Association of Campylobacter jejuni Cj0859c Gene (fspA) Variants with Different C. jejuni Multilocus Sequence Types

Abstract

Cj0859c variants fspA1 and fspA2 from 669 human, poultry, and bovine Campylobacter jejuni strains were associated with certain hosts and multilocus sequence typing (MLST) types. Among the human and poultry strains, fspA1 was significantly (P < 0.001) more common than fspA2. FspA2 amino acid sequences were the most diverse and were often truncated.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and responsible for more than 90% of Campylobacter infections (7). Case-control studies have identified consumption or handling of raw and undercooked poultry meat, drinking unpasteurized milk, and swimming in natural water sources as risk factors for acquiring domestic campylobacteriosis in Finland (7, 9). Multilocus sequence typing (MLST) has been employed to study the molecular epidemiology of Campylobacter (4) and can contribute to virulotyping when combined with known virulence factors (5). FspA proteins are small, acidic, flagellum-secreted nonflagellar proteins of C. jejuni that are encoded by Cj0859c, which is expressed by a σ²⁸ promoter (8). Both FspA1 and FspA2 were shown to be immunogenic in mice and protected against disease after challenge with a homologous strain (1). However, FspA1 also protected against illness after challenge with a heterologous strain, whereas FspA2 failed to do the same at a significant level. Neither FspA1 nor FspA2 protected against colonization (1). On the other hand, FspA2 has been shown to induce apoptosis in INT407 cells, a feature not exhibited by FspA1 (8). Therefore, our aim was to study the distributions of fspA1 and fspA2 among MLST types of Finnish human, chicken, and bovine strains.

In total, 367 human isolates, 183 chicken isolates, and 119 bovine isolates (n = 669) were included in the analyses (3). PCR primers for Cj0859c were used as described previously (8). Primer pgo6.13 (5′-TTGTTGCAGTTCCAGCATCGGT-3′) was designed to sequence fspA1. Fisher's exact test or a chi-square test was used to assess the associations between sequence types (STs) and Cj0859c. The SignalP 3.0 server was used for prediction of signal peptides (2).

The fspA1 and fspA2 variants were found in 62.6% and 37.4% of the strains, respectively. In 0.3% of the strains, neither isoform was found. Among the human and chicken strains, fspA1 was significantly more common, whereas fspA2 was significantly more frequent among the bovine isolates (Table 1). Among the MLST clonal complexes (CCs), fspA1 was associated with the ST-22, ST-45, ST-283, and ST-677 CCs and fspA2 was associated with the ST-21, ST-52, ST-61, ST-206, ST-692, and ST-1332 CCs and ST-58, ST-475, and ST-4001. Although strong CC associations of fspA1 and fspA2 were found, the ST-48 complex showed a heterogeneous distribution of fspA1 and fspA2. Most isolates carried fspA2, and ST-475 was associated with fspA2. On the contrary, ST-48 commonly carried fspA1 (Table 1). In our previous studies, ST-48 was found in human isolates only (6), while ST-475 was found in both human and bovine isolates (3, 6). The strict host associations and striking difference between fspA variants in human ST-48 isolates and human/bovine ST-475 isolates suggest that fspA could be important in host adaptation.

TABLE 1.

Percent distributions of fspA1 and fspA2 variants among 669 human, poultry, and bovine Campylobacter jejuni strains and their associations with hosts, STs, and CCs

