Differential Distribution of the wlaN and cgtB Genes, Associated with Guillain-Barré Syndrome, in Campylobacter jejuni Isolates from Humans, Broiler Chickens, and Wild Birds

Campylobacter jejuni causes campylobacteriosis, a bacterial gastroenteritis with high incidence worldwide. Moreover, C. jejuni infection can trigger the polyneuropathic disorder denominated Guillain-Barré syndrome (GBS). The C. jejuni strains that can elicit GBS carry either wlaN or cgtB, coding both genes for a β-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide (LOSSIAL). We described a differential prevalence of the genes wlaN and cgtB in C. jejuni isolates from three different ecological niches: humans, broiler chickens, and wild birds. The distribution of both genes, which is similar between broiler chicken and human isolates and distinct when compared to the wild bird isolates, suggests a host-dependent distribution. Moreover, the prevalence of the wlaN and cgtB genes seems to be restricted to some clonal complexes. Gene sequencing identified the presence of new variants of the G- homopolymeric tract within the wlaN gene. Furthermore, we detected two variants of a G rich region within the cgtB gene, suggesting that, similarly to wlaN, the G-tract in the cgtB gene mediates the phase variation control of cgtB expression. Caco-2 cell invasion assays indicate that there is no evident correlation between the production of LOSSIAL and the ability to invade eukaryotic cells.

The C. jejuni strain collection, previously described [15], is composed of 150 isolates obtained from faeces of three different sources: human patients suffering from symptomatic gastroenteritis (50 isolates), broiler chickens (50 isolates), and wild birds (50 isolates). Human isolates were obtained from the Santa Creu i Sant Pau Hospital (Barcelona) strain collection. Broiler chicken isolates belong to a Campylobacter strain collection at IRTA-CReSA, collected at different slaughterhouses located in Barcelona, Lleida, and Tarragona (Catalonia). The wild bird samples, collected in Catalonia and Alboran Island, were obtained from six different species (Spatula clypeata, Ciconia ciconia, Corvus corax, Columba livia, Larus michahellis, and Larus audouinii). The strain 81-176 (ATCC BAA-2151) was used as the reference strain [13]. Complete genomes of Campylobacter jejuni strains were recovered from an NCBI database.

PCR Amplification
Genomic DNA was extracted from cultures grown onto CBA plates using the InstaGene Matrix Kit (Bio-Rad Laboratoires). PCR reactions (PCR Master Mix x2, Thermo Scientific) were performed using 35 ng of DNA as a template and the presence of wlaN and cgtB genes were tested using the specific primers indicated in Table 1. Primers used to amplify the housekeeping gene gltA were included in the PCR mixtures as an internal control of the PCR reaction. Amplified fragments of wlaN and cgtB were purified and sequenced with the same primers that were used in the PCR reaction.

Analysis of LOS
Cell biomass from one CBA plate was resuspended in 1 ml of PBS and the OD 600 of the cell suspension determined. After centrifugation, the cells were resuspended in Laemmli buffer, adjusting the final volume accordingly to the OD 600 of the cell suspension (OD 600 × 0.5) (whole cell extracts). LOS was isolated as described previously [18]. Briefly, samples were incubated at 100 • C for 10 min and centrifuged at 10,000× g for 10 min. A 50 µl aliquot of the supernatant was treated with proteinase K (20 mg/ml; 2 h at 65 • C). LOS samples were fractionated by 15% SDS-PAGE and visualized by a carbohydrate specific silver staining method [16]. LOS SIAL was detected by Western blot using a peroxidase-conjugated labelled cholera toxin B (HRP-CT), as previously described [12]. Coomasie staining of whole cell extracts was used as the loading control.

