Campylobacter jejuni is one of the leading infectious causes of food-borne illness around the world. Its ability to persistently colonize the intestinal tract of a broad range of hosts, including food-producing animals, is central to its epidemiology since most infections are due to the consumption of contaminated food products. Using a highly saturated transposon insertion library combined with next-generation sequencing and a mouse model of infection, we have carried out a comprehensive genome-wide analysis of the fitness determinants for growth in vitro and in vivo of a highly pathogenic strain of C. jejuni. A comparison of the C. jejuni requirements to colonize the mouse intestine with those necessary to grow in different culture media in vitro, combined with isotopologue profiling and metabolic flow analysis, allowed us to identify its metabolic requirements to establish infection, including the ability to acquire certain nutrients, metabolize specific substrates, or maintain intracellular ion homeostasis. This comprehensive analysis has identified metabolic pathways that could provide the basis for the development of novel strategies to prevent C. jejuni colonization of food-producing animals or to treat human infections.
Campylobacter jejuni subsp. jejuni (C. jejuni) is one of the most common causes of infectious food-borne illness in industrialized countries [1,2]. The high incidence of this pathogen is due to its ability to persistently colonize the intestinal tract of food-producing animals. Contaminated food products—in particular, poultry meat—become a source of C. jejuni infection when improperly handled or undercooked [3]. While asymptomatic in most vertebrates, in humans, C. jejuni infection often leads to acute, although self-limiting, gastroenteritis [4]. Rarely, infections with C. jejuni lead to a sequelae known as Guillain-Barre syndrome, which is characterized as a serious neurodegenerative disorder [5]. A characteristic feature of C. jejuni that distinguishes it from other common enteropathogenic bacteria is the paucity of homologs of virulence factors that in other pathogens are engaged in specific interaction with the host [6,7]. In fact, C. jejuni has arguably more in common with commensal intestinal microbiota than with enteric pathogens. This is consistent with the observation that, other than in humans, the persistent presence of C. jejuni in the gut does not lead to pathology [8]. Why and how C. jejuni infection in humans leads to disease is very poorly understood, but it is expected that its ability to colonize and replicate within the intestinal tract to reach significant numbers must be central to its pathogenesis. Several studies have identified genes that are important for C. jejuni intestinal colonization using different animal models of infection. Other than genes required for motility or the modification of surface structures (e. g. , protein glycosylation), the vast majority of genes identified to date as required for colonization are involved in the acquisition and metabolism of essential nutrients [7,9–12]. Therefore, deciphering the metabolic requirements of C. jejuni is central to the understanding of its ability to colonize a host and potentially cause disease. In fact, the understanding of the metabolism of bacterial pathogens during infection is quickly emerging as an extremely important area of research. While the basic metabolism of model bacteria during their growth in vitro has been extensively studied, knowledge of the metabolic requirements of bacterial pathogens during infection has lagged behind [13]. Indeed, it is becoming increasingly clear that the functional purpose of some virulence factors that specifically target host processes is to increase the availability of crucial nutrients or to facilitate their acquisition for bacterial growth. Therefore, the concept of “nutritional virulence factors” has been proposed to describe such pathogenic determinants [14–17]. Although most of the C. jejuni colonization determinants known to date have been discovered by candidate-mutant experimental approaches, there have been some attempts to comprehensively identify such determinants using genome-wide approaches [18–20]. However, because of technical limitations in the approaches or animal models used, those studies have not been comprehensive. The availability of high-throughput nucleotide sequencing technologies coupled to transposon mutagenesis has provided a powerful tool to interrogate highly saturated mutant libraries of insertion mutants for specific phenotypes [21–23]. This approach allows not only the comprehensive identification of virulence or colonization determinants but also the simultaneous measurement of the relative fitness cost resulting from the inactivation of potentially every nonessential gene under various environmental conditions. When applied to the understanding of metabolic requirements, such comparative analysis can provide a much more encompassing view of the relative importance of specific metabolic pathways under the conditions examined. An essential prerequisite for the application of high-throughput approaches to interrogate genomic libraries is the absence of bottlenecks that could limit the depth of coverage of the mutagenesis screen. Since in most animal models of infection C. jejuni cannot be recovered in large numbers [24], this has been a major limitation in previous attempts to broadly interrogate mutant libraries for their ability to colonize the intestine [19,25]. Recently, a mouse model of infection has been described that allows the replication of C. jejuni to large numbers [26], thereby overcoming previous limitations for the application of high-throughput genome-wide analyses. We report here the use of this animal model in combination with transposon mutagenesis and next-generation sequencing to comprehensively interrogate a highly saturated transposon insertion library of C. jejuni 81–176 for its ability to colonize the intestine. To provide a more robust framework for the interpretation of these results, we have also examined the mutant library after growth under different in vitro conditions. The combination of this extensive genetic approach with isotopologue profiling, metabolic flow examination, and the analysis of specific mutant strains amounted to the most complete analyses of the metabolic determinants necessary for a bacterial pathogen to colonize a mammalian host to date. Importantly, this analysis has identified key metabolic pathways that could be targeted for the development of novel strategies to prevent C. jejuni infection.
We recently reported the construction of a highly saturated and randomly dispersed transposon mutant library composed of approximately 50,000 individual insertions with an average density of 31 insertions/kb across the C. jejuni 81–176 genome [27]. Analysis of the library indicated that 90% of the 1,758 predicted open reading frames in the genome and its plasmids pTet and pVir harbored transposon insertions, whereas the remaining 10% of the genes were presumably essential under the conditions used to generate the library [27]. We used this transposon mutant library to interrogate the fitness of the different C. jejuni mutants after growth in solid rich (blood agar) or defined liquid medium supplemented with asparagine (Asn), glutamine (Gln), or serine (Ser) as the main carbon sources (Fig 1). Comparison of the relative number of each transposon mutant remaining after 48 hours of growth under the different conditions with the number in the original inoculum identified 58 genes whose inactivation resulted in a growth defect under all conditions tested (Fig 1C, S1 and S2 Tables). Most of these genes are involved in basic metabolic processes such as nutrient transport and utilization, respiration, response to oxidative stress, cell envelope biogenesis, DNA replication and repair, translation, and protein turnover. This group of mutants also includes insertions in 13 genes whose functions are unknown. Insertions in 46 genes resulted in mutants with growth defects in defined liquid but not in solid rich media (Fig 1C and S2 Table). Among those, we identified mutations in genes involved in proline (proABC), biotin (bioAFD), and purine and pyrimidine (carB, pyrBF, and purEFHLMNQS) biosynthesis. These observations validated our experimental approach since the defined medium used in the screen lacked these substrates. The analysis of the results described below combined with isotopologue profiling and metabolic flow analysis provides a comprehensive view of the metabolic requirements for C. jejuni to grow in rich and defined media. It was recently reported that mice, rendered dysbiotic by antibiotic treatment, could be stably colonized by C. jejuni [26]. We found that, predictably, antibiotic treatment drastically reduced though did not eliminate the bacterial load in the intestinal tract (S4 Fig). More importantly, we found that, consistent with previous reports [26], C. jejuni strain 81–176 can robustly colonize the intestinal tract of dysbiotic mice for up to 3 weeks after oral infection (Fig 3A). Large