuni
The observed growth curves indicated that the inactivation of flhF does not influence the normal growth of C. jejuni. When cultured in MH broth, the OD600 values for C. jejuni NCTC 11168, the flhF mutant and the complemented strains were nearly same at different time points and no significant differences were observed, suggesting that these strains have a similar growth rate (Fig. 3). The biochemical characteristics of the C. jejuni strains were analyzed using an NH card with a VITEK 2 compact instrument. The NH card contains 30 biochemical tests that includes five alkalization tests, 10 acidification tests, 11 glycosidase and peptidase tests and four miscellaneous tests. For the C. jejuni flhF mutant and the complemented strain, all the indexes tested were the same as the NCTC 11168 strain except for OPS (phenylphosphonate) (Additional file 1: Table S3), indicating that the primary biochemical characteristics of C. jejuni were unaffected in the flhF mutant.
C. jejuni flhF mutant shows a decreased ability to infect cultured epithelial cells
To characterize the infectivity of C. jejuni after inactivation of flhF, the ability of the strains to invade cultured epithelial cells was assessed. As shown in Fig. 4, inactivation of flhF significantly influenced the ability of C. jejuni to infect intestinal epithelial cells. Despite the effort made to minimize motility-dependent effects via centrifuging cells, the relative percentage of the flhF mutant strain to adhere and invade Caco-2 cells was 52 and 27%, respectively, compared to the NCTC 11168 strain. Complementation with flhF restored this defect in the mutant, and the infection ability of the complemented strain was nearly same as that observed for the wild-type strain (129 and 97% for adherence and invasion, respectively). These results suggest that the adherence to the epithelium and the subsequent invasion during C. jejuni infection is hampered by the inactivation of flhF.
The autoagglutination and biofilm-formation abilities of C. jejuni are decreased by the inactivation of flhF
The AAG ability of C. jejuni strains was compared after they were statically incubated for a short (3, 6, and 9 h) and long (21, 24, and 27 h) time spans (Fig. 5a). The AAG phenotype of each tested strain became more prominent over time. During the first 6 h, no significant difference in the AAG ability was observed between the C. jejuni NCTC 11168 and flhF mutant strains. The influence of flhF on C. jejuni AAG became noticeable after the strains were incubated for 9 h. The measured OD600 of C. jejuni NCTC 11168 was significantly lower than that of the flhF mutant strain (P < 0.05), and this effect increased over time, as the observed difference was more significant at 21–27 h (P < 0.01).
The biofilms formed by the strains were quantified after being statically cultured for 48–96 h. As shown in Fig. 5b, the biofilms formed for each strain continuously increased over time, but the biofilm-forming potential was different between the strains. The flhF mutant strain showed a defect in biofilm formation, as the OD570 values of the tested strains across all assayed time points were significantly decreased compared to those observed for the NCTC 11168 strain (P < 0.01).
The biofilm-formation defect was partially restored after complementation of flhF, the AAG ability was also increased somewhat in the complemented strain. The detected values for the biofilms formed and AAG ability of the complemented strain were all higher than the mutant at all the assayed time points, although these values did not reach the level of the wild-type strain.
flhF is continuously upregulated during C. jejuni infection and influences the transcription of other infection-related genes
To analyze whether flhF is associated with C. jejuni infection in vivo, the NCTC 11168 strain was cultured in vitro and used to infect Caco-2 cell for 4, 8, 16 or 24 h, respectively. The RNA was extracted from samples at each time point, and a qRT-PCR assay was performed to compare the relative expression of flhF at the different time points. Compared to the in vitro groups, flhF was observed to be upregulated during the infection process at all the assayed time points. The observed fold-changes in expression were all over 3, and specifically for the 4, 8, 16 and 24 h time points were increased by 3.19-, 5.10-, 3.98- and 4.74-fold, respectively (Fig. 6a). This result suggests that flhF is a virulence-associated gene that may have a continuous role during C. jejuni infection.