Invasion Assay
Adherence and invasion assays were performed using human colonic carcinoma (Caco-2) cells, as previously described [19]. Caco-2 cells were seeded in 24-well plates at 2 × 10 4 cells per well and incubated for 8 days at 37 • C. Bacteria, grown on CBA plates under microaerophilic conditions for 24 h at 37 • C, were resuspended in PBS plus 1% inactivated-FBS (PBS-F) and the bacterial concentration was adjusted at approximately 2 × 10 8 cfu/ml (OD 600 of 0.04). Confluent monolayers of Caco-2 cells were washed once with PBS and infected with 0.5 ml of the bacterial suspension. To allow bacterial adherence and internalization, monolayers and bacteria were incubated for 3 h at 37 • C and 5% CO 2 in a humified atmosphere. For total cell-associated bacteria (intracellular and adhered) quantification, the unbound bacteria were removed from cell monolayers by washing with PBS, the cells were lysed with 0.5 ml Triton X-100 (1%) for 10 min, and the total cell-associated bacteria was determined by serial dilutions on CBA plates. For intracellular bacteria quantification, infected monolayers were washed with PBS and incubated in 0.5 ml of fresh PBS-F with 150 ml/ml gentamycin (Sigma) to kill extracellular bacteria. After 2 h at 37 • C, cells were washed and lysed following the same procedure as for the total cell-associated bacteria. The amount of both intracellular and total cell-associated bacteria (intracellular and adhered) was determined in triplicate assays. The invasion index was calculated as the percentage of intracellular bacteria relative to the total cell-associated bacteria.

Differential Prevalence of wlaN and cgtB Genes in C. jejuni Isolates from Human Patients, Broiler Chickens, and Wild Birds
The presence of the wlaN and cgtB genes in a collection of 150 C. jejuni strains from human patients (50), broiler chickens (50), and wild birds (50) was determined by PCR. A similar percentage of strains were positive for LOS SIAL related genes (wlaN + and cgtB + ) among human (28%) and broiler chicken (22%) strains. The percentage increased to 40% among wild bird strains. Interestingly, more striking differences exist in the prevalence of each specific gene depending on the origin of the strains (Figure 1). The wlaN gene was more frequently detected among human and broiler chicken strains (20% and 16%, respectively) than the cgtB gene (8% and 6%, respectively). In contrast, an inverse distribution was found among wild bird strains, with 34% of the strains cgtB + and only 6% wlaN + . Therefore, among the LOS SIAL proficient strains, the wlaN gene was responsible for the LOS modification in 72% of the human and broiler chicken strains, whereas the cgtB gene was responsible in 85% of the wild bird strains.

Differential Prevalence of wlaN and cgtB Genes in C. jejuni Isolates from Human Patients, Broiler Chickens, and Wild Birds
The presence of the wlaN and cgtB genes in a collection of 150 C. jejuni strains from human patients (50), broiler chickens (50), and wild birds (50) was determined by PCR. A similar percentage of strains were positive for LOS SIAL related genes (wlaN + and cgtB + ) among human (28%) and broiler chicken (22%) strains. The percentage increased to 40% among wild bird strains. Interestingly, more striking differences exist in the prevalence of each specific gene depending on the origin of the strains (Figure 1). The wlaN gene was more frequently detected among human and broiler chicken strains (20% and 16%, respectively) than the cgtB gene (8% and 6%, respectively). In contrast, an inverse distribution was found among wild bird strains, with 34% of the strains cgtB + and only 6% wlaN + . Therefore, among the LOS SIAL proficient strains, the wlaN gene was responsible for the LOS modification in 72% of the human and broiler chicken strains, whereas the cgtB gene was responsible in 85% of the wild bird strains.