numbers of C. jejuni colony-forming units (CFUs) were recovered from the large intestine and cecum of infected animals as early as 4 days after infection and up to 21 days post infection, although colonization of systemic tissues was inefficient. Consequently, this infection model is suitable to screen a library with a large number of transposon insertion mutants for their ability to colonize the intestine as a strategy to identify metabolic determinants necessary for C. jejuni growth in this compartment. After oral inoculation of dysbiotic C57BL/6 mice with the C. jejuni 81–176 transposon insertion mutant library, the infected animals were killed 4,7, or 21 days after infection, and C. jejuni CFUs were recovered from the cecum for INSeq analysis. About 1010 C. jejuni CFUs were recovered from the cecum of infected mice 4 days after infection, and between 108 and 109 CFUs were recovered 21 days after infection (Fig 3B). INSeq analysis of the inoculum revealed transposon insertions in 1,323 out of 1,758 predicted genes of C. jejuni 81–176. Since only insertions within the first 80% of the coding sequence were considered in the analysis, it is likely that all these insertions resulted in inactivation of the targeted genes. Analysis of the mutants recovered 21 days post infection revealed a marked increase in insertions within the pVir and pTet plasmids, with a marked reduction in the representation of the mutant pool (S5 Fig). The reasons for the “blooming” of these mutants late in infection are unclear, although our library already contained a preponderance of insertions within the resident plasmids, presumably because differences in DNA superhelicity may favor insertions into plasmid genes. Nevertheless, the blooming of plasmid insertion mutants observed at day 21 precluded the use of this time point to analyze C. jejuni colonization determinants. Examination of the mutant pool isolated 4 days after infection revealed the presence of an average of 935 mutants per animal (approximately 70% of the inoculum pool), and no indication of blooming since the relative number of insertions within plasmid or chromosomal genes recovered from the intestine was similar to their ratio in the inoculum (S5 Fig and S3 Table). Therefore, we chose this time point to identify genes involved in the initiation of C. jejuni colonization of the intestinal tract. The distribution of log2 values for the number of insertions in most genes showed an overall reduction in comparison with the inoculum (Fig 3C). We observed a similar distribution even when the data were normalized by an alternative approach (S6 Fig and S4 Table) (see “Materials and methods”). Although the reasons for this downshift in the population are not known and likely to be multifactorial, it is not unprecedented since it has been observed in previous similar studies [37]. Consequently, the fitness defect of a given mutant could not be confidently derived by a given cutoff in the log2 ratio of the difference between the number of insertions recovered from the intestine versus the number in the original inoculum. Instead, we identified genes with fitness defects by applying statistical analysis to determine the likelihood that the differences in log2 ratios for any specific gene could have happened by chance (see “Materials and methods”). This analysis identified 143 genes that showed a significantly decreased number of insertions within the mutant pool recovered from the intestine relative to the inoculum (Fig 3C and 3D and Table 1). We found that insertions located immediately downstream of 126 out of the 143 genes identified did not result in attenuating phenotypes indicating that the transposon insertions do not result in polar effects on downstream genes (S5 Table). Mutations in 101 of these genes did not result in a fitness reduction when grown in rich medium (Table 1), suggesting that they may be specifically required for initiation of intestinal colonization. Mutations in the remaining 42 genes resulted in different degrees of fitness loss when C. jejuni was grown in vitro (Table 1). Our INSeq screen identified attenuating transposon mutations in most of the genes that previous studies have shown to be required for intestinal colonization of C. jejuni 81–176 [19,33,38–42], which demonstrates the comprehensive nature of our approach. Thus, C. jejuni 81–176 mutants harboring transposon insertions within genes involved in motility, lipooligosaccharide and capsule biosynthesis, or N- and O-protein glycosylation, as well as metabolic traits, were readily detected among the