Use of In Vivo-Induced Antigen Technology to Identify In Vivo-Expressed Genes of Campylobacter jejuni During Human Infection

Abstract

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.

Introduction

Campylobacter jejuni has emerged as the leading cause of human bacterial foodborne diarrheal disease worldwide [17]. It can colonize the intestines of many hosts, including chickens, cattle, sheep, dogs, and wild birds. The pathogenic bacteria reside in the lower intestinal tract in humans and give rise to self-limiting diarrhea [25]. Owing to the wide distribution of C. jejuni in poultry and the broad consumption of poultry meat products [7], there is increasing pressure to control C. jejuni infections, although most cases of infection are sporadic [3]. An effective vaccination for C. jejuni, which may help to control infections, is lacking [11]. To identify targets for a vaccine to prevent C. jejuni infection, a better understanding of the biological characteristics of this pathogen, particularly its virulence mechanisms, is needed.

The completion of the sequencing of the C. jejuni NCTC 11168 genome in 2000 opened the door for studying its pathogenicity mechanisms. The most prominent feature of the sequence is its heteromorphism, which contributes to its strain-to-strain variability [17]. Moreover, the interactions between the host and pathogenic bacteria likely play a significant role in Campylobacter infection [27]. To elucidate the role of C. jejuni virulence-associated genes in infection, animal models, including chickens, rodents, and monkeys, have been developed and applied to investigate colonization or transmission. Unfortunately, these models do not entirely show the pathogenicity of C. jejuni in humans [15].

IVIAT (in vivo-induced antigen technology) is a screening technique that identifies virulence-associated genes during host infection by different types of pathogens [20]. Using IVIAT, we can identify pathogenicity-related genes that are activated and expressed in vivo when a strong interaction occurs between the host and microbe. The advantage of IVIAT is that it contributes to the identification of virulenceassociated genes expressed specifically during infection instead of during growth in laboratory conditions. Virulenceassociated genes identified using IVIAT could play an important role in virulence or pathogenesis in the particular host-pathogen system, and proteins encoded may be novel diagnostic targets and subunit vaccine candidates. To date, IVIAT has been used for a number of pathogens, including Escherichia coli [22], Vibrio [28], Streptococcus [5], Salmonella [9], and Brucella [13]. Virulence genes from these microbes are expressed in vivo. Some of the identified proteins are candidate antigens for vaccines, whereas others may be diagnostic markers [5, 20].

This identification of in vivo-expressed genes will help to elucidate the pathogenic mechanisms of human infection by C. jejuni and to develop novel prevention strategies. In this study, a genomic expression library was constructed using random DNA fragments from the sequenced human C. jejuni strain NCTC 11168. In vivo-induced antigens were identified from human convalescent sera after being thoroughly absorbed with C. jejuni NCTC 11168 grown in vitro (scheme showed in Fig. 1).

Fig. 1.The scheme of IVIAT in this study.

 

Materials and Methods

Bacteria, Recombinant Proteins, and Culture Conditions

C. jejuni NCTC 11168 was kindly provided by Dr. Wangbang Sun from Zunyi Medical College in China. The proteins FlaA370 (N-terminal 370 aa of FlaA) and CjaA have been previously expressed in our laboratory. The strain used for genomic DNA preparation and serum adsorption in vitro was cultured on Campy blood-free selective medium (CCDA) (Oxoid Ltd., Basingtoke, Hampshire, UK) plates at 37℃ under anaerobic conditions for 30 h. For determining the protein expression profiles of these in vivo-expressed genes, the C. jejuni NCTC 11168 strain was cultured on CCDA medium plus 0.1% sodium deoxycholate (DOC; Sigma, USA) [14] (referred to as DOC plates).

Preparation of Antisera

In this study, human sera were collected and used for screening the genomic expression library. Equal volumes of human sera were obtained and pooled from seven clinical patients (Subei Hospital of Yangzhou, China) who had been infected by C. jejuni diagnosed through the culture-confirmed method. The antibody titer was determined with an indirect ELISA kit (SERION ELISA classic CAMPYLOBACTER JEJUNI IgG; Virion\Serion, Wurzburg, Germany), and seven selected sera were positive for the C. jejuni antibody. These patients were more than 50 years old; they suffered from repeated episodes of diarrhea, but they had not received any medical treatment before the collection of the sera. Human sera experiments were conducted in accordance with national guidelines, approved by the Subei Hospital of Yangzhou, and consented to by each patient.