The Presence of the cgtB and wlaN Genes is Associated with Certain MLST Clonal Complexes
The phylogenetic population structure of the C. jejuni collection used in this work has previously been reported [15]. The most frequent clonal complexes (CC) detected were ST-21, ST-1275, ST-45, and ST-257, each CC with 24, 16, 13, and 12 strains, respectively.
The LOS SIAL producing strains grouped within certain CC ( Table 2). The most predominant clonal complex, the ST-21 CC, which was only found among human and broiler strains, showed the highest occurrence (78%) of LOS SIAL related genes, with 14 wlaN + and 3 cgtB + isolates. Remarkably, the three cgtB + strains belong to the ST-883. Within the ST-1275 CC, which was only found in wild bird strains, 44% were LOS SIAL (3 wlaN + and 4 cgtB + ). It should be highlighted that the only three wlaN + strains identified among wild bird isolates belong to this CC.
The ST-45 CC is a multihost complex found among the three populations. However, all LOS SIAL strains from this clonal complex (54%) were isolated only from wild birds and they carry the cgtB gene. The strains belonging to the ST-257 CC, isolated from human patients and broiler chickens, were negative for the presence of LOS SIAL -related genes. Among the non-predominant CC (containing 3 to 10 strains per CC), the ST-179 showed the highest percentage of LOS SIAL strains (57.5%). This CC was only found among wild bird strains and accordingly, all LOS SIAL strains were cgtB + . The 37.5% of

The Presence of the cgtB and wlaN Genes is Associated with Certain MLST Clonal Complexes
The phylogenetic population structure of the C. jejuni collection used in this work has previously been reported [15]. The most frequent clonal complexes (CC) detected were ST-21, ST-1275, ST-45, and ST-257, each CC with 24, 16, 13, and 12 strains, respectively.
The LOS SIAL producing strains grouped within certain CC ( Table 2). The most predominant clonal complex, the ST-21 CC, which was only found among human and broiler strains, showed the highest occurrence (78%) of LOS SIAL related genes, with 14 wlaN + and 3 cgtB + isolates. Remarkably, the three cgtB + strains belong to the ST-883. Within the ST-1275 CC, which was only found in wild bird strains, 44% were LOS SIAL (3 wlaN + and 4 cgtB + ). It should be highlighted that the only three wlaN + strains identified among wild bird isolates belong to this CC.
The ST-45 CC is a multihost complex found among the three populations. However, all LOS SIAL strains from this clonal complex (54%) were isolated only from wild birds and they carry the cgtB gene. The strains belonging to the ST-257 CC, isolated from human patients and broiler chickens, were negative for the presence of LOS SIAL -related genes. Among the non-predominant CC (containing 3 to 10 strains per CC), the ST-179 showed the highest percentage of LOS SIAL strains (57.5%). This CC was only found among wild bird strains and accordingly, all LOS SIAL strains were cgtB + . The 37.5% of the ST-607 CC strains, found in humans and broiler chickens, carry LOS SIAL -related genes (1 wlaN + and 2 cgtB + ). None of the strains belonging to the ST-48, 61, 354, 464, and 952 CC were positive for LOS SIAL -related genes.
The fact that within the same sequence type we found strains carrying different LOS determinants is consistent with previous studies, suggesting that the LOS locus is one of the hypervariable regions within the C. jejuni genome [20].

Homopolymeric G-tract variants in wlaN and cgtB genes
As described earlier, the wlaN gene carries an intragenic homopolymeric G-tract [12]. It is assumed that the number of G-residues can vary after DNA replication by a slipped strand mechanism. It has been identified that wlaN alleles carry homolopymeric G-tract with different numbers of G residues. So far, G-tracts with 6, 8, and 9G-residues have been described [12,14]. From those, the 8G-tract variant is the only one rendering a full-length product (ON). The homopolymeric G-tract was characterized for all wlaN + strains of our collection (Figure 2A,B). Variants carrying the previously identified 8 and 9G-tracts were found in both broiler chicken and human isolates. Additionally, new G-tract variants were identified during our study. In the broiler chicken isolate B50, the wlaN sequence reveals a mixed population, with 10G-(OFF) and 11G-tracts (ON) ( Figure 2B). Strikingly, the three unique wlaN + isolates among the wild bird strains (W09, W20, and W25), belonging to the ST-1275 CC, carry a 5G-tract variant, which was not previously described ( Table 2). Detection of LOS SIAL in extracts of the W20 strain corroborate that the newly described wlaN 5G-tract variant renders expression of a functional β-1,3-galactosyltransferase ( Figure 2D). It is worth mentioning that the nucleotide sequence surrounding the G-tract has very high identity among the sequenced wlaN variants ( Figure S1). strains was performed. Representative results are shown in Figure 2D and Figure 3. LOS SIAL was detected in all extracts from either wlaN + or cgtB + strains carrying "ON" G-tracts and in most of the genotypically "OFF" characterized strains, as in the case of H63 strain (Figure 3). Exceptionally, in extracts from H11 and H58 strains, carrying "OFF" G-tracts were negative for LOS SIAL detection ( Figure 2D). LOS silver staining indicates that both strains indeed produce LOS structures, although it does not carry the modification recognized by the cholera toxin ( Figure 2E).