pool of mutants showing reduced colonization (Table 1 and S6 Table). Notably, however, several mutations in genes encoding cytolethal distending toxin components (CdtA, CdtB, and CdtC) and other previously described putative virulence factors involved in cell adhesion and invasion such as CiaB/C/I, CadF, FlpA, JlpA, PldA, or HtrA resulted in either no fitness cost or a level of attenuation significantly lower than many mutations affecting metabolic processes (see below) (S3 Table). These observations underscore the relative importance of metabolism in C. jejuni intestinal colonization. Our analysis also identified insertions in many genes that have not been previously linked to intestinal colonization. What follows is an analysis of these findings aided by the framework provided by the information obtained through studies of the requirements for C. jejuni growth under different in vitro culture conditions. It has become increasingly clear that the ability to secure nutrients is a key determinant of bacterial pathogenesis [14,16,80]. Our in vivo INSeq analysis has provided evidence that during the infection process C. jejuni relies more on specific metabolic adaptations than on specific virulence factors targeting host processes. In contrast to other intestinal pathogens, like Salmonella or pathogenic Escherichia coli [81–83], C. jejuni utilizes a limited number of carbon and energy sources. However, since low C. jejuni infectious doses are sufficient to cause disease [84,85], such metabolic restrictions do not seem to impede the ability of C. jejuni to overcome the microbiota-mediated colonization resistance. In fact, it is possible that C. jejuni may rely on the microbiota to provide essential nutrients to fuel its metabolism. We find that C. jejuni can utilize potential catabolic end products of the intestinal microbiota such as acetate or CO2-derived hydrogen carbonate and especially free amino acids and di-/ or oligopeptides, which are released from dietary or endogenous proteins. Although free amino acids and dipeptides are prominent in the mucus layer of the small intestine, our study suggests that the concentration of some amino acids may not be sufficient for C. jejuni since auxotrophic mutants unable to synthesize branched-chain and aromatic amino acids or Ser exhibit a colonization defect. We show that C. jejuni overcomes such substrate restriction through an active TCA cycle, gluconeogenesis, and nonoxidative PPP, which together facilitate its anabolic capacity and are crucial for its in vitro and in vivo growth. C. jejuni preferentially colonizes the mucus of the intestinal crypts [54–56], where it is spatially segregated from the luminal microbiota [86,87]. This spatial separation may allow C. jejuni to thrive despite its limited metabolic capacity. Indeed, while other enteropathogenic bacteria like Salmonella exhibit redundant catabolic pathways that allow the exploitation of multiple nutrients [88], our analyses clearly showed that the metabolic network of C. jejuni lacks comparable redundancy and harbors various bottlenecks, as it depends on the gluconeogenic activity of the EMP pathway and the nonoxidative PPP. These metabolic bottlenecks may allow the development of novel strategies to prevent colonization and novel anti-infective drugs.
The complete list of strains and plasmids used in this study is shown in S9 Table. The highly saturated Himar 1 Mariner transposon mutant library has been previously described [27]. The C. jejuni 81–176 strains were grown on brucella broth agar or on blood agar plates (Trypticase soy agar supplemented with 5% sheep blood) at 37°C in an incubator equilibrated to a 10% CO2 atmosphere. The C. jejuni transformants were selected on plates supplemented with 50 μg/ml kanamycin, 7. 5 μg/ml chloramphenicol, and 10 μg/ml erythromycin as indicated. For liquid cultures, C. jejuni strains were grown in brain heart infusion (BHI) medium with no antibiotics added in most cases except for the growth of the mutant library or defined C. jejuni mutant strains (see below). All C. jejuni strains were stored at −80°C in BHI broth containing 30% glycerol. For growth in rich-medium experiments, approximately 108 CFUs of the C. jejuni transposon mutant library were plated on blood agar in 15-cm petri dishes, and the bacterial cells were collected after 48 hours. Six replicates of this growth condition were processed as described below. For growth in liquid defined minimal medium, 108 CFUs of the C. jejuni transposon mutant library were added to 4 ml of DMEM (GIBCO; catalogue number 