Host or ST complex/ST (no. of isolates)	% of strains witha:		P valueb
	fspA1	fspA2
Host
All (669)	64.3	35.4
Human (367)	69.5	30.0	<0.001
Poultry (183)	79.2	20.8	<0.001
Bovine (119)	25.2	74.8	<0.0001
ST complex and STs
ST-21 complex (151)	2.6	97.4	<0.0001
ST-50 (76)	NF	100	<0.0001
ST-53 (19)	NF	100	<0.0001
ST-451 (9)	NF	100	<0.0001
ST-883 (11)	NF	100	<0.0001
ST-22 complex (22)	100	NF	<0.0001
ST-22 (11)	100	NF	<0.01
ST-1947 (9)	100	NF	0.03
ST-45 complex (268)	99.3	0.7	<0.0001
ST-11 (7)	100	NF	NA
ST-45 (173)	99.4	0.6	<0.0001
ST-137 (22)	95.5	4.5	0.001
ST-230 (14)	100	NF	<0.0001
ST-48 complex (18)	44.4	55.6	NA
ST-48 (7)	100	NF	NA
ST-475 (8)	NF	100	<0.001
ST-52 complex (5)	NF	100	<0.01
ST-52 (4)	NF	100	0.02
ST-61 complex (21)	NF	100	<0.0001
ST-61 (11)	NF	100	<0.0001
ST-618 (3)	NF	100	0.04
ST-206 complex (5)	NF	100	<0.01
ST-283 complex (24)	100	NF	<0.0001
ST-267 (23)	100	NF	<0.0001
ST-677 complex (59)	100	NF	<0.0001
ST-677 (48)	100	NF	<0.0001
ST-794 (11)	100	NF	<0.001
ST-692 complex (3)	NF	100	0.04
ST-1034 complex (5)	NF	80	NA
ST-4001 (3)	NF	100	0.04
ST-1287 complex/ST-945 (8)	100	NF	NA
ST-1332 complex/ST-1332 (4)	NF	100	0.02
Unassigned STs
ST-58 (6)	NF	100	<0.01
ST-586 (6)	100	NF	NA

^aIn 0.3% of the strains, neither isoform was found. NF, not found.

^bNA, not associated.

A total of 28 isolates (representing 6 CCs and 13 STs) were sequenced for fspA1 and compared to reference strains NCTC 11168 and 81-176. All isolates in the ST-22 CC showed the same one-nucleotide (nt) difference with both NCTC 11168 and 81-176 strains, resulting in a Thr→Ala substitution in the predicted protein sequence (represented by isolate FB7437, GenBank accession number HQ104931; Fig. 1). Eight other isolates in different CCs showed a 2-nt difference (isolate 1970, GenBank accession number HQ104932; Fig. 1) compared to strains NCTC 11168 and 81-176, although this did not result in amino acid substitutions. All 28 isolates were predicted to encode a full-length FspA1 protein.

FIG. 1.

Comparison of FspA1 and FspA2 isoforms. FspA1 is represented by 81-176, FB7437, and 1970. FspA2 is represented by C. jejuni strains 76763 to 1960 (GenBank accession numbers HQ104933 to HQ104946). Scale bar represents amino acid divergence.

In total, 62 isolates (representing 7 CCs and 35 STs) were subjected to fspA2 sequence analysis. Although a 100% sequence similarity between different STs was found for isolates in the ST-21, ST-45, ST-48, ST-61, and ST-206 CCs, fspA2 was generally more heterogeneous than fspA1 and we found 13 predicted FspA2 amino acid sequence variants in total (Fig. 1). In several isolates with uncommon and often unassigned (UA) STs, the proteins were truncated (Fig. 1), with most mutations being ST specific. For example, all ST-58 isolates showed a 13-bp deletion (isolate 3074_2; Fig. 1), resulting in a premature stop codon. Also, all ST-1332 CC isolates were predicted to have a premature stop codon by the addition of a nucleotide between nt 112 and nt 113 (isolate 1960; Fig. 1), a feature shared with two isolates typed as ST-4002 (UA). A T68A substitution in ST-1960 (isolate T-73494) also resulted in a premature stop codon. Interestingly, ST-1959 and ST-4003 (represented by isolate 4129) both lacked one triplet (nt 235 to 237), resulting in a shorter FspA2 protein. SignalP analysis showed the probability of a signal peptide between nt 22 and 23 (ACA-AA [between the underlined nucleotides]). An A24C substitution in two other strains, represented by isolate 76580, of ST-693 and ST-993 could possibly result in a truncated FspA2 protein as well.

In conclusion, our results showed that FspA1 and FspA2 showed host and MLST associations. The immunogenic FspA1 seems to be conserved among C. jejuni strains, in contrast to the heterogeneous apoptosis-inducing FspA2, of which many isoforms were truncated. FspA proteins could serve as virulence factors for C. jejuni, although their roles herein are not clear at this time.