Pooled human sera were adsorbed using C. jejuni NCTC 11168 whole cells cultured on CCDA plates on a rocking platform (Incubator Shaker; Crystal, China) at 4℃ for 1 h. The sera were adsorbed with C. jejuni whole cells six times and then with E. coli BL21 (DE3) whole cells six times. Adsorption was performed on a PVDF membrane coated with the ultrasonically disrupted lysates of C. jejuni or E. coli BL21 (DE3) cells, inactivated ultrasonically disrupted lysates of C. jejuni NCTC 11168 or E. coli BL21 (DE3) cells, secreted proteins from NCTC 11168 (prepared referencing to [21]), and lysis-induced BL21 (DE3) cells (Fig. 2). Indirect ELISAs were conducted to assess the adsorption of the sera by coating the plates with C. jejuni NCTC 11168 and E. coli BL21 (DE3) whole cells, lysates, CjaA, FlaA370 recombinant protein, and extracted flagellum protein. To evaluate the reactivity of the antibodies to the C. jejuni whole-cell lysates, successively diluted nickel affinity-purified lysate proteins were spotted onto a nitrocellulose (NC) membrane. A Dot-ELISA was conducted according to the standard protocol [16].

Fig. 2.The adsorption steps of human sera using several kinds of in vitro antigens.

In Vivo-Induced Antigen Screening and Functional Prediction

We constructed an inducible expression library of genomic proteins from sequenced C. jejuni NCTC 11168 according to standard protocol [20, 22]. The IVIAT process was performed based on previously described methods [6, 20] with a few modifications. Plasmids from the library were transformed into E. coli BL21 (DE3) cells containing 50 μg/ml of kanamycin to obtain 300 to 400 colonies per plate after culturing the bacteria at 37℃ overnight. These colonies were then replicated onto duplicate LB agar plates (containing 50 μg/ml of kanamycin and 1 mM of IPTG) using NC membranes and incubated for 5 h at 30℃ to induce expression of the inserted genes. The NC membranes were removed, and the colonies were lysed in chloroform vapors for 15 min in a hermetic container. The membranes were then saturated with 1% BSA in PBS plus 0.1% Tween-20 (PBST) and allowed to incubate for 1 h with mild agitation at room temperature. Next, the membranes were incubated with the adsorbed sera (1:1,000 in PBST). Colony immunoblotting was performed using the pooled adsorbed sera as the primary antibodies. Reactive colonies were detected using goat anti-human-horseradish peroxidase (HRP) secondary antibodies (Sigma, USA) and were visualized using a chemiluminescent ECL substrate (Thermo, USA). Secondary and tertiary screens were performed by means as described for the primary screen. Clones that maintained reactivity through the three rounds of screening were considered positive identifications and were subjected to DNA sequencing using T7 primers that flanked the multiple cloning sites within the vectors. The sequences of the in vivo-induced (IVI) genes were analyzed by BLASTx alignment and a Conserved Domain Database (CDD) domain search (http://www.ncbi.nlm.nih.gov/BLAST/).

Real-Time PCR Analysis

The in vivo gene expression profiles were evaluated by comparing the level of RNA transcription on DOC and CCDA plates with the expression of virulence genes induced with sodium deoxycholate [10, 14]. The total RNA was extracted using TRIzol (TaKaRa, Dalian, China) according to the manufacturer’s instructions after a 15 h culture of strain NCTC 11168 on CCDA or DOC plates. RNA was eluted with diethyl pyrocarbonate-treated water and treated with DNase I (TaKaRa). Next, the cDNA was synthesized from 500 ng of RNA using an RT-PCR kit (TaKaRa) according to the manufacturer’s instructions. The real-time RTPCR amplification of 2.0 μl of cDNA was performed using a reaction mixture containing SYBR Premix Ex Taq II (TaKaRa), 10 μM of the forward primer, 10 μM of the reverse primer, and diethyl pyrocarbonate-treated water. The real-time RT-PCR analysis was performed using a Gene Amp 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) with the following PCR parameters: 2 min at 50℃, followed by 40 cycles of denaturation at 95℃ for 30 sec and annealing at 60℃ for 34 sec. Ten IVI genes (primers are shown in Table 1) were selected, and their transcription levels in the NCTC 11168 strain cultured on DOC plates were compared with those on CCDA plates. Gene Cj0402 was used to normalize these samples because this housekeeping gene is expressed both on DOC and CCDA plates [14]. Duplicate reactions were performed, and three biological replicates were used for each sample. The threshold cycle values were determined using 7500 software, ver. 2.0.1 (Applied Biosystems).