11965) supplemented with 20 mM Asp, Gln, or Ser. The culture tubes were placed on a rotating wheel in 10% CO2 atmosphere for 48 hours, and the bacterial cells were collected by centrifugation. Three replicates for each of these growth conditions were processed as described below. DNA extraction and sequencing were carried out as previously described [27]. The sequencing data were analyzed using the INSeq_pipeline_v2 package [89]. For this analysis, the sequencing data of each sample were normalized to total counts per million (i. e. , the number of transposon insertions at each site was multiplied by 106 and then divided by the total number of insertions in the sample) [90,91]. This normalization step enabled comparison of the relative abundance change of each insertion mutant in the mutant library across different samples. Alternatively, the data were normalized using the median values of the number of reads. In this case, the median read number for all transposon insertions was calculated in each sample and normalized to a median read number of 50. To identify significant fitness genes under different growth conditions, a Z-test was applied to all gene mutants whose log-transformed output: input ratios were significantly different from the overall distribution across all biological replicates [89]. Consequently, only genes with a q-value of <0. 05 from the Z-test were considered significantly altered from the input. All deep-sequencing data filtering, normalization, mapping, and statistical analysis were conducted in Perl and R. All animal experiments were conducted according to protocols approved by the Yale University Institutional Animal Care and Use Committee. Six- to eight-week-old C57BL/6 mice were treated with antibiotics in drinking water for 4 weeks to eradicate their commensal gut flora and allow robust C. jejuni colonization as previously described [26]. Antibiotics were added to drinking water at the following concentration: ampicillin, 1 mg/ml; neomycin sulfate, 1 mg/ml; ciprofloxacin 200 μg/ml; metronidazole, 1 mg/ml; and vancomycin, 500 μg/ml. Antibiotics were removed from drinking water 2 days prior to the oral administration through stomach gavage of 100 μl of sodium bicarbonate (to neutralize the stomach pH) followed by the oral administration of 109 CFUs of C. jejuni in 100 μl of PBS. Mutants were reisolated 4 and 21 days after the mice were inoculated with the transposon insertion pools. Analysis of the mutants recovered 21 days post infection revealed a marked increase in insertions within the pVir and pTet plasmids with a drastic reduction in the representation of the mutant pool. Although the reasons for the “blooming” of these mutants late in infection are unclear, their emergence precluded the use of this time point to analyze C. jejuni colonization determinants. Consequently, the mutant library screen was performed by killing the mice and homogenizing their ceca with a tissue homogenized 4 days after infection. Bacteria were recovered from the cecum homogenates by plating on 15-cm blood agar petri dish plates (3 plates per cecum sample) containing Campylobacter selective supplements (Karmali, Oxoid SR0167). Colonies were scraped off the plates, and the genomic DNA was isolated as previously described [27]. The INseq DNA sample preparation, sequencing, and data analysis were carried out as described above. C. jejuni 81–176 knockout mutant strains were constructed as previously described [41]. Briefly, flanking regions of the selected open reading frames (ORFs) were amplified with specific primers, linked to either side of a kanamycin (aphA3), an erythromycin (erm), or a chloramphenicol (cat) resistance cassette, and cloned into the pBluescript II SK using the Gibson assembly protocol [92]. The resulting plasmids were used to move the mutated alleles into the chromosome of C. jejuni 81–176 by natural transformation and allelic recombination. Complementation of the mutant strains of C. jejuni was achieved by introducing a wild-type copy of the gene at the hsdM locus as previously described [93]. The plasmids used to generate the mutants are listed in S9 Table. Six- to eight-week-old C57BL/6 mice were infected by oral gavage with 109 CFUs of the different C. jejuni mutants mixed with an equal number of the wild-type parent strain. To enumerate bacterial loads in the cecum, mice were killed 4 days after infection, ceca were homogenized in 3 ml PBS containing 0. 