The genes not directly involved in flagellar assembly but that were reported to influence C. jejuni adhesion, invasion and colonization were summarized (Additional file 1: Table S2) and their relative expression after inactivation of flhF was analyzed using our previous microarray data. As shown in Fig. 6b, both the colonization- (40%, 20/50) and adhesion/invasion- (50%, 25/50) related genes were downregulated after the inactivation of flhF. The average influence on adhesion/invasion was slightly higher than that observed for colonization. Some genes that could influence both the adhesion/invasion and colonization ability of C. jejuni were identified (the overlapping genes are presented in the middle of the heatmap), and their average transcription was observed to be decreased more than that of the other two groups. Some genes were selected for further qRT-PCR verification. In addition to cadF and docC, which were slightly upregulated in the qRT-PCR assay (Fig. 6c), all the other assayed genes showed the same trends as the microarray, where the transcription of fedA, fedB, fedC and fedD (Feds) were apparently affected. Thus, besides the major role flhF plays in the flagellar system, it may also affect C. jejuni infection in other ways, but the underlying mechanism still requires further investigation.
The efficient establishment of colonization is a prerequisite for C. jejuni pathogenesis. It was originally hypothesized that motility plays an essential role in C. jejuni colonization, as only motile strains could be recovered from human volunteers . Subsequent studies revealed that flagella are an important colonization factor, as intact and motile flagella are required for efficient colonization . The flagellar filament accounts for the largest proportion of the synthesized flagella and was the earliest studied flagellar substructure. The C. jejuni flagellar filament is composed of two almost identical flagellin proteins. Flagellin A is predominantly responsible for the intact and fully motile flagellation phenotype and has a specific contribution to C. jejuni colonization regardless of the effects observed by a defect in motility or inactivation of flaB . Although Wassenaar et al. suggested that flagellin A, rather than motility, is essential for optimal colonization . However, the role of motility cannot be ignored, as the mutation of pflA, which results in nonmotile bacteria with paralyzed flagella, also influences C. jejuni colonization .
Subsequent studies focused on the role of other flagellar elements. For the flagellar biosynthesis regulators RpoN (σ54) and FliA (σ28), mutation of either of these regulators resulted in an apparent attenuation for caecal colonization in chicks, but the loss of fliA had a more obvious effect than that of rpoN, despite its lesser influence on C. jejuni motility . FlgR is a σ54-dependent response regulator that was observed to decrease the C. jejuni colonization ability by 10- to 500-fold in chicks . Although inactivation of the flagellar motor protein MotAB caused C. jejuni to be nonmotile, these cells expressed a full-length flagellum yet completely lost the ability to colonize chicks [24, 46]. FlgK is a flagellar hook-associated protein, and the colonization of an flgK mutant was five orders of magnitude lower than that observed for wild-type strain in chicks . The flagella secreted Campylobacter invasion antigen (Cia) proteins also play a role in colonization. Mutations in CiaI and CiaB lead to a defect in chick colonization compared to the wild-type strain [15, 22, 25]. Furthermore, genes associated with the flagellin O-linked glycosylation system also affect C. jejuni colonization and may be mediated by structural and surface changes in the glycan when interacting with the host [16, 23].
Although evaluation of bacterial colonization ability between studies may be difficult due to the diverse methods used by various laboratories, when assayed using the same method the flagellar elements were shown to truly have a various contributions to C. jejuni colonization . Thus, the role of FlhF on C. jejuni colonization was systematically analyzed in this study. C. jejuni is more commonly considered a commensal intestinal microbiota in chickens, where it has a persistent presence in the gut but does not lead to pathology . The chick colonization assay was first performed using both the reference strain NCTC 11168 and an isolate from monkey diarrhea. Different C. jejuni strains shows diverse infectivity, and the colonization level of the NCTC 11168 strain was not ideal compared to that of the fecal isolate, which may be due to it being a poor colonizer . In our study, inactivation of flhF in NCTC 11168 and the isolate both resulted in significantly decreased C. jejuni colonization at 1 and 7 days post inoculation, indicating that the commensal colonization of C. jejuni was severely hampered by the loss of flhF.