The production of LOS SIAL is Not Affected by Temperature
C. jejuni colonizes different hosts, including birds as broilers and mammals as humans. The LOS SIAL production at 42°C and 37°C, resembling the gastrointestinal tract temperature in broiler chickens and humans, respectively, was monitored. No difference was detected in the amount of LOS in extracts from the LOS SIAL (strains B24 and H63) or non-sialylated LOS (H33) from cultures grown at 42 °C and 37 °C (Figure 3). It has not yet been reported whether cgtB expression is under phase variation control. Interestingly, the cgtB gene carries a G-tract which is located in a different relative position within the coding sequence as compared to wlaN. Within the wlaN coding sequence, the G-tract is located at position 331 from 912 nt; whereas, within the cgtB coding sequence, the newly described G-tract is located at position 476 from 906 nt ( Figure S2). In most cgtB + C. jejuni sequenced strains, the cgtB gene carries a 5G-tract, which renders a full length protein ( Table 2, [21]). The length of the homopolymeric G-tract in the cgtB + isolates was determined. Although most cgtB + strains carry a 5G-tract (ON), the human isolate H58 carries a 6G-tract, which will generate a truncated protein ( Figure 2C). These results suggest that the homopolymeric G-tract which is present within the cgtB gene may also be mediating phase variation control. As for wlaN, the sequences surrounding the G tract of the cgtB variants show very high identity ( Figure S3).
The phase variation control involves phenotypic diversity (LOS and LOS SIAL ) within a clonal population. The presence of LOS SIAL on C. jejuni surface can be detected by its ability to bind cholera toxin subunit B (CT) [12]. Phenotypic characterization of the LOS SIAL production in wlaN + and cgtB + strains was performed. Representative results are shown in Figures 2D and 3. LOS SIAL was detected in all extracts from either wlaN + or cgtB + strains carrying "ON" G-tracts and in most of the genotypically "OFF" characterized strains, as in the case of H63 strain (Figure 3). Exceptionally, in extracts from H11 and H58 strains, carrying "OFF" G-tracts were negative for LOS SIAL detection ( Figure 2D). LOS silver staining indicates that both strains indeed produce LOS structures, although it does not carry the modification recognized by the cholera toxin ( Figure 2E).

Sialylation is Not Needed for Invasiveness
The role of LOS SIAL structures in C. jejuni pathogenicity is not fully understood. It was proposed that the LOS SIAL presence on the surface of C. jejuni might promote invasion of eukaryotic cells [14,22,23]. However, controversial data is reported by different authors [24,25].
The ability to invade Caco-2 cells by 38 C. jejuni strains (37 from our collection and the pathogenic 81-176 strain) was tested. Half of the strains were LOS SIAL proficient (wlaN + or cgtB + ). The invasion index was calculated and the results indicate that no correlation exists between the presence of LOS SIAL structures and invasiveness (Figure 4).

The production of LOS SIAL is Not Affected by Temperature
C. jejuni colonizes different hosts, including birds as broilers and mammals as humans. The LOS SIAL production at 42 • C and 37 • C, resembling the gastrointestinal tract temperature in broiler chickens and humans, respectively, was monitored. No difference was detected in the amount of LOS in extracts from the LOS SIAL (strains B24 and H63) or non-sialylated LOS (H33) from cultures grown at 42 • C and 37 • C (Figure 3).