Table 1.Primers used for real-time RT-PCR.

 

Results

Serum Selection and Adsorption

Seven human positive sera from patients were determined using a SERION ELISA IgG kit (ELISA data not shown). Pooled human sera were detected after each adsorption step using ELISA plates coated with C. jejuni NCTC 11168 and E. coli BL21 (DE3) whole cells, lysates, CjaA, FlaA370 recombinant protein, and extracted flagellum protein (Fig. 3A). These data showed that the immunoreactivity of human sera with in vitro-cultured C. jejuni antigen gradually decreased with the series of adsorptions. A Dot-ELISA showed that the levels of antibodies against in vitro antigens in human sera were drastically reduced (Fig. 3B).

Fig. 3.Evaluation of adsorption for pooled sera using ELISA. (A) The results of ELISA for human sera with seven coated antigens after each step of adsorption. The whole cells of C. jejuni NCTC 11168, whole cells of E. coli BL(DE3), lysates from C. jejuni, lysates from BL21(DE3), recombinant CjaA, FlaA370 protein, and extracted flagellum protein were used as coating antigens for assaying the adsorption of sera. (B) Dot-ELISA results of reactivities of pooled unadsorbed (I) and adsorbed (II) human sera against C. jejuni whole-cell lysates. The proteins of C. jejuni whole-cell lysates were quantified using a UV spectrophotometer and diluted from 5 μg to 1 μg for each colony.

Identification of C. jejuni Antigens by IVIAT

In the primary screening, approximately 25,000 clones from the C. jejuni NCTC 11168 genomic expression library were probed using extensively absorbed human sera. In total, 157 immunoreactive clones were identified by PCR using pET30-specific primers, and the encoded proteins of the selected clones were analyzed in the SDS-PAGE assay after the primary screening. Of the initial clones, 31 were positive upon secondary screening. Finally, 24 unique protein-expressing ORFs were ascertained by nucleotide sequencing and homology analysis.

Nucleotide sequencing was performed in both directions, and homology and functional analyses were performed on the inserted sequence based on the published nucleotide sequence and the known functions of the proteins from the C. jejuni NCTC 11168 strain [17]. The majority of these 24 proteins have a defined or suspected role in the pathogenesis of C. jejuni infection in vivo. These proteins are implicated in metabolism, molecular biosynthesis, genetic information processing, transport, and other processes (Table 2).

Table 2.Twenty-four genes of C. jejuni identified by IVIAT from human hosts.

Analysis of In Vivo Gene Expression Profiles

To evaluate the in vivo expression of the C. jejuni genes identified using the IVIAT method, we selected six genes according to their functional category to compare their expression levels in vivo and in vitro using real-time RTPCR. An in vitro DOC plate culturing method was used for analyzing the RNA transcription, to mimic in vivo conditions. The real-time PCR results showed that these genes were upregulated under in vivo-like conditions (>1-fold) (Fig. 4).

Fig. 4.In vivo gene expression relative to the level of expression in vitro by real-time PCR. C. jejuni NCTC 11168 was cultured in plates of CCDA (control in vitro) and DOC (in vivo-like condition) for 15 h, and the transcription levels of mRNA were assessed by real-time PCR. The levels of transcription above 1-fold were considered upregulated in contrast with in vitro.