In contrast to chicks, C. jejuni infection in humans often leads to a series of illnesses, but the underlying mechanism is not fully understood . To understand whether the importance of flhF on commensal colonization was consistent in mammals, mice were used for further assays. The reference strain NCTC 11168 failed to colonize the mice in our assay. Similar findings were also reported in a previous study, where NCTC 11168 was observed to colonize mice at very low efficiency, although after this strain was reisolated from mice it could then colonize mice with a high efficiency . Thus, this phenomenon may be caused by the subculture of this genome-sequenced strain in the laboratory or the need for genetic adaptations in the intestinal tracts to promote its growth [48, 49]. The C. jejuni isolate used in our study could stably colonize the assayed mice. Compared to the chicks, the initial establishment of colonization in mice was not severely influenced by the inactivation of flhF, which may be due to the diverse characteristics of C. jejuni with respect to infecting different hosts. At 7-days post inoculation, all of the isogeneic flhF mutants were cleared from the mice, indicating its important role in promoting mice colonization.
The possible factors that may contribute to the colonization defect were investigated. C. jejuni growth and metabolism were observed to not be influenced by the mutation of flhF, eliminating the possible effects of the flhF mutation on bacterial multiplication. Previous studies showed that the motility and flagellar biosynthesis of C. jejuni were severely affected by the inactivation of flhF [34, 35]. The flagella of C. jejuni are involved in not just motility , so some of the flagella-associated phenotypes which might play a role in the bacterial colonization were subsequently analyzed. Adherence to the epithelium helps bacteria resist intestinal peristalsis and expulsion , and the subsequent invasion is also important. These factors are key determinants for C. jejuni colonization . The potential infectivity of the assayed C. jejuni strains on Caco-2 cells was assessed. Despite efforts to minimize the influence caused by motility, both the adhered and invaded bacteria were observed to be significantly decreased after inactivation of flhF, indicating that the colonization was hampered at this step. Autoagglutination is the basis of bacterial aggregation, which is involved in biofilm formation . Biofilm formation is essential for both environmental survival and successful infections [52, 53], and this mode of bacterial growth is also required for C. jejuni colonization . The autoagglutination and biofilm-formation abilities of the C. jejuni flhF mutant strain were observed to be decreased in our study, contributing to the observed colonization defect of this strain. The complementation of flhF completely or partially restored the defects of adhesion, invasion and biofilm-formation abilities of the flhF mutant strain. However, limited restoration of the AAG ability was observed. Previous studies showed that the presence of the flagellin and its glycosylation modification is crucial for C. jejuni AAG . We has observed the intact flagellin in the flhF complemented strain in our previous study , so flagellin glycosylation may not be fully restored after complementation of flhF.
In addition to the above mentioned phenotypic changes, the mechanism by which flhF affects C. jejuni colonization was also explored at the gene expression level. Although flhF is a class I flagellar gene that is stably expressed both in vitro and in vivo , when the two sets of expression data were compared, we observed that flhF is continuously upregulated during the infection process. This observation suggests a close relationship between this gene and C. jejuni infectivity, maybe an intact flagellum is required to maintain the secretion of virulence proteins during the infection process , or FlhF may function continuously to regulate other virulence factors in an unclarified-method. Inactivation of flhF globally affects flagellar gene transcription . Unlike many other flagellated species whose flagellar genes are clustered into operons, the flagellar genes in C. jejuni are scattered throughout the genome [34, 54]. Many of these genes are grouped into operons that are involved in other pathways , and some genes are also co-expressed with flagellar genes . Thus, we were interested in determining whether flhF could affect other infective factors not directly involved in flagellar assembly in this bacterium. Some representative genes were identified based on the literature, and using our previously obtained microarray data . We observed that 45% (39/86) of the colonization and adhesion/invasion related genes were downregulated, among which the expression of flagellar coexpressed determinants (Feds) was apparently affected. This phenomenon may be caused by the Feds genes that belong to the flagellar σ28 regulon , which are significantly affected by the inactivation of flhF .