Sialylation is Not Needed for Invasiveness
The role of LOS SIAL structures in C. jejuni pathogenicity is not fully understood. It was proposed that the LOS SIAL presence on the surface of C. jejuni might promote invasion of eukaryotic cells [14,22,23]. However, controversial data is reported by different authors [24,25].
The ability to invade Caco-2 cells by 38 C. jejuni strains (37 from our collection and the pathogenic 81-176 strain) was tested. Half of the strains were LOS SIAL proficient (wlaN + or cgtB + ). The invasion index was calculated and the results indicate that no correlation exists between the presence of LOS SIAL structures and invasiveness (Figure 4).

Discussion
The presence of LOS SIAL -related genes-wlaN and cgtB-coding for β-1,3-glycosyltransferases is correlated with the ability to trigger GBS in patients suffering from campylobacteriosis. In our study, the prevalence of these genes was determined among C. jejuni isolates from human patients and broiler chickens since the consumption of undercooked chicken meat is the most common transmission route of C. jejuni to humans. C. jejuni isolates from wild birds were also analyzed since circulation of C. jejuni strains among poultry and wild birds has been reported [26]. Furthermore, wild birds are potential sources of human campylobacteriosis [4,27]. Previous studies focused mostly on comparing human and broiler chicken isolates and no correlation between the origin of the strains and the presence of wlaN and cgtB genes was found. It has been described that 40% to 60% of human isolates and 28% to 90% of broiler chicken isolates carry LOS SIAL related genes [21,[28][29][30][31][32]. Similarly, in our collection, no significant differences were found in the prevalence of LOS SIAL -related genes among human and broiler chicken isolates (28% and 22%, respectively). Remarkably, our data indicate that the prevalence of LOS SIAL -related genes is higher among the wild bird isolates (40%).
Among the LOS SIAL proficient strains, a differential distribution of the cgtB and wlaN genes between the human/broiler chicken isolates and those from wild birds was found. The wlaN gene is detected in 72% of the LOS SIAL strains from humans and broiler chickens, whereas the most prevalent LOS SIAL -related gene in wild bird isolates is cgtB (85%). The information available on the prevalence of LOS SIAL -related genes among wild bird isolates is limited. No previous data exist on the prevalence of the cgtB gene, and the presence of the wlaN gene has been estimated between 11% and 17% of wild bird isolates [33,34]. Although several reports indicated that cgtB and wlaN may coexist [14,31], our data suggest the contrary, since none of the isolates carry both genes.
A search for the presence of LOS SIAL -related genes among 136 C. jejuni genome sequences available in the NCBI database was performed (Table S1). In agreement with our data, the presence of LOS SIAL -related genes was confirmed in 40% of the strains, with wlaN and cgtB present in 22% and 18%, respectively. It should be noted that 87% of LOS SIAL positive strains were isolated from human or broiler chicken hosts and the remaining 13% from other domestic animals. Moreover, none of the genome sequences were found to simultaneously carry wlaN and cgtB.
The wlaN gene is under phase variation control [12], meaning that within a clonal population, cells can express sialylated and unsialylated LOS. This phenomenon is achieved since the number of G residues in a G-tract within the wlaN coding sequence can be randomly altered during replication, rendering a truncated or a fully functional protein. Three variants were previously described (6G, 8G,

