Distribution of flagella secreted protein and integral membrane protein among Campylobacter jejuni isolated from Thailand
© Pootong et al; licensee BioMed Central Ltd. 2011
Received: 3 June 2011
Accepted: 12 July 2011
Published: 12 July 2011
Campylobacter jejuni, a gram-negative bacterium, is a frequent cause of gastrointestinal food-borne illness in humans throughout the world. There are several reports that the virulence of C. jejuni might be modulated by non-flagellar proteins that are secreted through the filament. Recently, FspA (Flagella secreted proteins) have been described. Two alleles of fspA (fspA1 and fspA2) based on sequence analysis were previously reported and only the fspA2 allele was found in Thai isolates. The aim of this study is to analyze the deduced amino acid sequences fspA and the adjacent putative integral membrane protein from 103 Thai C. jejuni isolates.
A total of 103 representative C. jejuni isolates were amplified by PCR for the fspA gene and the adjacent integral membrane protein gene. Two PCR product sizes were amplified using the same primers, an approximately 1600-bp PCR product from 19 strains that contained fspA and integral membrane protein genes and an approximately 800-bp PCR product from 84 strains that contained only the fspA gene. DNA sequencing was performed on the amplified products. The deduced amino acid sequences of both genes were analyzed separately using CLC Free Workbench 4 software. The analysis revealed three groups of FspA. Only FspA group 1 sequences (19/103) (corresponding to fspA1) consisting of 5 subgroups were associated with the adjacent gene encoding the integral membrane protein. FspA group 2 was the largest group (67/103) consisting of 9 subgroups. FspA group 2p (17/103) consisting of 7 subgroups was found to contain stop codons at a position before the terminal 142 position.
This study reveals greater heterogeneity of FspA (group 1, 2 and 2p) among Thai C. jejuni isolates than previously reported. Furthermore, the subgroups of FspA groups 1 were associated with groups of integral membrane protein. The significance of these different FspA variants to virulence requires further study.
Campylobacter jejuni is a major cause of gastroenteritis worldwide especially in children, travelers, military personnel deployed to developing countries. Although these pathogens are generally considered invasive, the level of invasion of intestinal epithelial cells in vitro varies among strains . Despite the high incidence of human disease and multiple genome sequences [2–5], understanding about the pathogenesis of diarrheal disease at the molecular level is limited. Genomic studies have indicated that C. jejuni strains lack specialized type III secretion systems that are essential to virulence of many other enteric pathogens [6, 7]. There are several reports that flagella can function to secrete non-flagellar proteins that might modulate virulence.
The Cia (Campylobacter invasion antigen) proteins [8–10] and FlaC (Flagellin C protein)  are non-flagellar proteins that are secreted through the filament. Recently, a third secreted protein, FspA (Flagella secreted protein) has been described  and additional candidates have also been reported recently . Similar to FlaC, FspA is a small acidic protein is secreted into the supernatant of broth-grown bacteria. Two alleles of fspA (fspA1 and fspA2) based on sequence analysis were found in previous reports that examined strains from diverse geographical areas. The entirety of the Thai isolates tested in this previous study contained fspA2 alleles. Recombinant proteins encoded by an fspA1 allele from strain 81-176 and by an fspA2 allele from strain CG8486, a clinical isolate from Thailand  were studied previously. These studies revealed that recombinant FspA2 bound to intestinal epithelial cells in vitro and also induced apoptosis, whereas recombinant FspA1 did not . Thus, only one form of the FspA protein appeared toxic to eukaryotic cells, but the mechanism and significance to virulence remains to be determined. Moreover, the study of immunogenicity and protective efficacy of FspA1 and FspA2 were compared in a mouse intranasal model. Immunization with FspA1 resulted in higher protection against homologous challenge with 81-176, which expresses the homologous FspA1, and CG8486, which expresses FspA2; immune protection with FspA2 was less robust against both strains . Genomes presenting fspA2 allele (encoded from Cj0859c gene in reference strain NCTC11168) have been reported to systematically lack the adjacent gene Cj0860, a putative integral membrane protein. In contrast, the fspA1 allele was adjacent to the integral membrane protein gene . However, no functional relation has been reported between these two proteins. Additionally, another report  showed FspA variants and MLST associations among human, poultry and bovine Campylobacter jejuni strains.