In this study, we systematically analyzed the influence of FlhF on C. jejuni colonization and the possible mechanisms that may contribute to this defect, including the associated virulence phenotypes and a preliminary exploration of changes at the gene expression level. The pathogenesis of C. jejuni is not yet well elucidated compared to other analogous species that can also cause diarrheal diseases. C. jejuni lacks many of the classic virulence factors [1, 3, 6]. It is generally believed that C. jejuni has a regulatory mechanism that co-regulates its virulence. For example, the polar flagella are not only required for motility but also functions as a type III secretion system (T3SS) [26, 54]. The flhF gene is continuously upregulated during the infection process and influences the expression of other infection-related factors, which indicates a close relationship between this gene and C. jejuni infectivity. It is unknown whether this is an isolated case or a common phenomenon for other flagellar genes or whether other aspects related to C. jejuni pathogenesis are affected. These questions still need to be further investigated, which will provide insights into the role of this gene as well as the mechanism of the co-regulation of virulence of C. jejuni flagella.
In this study, the influence of flhF on C. jejuni colonization was systematically evaluated. Inactivation of flhF resulted in severe defects in the commensal colonization of chicks and resulted in a complete loss in the ability of this bacterium to continuously colonize mice. A combination of phenotypic and genetic characterizations revealed that there is a decreased ability of flhF mutant strain to adhere and invade epithelial cells, and autoagglutination and biofilm formation, which may contribute to the colonization defect. The flhF gene was observed to be continuously upregulated during the infection process and affects the transcription of other infection-related genes that are not directly involved in flagellar assembly, such as the flagellar coexpressed determinants (Feds). This result makes sense, but their definite relationship with flhF and the underlying mechanisms that influence C. jejuni pathogenesis still requires further investigation.
American Type Cell Culture
Cytolethal distending toxin
Campylobacter invasion antigen
Dulbecco’s minimal essential medium
Fetal bovine serum
Gene Expression Omnibus
Phosphate buffered saline
quantitative real-time polymerase chain reaction
Type III secretion system
Du X, Wang N, Ren F, Tang H, Jiao X, Huang J. cj0371: A Novel Virulence-Associated Gene of Campylobacter jejuni. Front Microbiol. 2016;7:1094.
Zang XQ, Tang HY, Jiao XN, Huang JL. Can a visual loop-mediated isothermal amplification assay stand out in different detection methods when monitoring Campylobacter jejuni from diverse sources of samples? Food Control. 2017;75:220–7.
Dasti JI, Tareen AM, Lugert R, Zautner AE, Gross U. Campylobacter jejuni: a brief overview on pathogenicity-associated factors and disease-mediating mechanisms. Int J Med Microbiol. 2010;300(4):205–11.
Young KT, Davis LM, Dirita VJ. Campylobacter jejuni: molecular biology and pathogenesis. Nat Rev Microbiol. 2007;5(9):665–79.
McCarthy ND, Gillespie IA, Lawson AJ, Richardson J, Neal KR, Hawtin PR, Maiden MC, O’Brien SJ. Molecular epidemiology of human Campylobacter jejuni shows association between seasonal and international patterns of disease. Epidemiol Infect. 2012;140(12):2247–55.
Burnham PM, Hendrixson DR. Campylobacter jejuni: collective components promoting a successful enteric lifestyle. Nat Rev Microbiol. 2018;16(9):551–65.
Gao B, Vorwerk H, Huber C, Lara-Tejero M, Mohr J, Goodman AL, Eisenreich W, Galan JE, Hofreuter D. Metabolic and fitness determinants for in vitro growth and intestinal colonization of the bacterial pathogen Campylobacter jejuni. PLoS Biol. 2017;15(5):e2001390.
Silva J, Leite D, Fernandes M, Mena C, Gibbs PA, Teixeira P. Campylobacter spp. as a Foodborne Pathogen: A Review. Front Microbiol. 2011;2:200.
Jones MA, Marston KL, Woodall CA, Maskell DJ, Linton D, Karlyshev AV, Dorrell N, Wren BW, Barrow PA. Adaptation of Campylobacter jejuni NCTC11168 to high-level colonization of the avian gastrointestinal tract. Infect Immun. 2004;72(7):3769–76.