05% sodium deoxycholate, and the dilutions were plated on blood agar plates with Campylobacter selective supplements with and without the selection antibiotics to determine the CFUs of the wild-type and mutant strains. Cultivation of C. jejuni for isotopologue analysis was performed as described in S1 Text. Sample preparation for isotopologue analysis by GC/MS was performed as previously described [94]. During sample preparation, the acidic hydrolysis of cellular proteins did not allow the determination of Asn, Cys, Gln, Met, and Trp: Cys and Met are destroyed during the hydrolysis procedure, whereas Asn and Gln are converted to Asp and Glu, respectively. Consequently, the values for Asp and Glu correlate with the averages of Asn/Asp and Gln/Glu [95,96]. C. jejuni 81–176 was grown in 50 ml Hank’s Balanced Salt Solution supplemented with iron (II) ascorbate (Sigma), MEM Vitamin Solution (Invitrogen), and 1% Casamino acids (Roth) as a carbon source. The liquid cultures were incubated under reduced-oxygen atmosphere using Anaerocult C-Packs (Merck) at 37°C under shaking (150 rpm) conditions. The uptake of amino acids through C. jejuni 81–176 was investigated by analyzing the changes in the amount of the amino acids in the culture supernatants. Samples of 2 ml supernatant were taken at the indicated time points, the bacterial cells were removed by centrifugation (17,000 g, 5 min, 4°C), and an aliquot of the filtrated supernatant was used for GC/MS analysis. The 100 μl aliquot of the supernatant was diluted in 500 μl water, containing 4 μg of ribitol as an internal standard. The samples were mixed, dried under vacuum at room temperature, and stored at −20°C. Derivatization of the samples was done with 40 μl pyridine, containing methoxyamine hydrochloride (20 mg/ml) and 60 μl N-Methyl-N-trimethylsilyltrifluoro-acetamide (MSTFA). The GC/MS analysis was done on a Thermo GC Ultra coupled to a DSQII mass spectrometer equipped with an AS3000 autosampler (ThermoScientific, Dreieich, Germany) as described before [97], with the following exceptions: helium flow was set to 1. 1 ml/min, and the temperature was increased to a final 325°C. The solvent delay time was 5. 80 min. Data analysis was performed with Metabolite Detector (version 2. 07; [98]), and quantification was done using 1 unique fragment ion for each metabolite. For statistical analysis, data were first normalized by dividing the peak area of every detected compound in each sample by the peak area of the respective internal standard ribitol. Afterwards, the mean and the standard deviation were calculated from 3 biological samples with 3 technical replicates each. To investigate the growth properties of amino-acid auxotrophic C. jejuni 81–176 mutants, the different strains were cultivated in a defined DAAM based on Hank’s Balanced Salt Solution with each amino acid present in 2 mM concentration supplemented with vitamin mix, 10 μM ferrous ascorbate, and 20 mM lactate as described before [61]. To compare the growth of the C. jejuni ktr and kdp mutants with the wild-type strain in the presence or absence of K+, strains were inoculated (starting OD600 of 0. 02) into 4 ml of liquid defined rich medium (S10 Table) with or without the addition of 0. 5 or 5 mM KCl. Cultures were incubated on a rotating wheel under 10% CO2 atmosphere, and the OD600 were measure at the indicated times after inoculation. All experiments were performed at least 3 times. For the motility assay, the optical density of the bacterial cultures was adjusted to an OD600 of 0. 3, and 10 μl was spotted onto soft agar (0. 5%, wt/vol). The plates were incubated for 24 hours at 37°C, and the swarming diameters of the different strains were compared to the wild type and the nonmotile C. jejuni ΔmotA mutant strain.
There is accumulating evidence that in addition to canonical virulence factors such as toxins, adhesins, or invasins, bacterial pathogens utilize specific metabolic traits to colonize and proliferate within their hosts, a concept that is increasingly referred to as “nutritional virulence”. We have used transposon insertion mutagenesis combined with next-generation sequencing, a mouse model of infection, isotopologue profiling, and metabolic flow analysis to obtain a comprehensive view of the metabolic requirements for the intestinal colonization of C. jejuni, a leading cause of food-borne gastroenteritis in industrialized countries. This information could provide the basis to control C. jejuni colonization of food-producing animals or the human host.
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Metabolic and fitness determinants for in vitro growth and intestinal colonization of the bacterial pathogen Campylobacter jejuni