Discussion
The presence of LOS SIAL -related genes-wlaN and cgtB-coding for β-1,3-glycosyltransferases is correlated with the ability to trigger GBS in patients suffering from campylobacteriosis. In our study, the prevalence of these genes was determined among C. jejuni isolates from human patients and broiler chickens since the consumption of undercooked chicken meat is the most common transmission route of C. jejuni to humans. C. jejuni isolates from wild birds were also analyzed since circulation of C. jejuni strains among poultry and wild birds has been reported [26]. Furthermore, wild birds are potential sources of human campylobacteriosis [4,27]. Previous studies focused mostly on comparing human and broiler chicken isolates and no correlation between the origin of the strains and the presence of wlaN and cgtB genes was found. It has been described that 40% to 60% of human isolates and 28% to 90% of broiler chicken isolates carry LOS SIAL related genes [21,[28][29][30][31][32]. Similarly, in our collection, no significant differences were found in the prevalence of LOS SIAL -related genes among human and broiler chicken isolates (28% and 22%, respectively). Remarkably, our data indicate that the prevalence of LOS SIAL -related genes is higher among the wild bird isolates (40%).
Among the LOS SIAL proficient strains, a differential distribution of the cgtB and wlaN genes between the human/broiler chicken isolates and those from wild birds was found. The wlaN gene is detected in 72% of the LOS SIAL strains from humans and broiler chickens, whereas the most prevalent LOS SIAL -related gene in wild bird isolates is cgtB (85%). The information available on the prevalence of LOS SIAL -related genes among wild bird isolates is limited. No previous data exist on the prevalence of the cgtB gene, and the presence of the wlaN gene has been estimated between 11% and 17% of wild bird isolates [33,34]. Although several reports indicated that cgtB and wlaN may coexist [14,31], our data suggest the contrary, since none of the isolates carry both genes.
A search for the presence of LOS SIAL -related genes among 136 C. jejuni genome sequences available in the NCBI database was performed (Table S1). In agreement with our data, the presence of LOS SIAL -related genes was confirmed in 40% of the strains, with wlaN and cgtB present in 22% and 18%, respectively. It should be noted that 87% of LOS SIAL positive strains were isolated from human or broiler chicken hosts and the remaining 13% from other domestic animals. Moreover, none of the genome sequences were found to simultaneously carry wlaN and cgtB.
The wlaN gene is under phase variation control [12], meaning that within a clonal population, cells can express sialylated and unsialylated LOS. This phenomenon is achieved since the number of G residues in a G-tract within the wlaN coding sequence can be randomly altered during replication, rendering a truncated or a fully functional protein. Three variants were previously described (6G, 8G, and 9G) [12,13]. Here, we revealed three new variants: a 5G-tract found in three wild bird isolates and 10-and 11G-tracts, which were both found in a broiler chicken isolate ( Figure 2B and Figure S1). Remarkably, the cgtB gene carries a G-tract within the coding sequence, but a potential phase variation regulation was not reported. Here, two cgtB variants were found ( Figure 2C, Figure S3): a 5G-tract, detected in most cgtB + strains, rendering a full protein and a 6G-tract, detected only in one isolate, rendering a truncated peptide. Interestingly, the genomic sequence of the well-characterized strain 81-176 indicates the presence of a truncated cgtB gene with a 6G-tract. Overall, our data suggest that, similar to the wlaN gene, cgtB is under phase variation regulation by altering the number of residues in the G-tract. Further studies will be required to fully characterize the mechanism behind the described phenomenon.
Despite the well-established role of the cgtB and wlaN genes in triggering GBS [6], the relevance of these genes in the pathogenesis of C. jejuni during gastrointestinal infection remains unclear. The lack of thermoregulation of LOS and/or LOS SIAL suggests that its production is neither promoted nor repressed in a specific host temperature. Some authors reported a link between LOS sialylation and the ability to invade eukaryotic cells [14,22,23]. In agreement with other reports [24,25], our data suggest that no correlation exists between these two processes since no differences in the invasion index were detected between LOS SIAL and non-sialylated strains.
Overall, our data reveal a closer relationship between human and broiler chicken isolates as compared to wild bird isolates, which is in agreement with the previous characterization of the strain collection in terms of population structure, drug resistance, and virulence factor profiling [15]. The differential distribution of wlaN and cgtB genes may also indicate a host-dependent distribution of the LOS SIAL -related genes, with wlaN positively selected among broiler chickens and consequently, also among human isolates, and cgtB positively selected among wild birds.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/3/325/s1, Figure S1: wlaN nucleotide sequence surrounding the position of G-tract in the wlaN positive strains, Figure S2: Alignment of wlaN and cgtB nucleotide sequences, Figure S3: cgtB nucleotide sequence surrounding the position of the G-tract in the cgtB positive strains, Table S1: Presence of the wlaN and cgtB genes in complete genome sequences of C. jejuni from the NCBI database.