Velayudhan J, Jones MA, Barrow PA, Kelly DJ. L-serine catabolism via an oxygen-labile L-serine dehydratase is essential for colonization of the avian gut by Campylobacter jejuni. Infect Immun. 2004;72(1):260–8.
Bingham-Ramos LK, Hendrixson DR. Characterization of two putative cytochrome c peroxidases of Campylobacter jejuni involved in promoting commensal colonization of poultry. Infect Immun. 2008;76(3):1105–14.
Davis LM, Kakuda T, DiRita VJ. A Campylobacter jejuni znuA orthologue is essential for growth in low-zinc environments and chick colonization. J Bacteriol. 2009;191(5):1631–40.
Flanagan RC, Neal-McKinney JM, Dhillon AS, Miller WG, Konkel ME. Examination of Campylobacter jejuni putative adhesins leads to the identification of a new protein, designated FlpA, required for chicken colonization. Infect Immun. 2009;77(6):2399–407.
Ribardo DA, Hendrixson DR. Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport. J Bacteriol. 2011;193(22):6233–43.
Barrero-Tobon AM, Hendrixson DR. Identification and analysis of flagellar coexpressed determinants (Feds) of Campylobacter jejuni involved in colonization. Mol Microbiol. 2012;84(2):352–69.
Hendrixson DR, DiRita VJ. Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract. Mol Microbiol. 2004;52(2):471–84.
Jeon B, Muraoka WT, Zhang Q. Advances in Campylobacter biology and implications for biotechnological applications. Microb Biotechnol. 2010;3(3):242–58.
Fernando U, Biswas D, Allan B, Willson P, Potter AA. Influence of Campylobacter jejuni fliA, rpoN and flgK genes on colonization of the chicken gut. Int J Food Microbiol. 2007;118(2):194–200.
Baldvinsson SB, Sorensen MC, Vegge CS, Clokie MR, Brondsted L. Campylobacter jejuni motility is required for infection of the flagellotropic bacteriophage F341. Appl Environ Microbiol. 2014;80(22):7096–106.
Barrero-Tobon AM, Hendrixson DR. Flagellar biosynthesis exerts temporal regulation of secretion of specific Campylobacter jejuni colonization and virulence determinants. Mol Microbiol. 2014;93(5):957–74.
Sommerlad SM, Hendrixson DR. Analysis of the roles of FlgP and FlgQ in flagellar motility of Campylobacter jejuni. J Bacteriol. 2007;189(1):179–86.
Biswas D, Fernando UM, Reiman CD, Willson PJ, Townsend HG, Potter AA, Allan BJ. Correlation between in vitro secretion of virulence-associated proteins of Campylobacter jejuni and colonization of chickens. Curr Microbiol. 2007;54(3):207–12.
Howard SL, Jagannathan A, Soo EC, Hui JP, Aubry AJ, Ahmed I, Karlyshev A, Kelly JF, Jones MA, Stevens MP, et al. Campylobacter jejuni glycosylation island important in cell charge, legionaminic acid biosynthesis, and colonization of chickens. Infect Immun. 2009;77(6):2544–56.
Chang C, Miller JF. Campylobacter jejuni colonization of mice with limited enteric flora. Infect Immun. 2006;74(9):5261–71.
Ziprin RL, Young CR, Byrd JA, Stanker LH, Hume ME, Gray SA, Kim BJ, Konkel ME. Role of Campylobacter jejuni potential virulence genes in cecal colonization. Avian Dis. 2001;45(3):549–57.
Guerry P. Campylobacter flagella: not just for motility. Trends Microbiol. 2007;15(10):456–61.
Pandza S, Baetens M, Park CH, Au T, Keyhan M, Matin A. The G-protein FlhF has a role in polar flagellar placement and general stress response induction in Pseudomonas putida. Mol Microbiol. 2000;36(2):414–23.