 

Discussion

IVIAT is a rigorous method that can identify virulenceassociated genes, which are expressed instantaneously in vivo when hosts are infected by pathogenic bacteria [20]. We used IVIAT to identify in vivo-induced genes of C. jejuni to better understand the pathogenicity mechanism of Campylobacter in humans. C. jejuni NCTC 11168 was used to construct a genomic expression library because this isolate was originally derived from infected humans. Moreover, the sequence of NCTC 11168 has been annotated. This information supports our IVIAT screens and analysis of in vivo-induced genes. We identified two genes, including Cj0100 and Cj0788, through IVIAT, that were found to have products that were upregulated in the C. jejuni F38011 strain in the presence of 0.1% sodium DOC. These culture conditions have been reported to mimic the in vivo environment [14].

Twelve in vivo-induced genes that were classified with a metabolic function were identified in this study and may have important roles in bacterial growth or pathogenicity in vivo. Several of the genes, including leuC, ptmB, eno, and fcl, participate in special carbohydrate metabolism, such as C5-branched dibasic acid metabolism, amino sugar and nucleotide sugar metabolism, and glycolysis and fructose mannose metabolism. Previous studies [8] have shown that some strains of C. jejuni can utilize glutamine or glutathione to enhance their ability to colonize the intestines of their hosts. Because serine catabolism is required for colonization of the intestinal tract [8], these results indicate that the serC and Cj1365c genes identified by IVIAT may contribute to the colonization of C. jejuni. Cj1365c, a putative secreted serine protease, could be a significant marker for strains following environmental transmission because of its primary distribution in clinical and livestock isolates [26]. The protein FabH could catalyze the condensation of acetyl-CoA with malonyl-ACP to initiate cycles of fatty acid elongation [24]. The gene fabH (Cj0328c) was upregulated (3.09-fold by microarray) during chicken colonization, indicating that the protein encoded by fabH may be an important virulence-associated gene in vivo [24].

Five genes, including glf, tufB, moeA, flhF, and Cj1200, were classified as having a molecular biosynthesis function. Gene tufB, which encodes outer membrane translation elongation factor TU (Ef-Tu), was more abundant in the Cj0596 mutant (upregulated 2.4-fold). These results indicate that the fraction of outer membrane localization of Ef-Tu was increased. The altered outer membrane protein suggests that Ef-Tu is possibly involved in the pathogenesis of C. jejuni [12, 18]. The gene flhF encodes the flagellar biosynthesis regulator FlhF, which is a putative GTPase that is necessary for the development of the flagellar organelles in polarly flagellated bacteria [1]. FlhF is essential for σ54-dependent flagellar gene expression and flagellar biosynthesis but participates in an independent pathway that converges with or works downstream of the flagellar export apparatus-FlgSR pathway [1, 2]. The characteristics of the other genes remain unknown.

Three in vivo-induced genes (aspS, recG, and Cj1710c) were assigned to a genetic information processing function, but their specific functions have not been determined. All bacteria have DNA repair mechanisms that act to reduce DNA damage and maintain genetic structures and stability [4]. For C. jejuni, genomic polymorphisms are important for its adaptation to diverse environments. However, DNA repair is essential for its survival in response to significant environmental stresses. We hypothesize that these nine DNA repair genes identified as in vivo-associated genes may be important when C. jejuni lives in human or chicken tissues.

We identified two virulence-associated genes, ctsE and Cj1587c, that were assigned to a transportation function. The gene ctsE, which encodes a putative type II secretion system E protein and is essential for DNA uptake and natural transformation, was classified as having a Campylobacter transformation function [19]. This protein is an important virulence-associated factor because its encoding gene is similar to several genes responsible for the transport of pilus subunits, toxins, and other exoenzymes [23].

In this study, real-time PCR was performed to assess the expression profiles of IVI-virulence genes by comparing the transcription levels of genes in DOC and CCDA cultures. All six genes selected were upregulated under the DOC culture conditions, which were considered to be in vivo-like conditions. Out of these six genes selected, Cj0100 and Cj0788 were previously found to be upregulated in the presence of 0.1% DOC by a microarray analysis [14], which is consistent with the results of this study. IVIAT identified 24 C. jejuni NCTC 11168 proteins expressed in vivo during the human infection with C. jejuni. This study is the first profile of virulence-associated genes of interaction between human hosts and C. jejuni. Several of the genes identified by IVIAT were closely correlated with the virulence and pathogenesis of C. jejuni, and the coded proteins may be candidates for vaccines.