Kusumoto A, Kamisaka K, Yakushi T, Terashima H, Shinohara A, Homma M. Regulation of polar flagellar number by the flhF and flhG genes in Vibrio alginolyticus. J Biochem. 2006;139(1):113–21.
Salvetti S, Ghelardi E, Celandroni F, Ceragiol M, Gianness F, Senes S. FlhF, a signal recognition particle-like GTPase, is involved in the regulation of flagellar arrangement, motility behaviour and protein secretion in Bacillus cereus. Microbiology. 2007;153:2541–52.
Kusumoto A, Shinohara A, Terashima H, Kojima S, Yakushi T, Homma M. Collaboration of FlhF and FlhG to regulate polar-flagella number and localization in Vibrio alginolyticus. Microbiology. 2008;154:1390–9.
Kusumoto A, Nishioka N, Kojima S, Homma M. Mutational analysis of the GTP-binding motif of FlhF which regulates the number and placement of the polar flagellum in Vibrio alginolyticus. J Biochem. 2009;146(5):643–50.
Schniederberend M, Abdurachim K, Murray TS, Kazmierczak BI. The GTPase activity of FlhF is dispensable for flagellar localization, but not motility, in Pseudomonas aeruginosa. J Bacteriol. 2013;195(5):1051–60.
Kazmierczak BI, Hendrixson DR. Spatial and numerical regulation of flagellar biosynthesis in polarly flagellated bacteria. Mol Microbiol. 2013;88(4):655–63.
Ren F, Lei T, Song Z, Yu T, Li Q, Huang J, Jiao XA. Could FlhF be a key element that controls Campylobacter jejuni flagella biosynthesis in the initial assembly stage? Microbiol Res. 2018;207:240–8.
Balaban M, Joslin SN, Hendrixson DR. FlhF and its GTPase activity are required for distinct processes in flagellar gene regulation and biosynthesis in Campylobacter jejuni. J Bacteriol. 2009;191(21):6602–11.
Huang JL, Xu HY, Bao GY, Zhou XH, Ji DJ, Zhang G, Liu PH, Jiang F, Pan ZM, Liu XF, et al. Epidemiological surveillance of Campylobacter jejuni in chicken, dairy cattle and diarrhoea patients. Epidemiol Infect. 2009;137(8):1111–20.
Mansfield LS, Bell JA, Wilson DL, Murphy AJ, Elsheikha HM, Rathinam VA, Fierro BR, Linz JE, Young VB. C57BL/6 and congenic interleukin-10-deficient mice can serve as models of Campylobacter jejuni colonization and enteritis. Infect Immun. 2007;75(3):1099–115.
Valenza G, Ruoff C, Vogel U, Frosch M, Abele-Horn M. Microbiological evaluation of the new VITEK 2 Neisseria-Haemophilus identification card. J Clin Microbiol. 2007;45(11):3493–7.
Christensen JE, Pacheco SA, Konkel ME. Identification of a Campylobacter jejuni-secreted protein required for maximal invasion of host cells. Mol Microbiol. 2009;73(4):650–62.
Naito M, Frirdich E, Fields JA, Pryjma M, Li J, Cameron A, Gilbert M, Thompson SA, Gaynor EC. Effects of sequential Campylobacter jejuni 81-176 lipooligosaccharide core truncations on biofilm formation, stress survival, and pathogenesis. J Bacteriol. 2010;192(8):2182–92.
Deng W, Wang Y, Liu Z, Cheng H, Xue Y. HemI: a toolkit for illustrating heatmaps. PLoS One. 2014;9(11):e111988.
Black RE, Levine MM, Clements ML, Hughes TP, Blaser MJ. Experimental Campylobacter jejuni infection in humans. J Infect Dis. 1988;157(3):472–9.
Nachamkin I, Yang XH, Stern NJ. Role of Campylobacter jejuni flagella as colonization factors for three-day-old chicks: analysis with flagellar mutants. Appl Environ Microbiol. 1993;59(5):1269–73.
Wassenaar TM, van der Zeijst BA, Ayling R, Newell DG. Colonization of chicks by motility mutants of Campylobacter jejuni demonstrates the importance of flagellin A expression. J Gen Microbiol. 1993;139(Pt 6):1171–5.
Hendrixson DR. A phase-variable mechanism controlling the Campylobacter jejuni FlgR response regulator influences commensalism. Mol Microbiol. 2006;61(6):1646–59.
Mertins S, Allan BJ, Townsend HG, Koster W, Potter AA. Role of motAB in adherence and internalization in polarized Caco-2 cells and in cecal colonization of Campylobacter jejuni. Avian Dis. 2013;57(1):116–22.
Hermans D, Pasmans F, Heyndrickx M, Van Immerseel F, Martel A, Van Deun K, Haesebrouck F. A tolerogenic mucosal immune response leads to persistent Campylobacter jejuni colonization in the chicken gut. Crit Rev Microbiol. 2012;38(1):17–29.
Wilson DL, Rathinam VAK, Qi WH, Wick LM, Landgraf J, Bell JA, Plovanich-Jones A, Parrish J, Finley RL, Mansfield LS, et al. Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL10-deficient mice. Microbiology. 2010;156:2046–57.
Gaynor EC, Cawthraw S, Manning G, MacKichan JK, Falkow S, Newell DG. The genome-sequenced variant of Campylobacter jejuni NCTC 11168 and the original clonal clinical isolate differ markedly in colonization, gene expression, and virulence-associated phenotypes. J Bacteriol. 2004;186(2):503–17.
Hermans D, Van Deun K, Martel A, Van Immerseel F, Messens W, Heyndrickx M, Haesebrouck F, Pasmans F. Colonization factors of Campylobacter jejuni in the chicken gut. Vet Res. 2011;42:82.
Guerry P, Ewing CP, Schirm M, Lorenzo M, Kelly J, Pattarini D, Majam G, Thibault P, Logan S. Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence. Mol Microbiol. 2006;60(2):299–311.
Kalmokoff M, Lanthier P, Tremblay TL, Foss M, Lau PC, Sanders G, Austin J, Kelly J, Szymanski CM. Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation. J Bacteriol. 2006;188(12):4312–20.
Pascoe B, Lappin-Scott H, Sheppard SK, Asakura H. Does Biofilm Formation Aid Colonization and Infection in Campylobacter? In: Sheppard SK, Meric GUK, editors. Campylobacter Ecology and Evolution. Norwich: Caister Academic Press; 2014. p. 177–88. https://researchportal.bath.ac.uk/en/publications/doesbiofilm-formation-aid-colonization-and-infection-in-campylob.
Hendrixson DR. Regulation of flagellar gene expression. In: Nachamkin I, Szymanski CM, Blaser MJ, editors. Campylobacter. 3rd ed. Washington, D.C.: ASM Press; 2008. p. 545–59.
We thank Qijing Zhang at Iowa State University for providing the Campylobacter shuttle vectors. We are grateful to all members of the Jiangsu Key Laboratory of Zoonosis for kind comments and suggestions.
This work was supported by the National Natural Science Foundation of China (31872493, 31320103907), the National Key Research and Development Program of China (2018YFD0500500, 2014BAD13B02), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Six Talent Peaks Project in Jiangsu Province (2015-SWYY-02), BE2017341, CX(16)1028. We declare that none of these funding sources were involved in the design of this study, nor in the collection, analysis, and interpretation of data as well as in writing of the manuscript.
Availability of data and materials
The whole transcriptome microarray data were deposited in the NCBI Gene Expression Omnibus (GEO) database with the accession number GSE 87852. The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
All experimental and animal management procedures were approved by the Animal Welfare and Ethics Committees of Yangzhou University and complied with the guidelines of the institutional administrative committee and ethics committee of laboratory animals (SYXK [Su] 2016–0020).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Figure S1. Identification of the C. jejuni isolate. Table S1. Primers used in this study. Table S2. The relative mRNA expression of other infection-related genes in the microarray assay. Table S3. The biochemical characteristics of C. jejuni strains. (DOCX 243 kb)