A significant high isolation rate of C. jejuni in Thailand and potential role of the FspA protein as one of vaccine candidate lead us to further characterize the deduced amino acid sequences fspA and the adjacent putative integral membrane protein from additional Thai C. jejuni isolates.
Results and Discussion
The 103 C. jejuni isolates representing clusters from a previous PFGE (Pulsed Field Gel Electrophoresis) cluster analysis of C. jejuni isolates from the diarrhea studies on adults in Thailand were selected for this study . We investigated the variation of FspA and integral membrane protein by DNA sequencing of PCR products and analyzed their deduced amino acid sequences.
PCR and sequencing
Cj0859c and Cj0860, if present, were amplified from DNA samples of the strains. Approximate 1600-bp PCR products containing both genes were obtained from 19 strains and approximate 800-bp PCR products containing only Cj0859c were obtained from 84 strains. Both PCR product sizes were amplified using the same pg06.14 and pg06.15 primers. The deduced amino acid sequences of both genes were analyzed separately.
Analysis of the deduced amino acid sequences of FspA
Alignment of the deduced amino acid sequences of FspA
Distribution and alignment of the deduced amino acid sequence fspA of 103 C. jejuni strains
Alignment with reference C. jejuni*
Amino acid change†
N75S, V80G, K124I
E93G, V129I, A142T
K8T, H28R, T47A, N75S, G78D
D20N, V24A, T47A, N75S, V80G, K124I
D20N, V24A, T47A, N75S, V80G, I88M, K124I
E93G, V129I, A142T
E93G, T115D, F116I, Q117S, Q119T, I120N
V80I, E93G, T115D, F116I, Q117S, Q119T, I120N
Q70K, L95P, A96F, E100K, L101V
K58Q, V59M, S60K, K63V
D20N, V24A, K58F, V59R
For FspA group 1, percent identity within the group without reference sequence was 97.18%, except for strain BHC-01-360. The BHC-01-360 strain was aligned with C. jejuni 81-176 and showed 25 deleted amino acids. The percent identity within FspA group 2 without reference sequence was 88.73%. The results imply that FspA group 1 is more conserved than FspA group 2. This is in contrast to the results in preceding report that FspA1alleles were less conserved than FspA2 .
The FspA group 1 consensus sequence encoded a predicted soluble cytoplasmic protein of 15.5 kDa, pI 4.84, and the FspA group 2 consensus sequence encoded a predicted soluble cytoplasmic protein of 16.0 kDa, pI 5.96. Additionally, the two variants were only 40.14% identical at the protein level (Figure 2). Therefore, FspA group 1 is obviously distinguishable from FspA group 2, as reported previously.
Analysis of the deduced Cj0860 protein sequences
Alignment of the deduced Cj0860 protein sequences
Distribution and alignment of the deduced integral membrane protein sequence of 19 C. jejuni strains
Alignment with C. jejuni 81-176
Amino acid change*
I33V, V145I, G216S, V274A
I33V, A52T, T59A, M69I
Association between subgroups of fspA group 1 and Cj0860 alleles
As reported previously, only fspA group 1 alleles were associated with Cj0860 alleles. Furthermore, each subgroup of FspA group 1 was associated with each group of integral membrane protein. Subgroup 1A of FspA group 1 included 1E that has only one amino acid different from 1A, were associated with group A of integral membrane protein. Subgroup 1B, 1C and 1D of FspA group1 were associated with group B, C and D of integral membrane protein, respectively. The associations are shown in Figure 1 and 3.
It has been known that only FspA group 1 encoding gene was adjacent to integral membrane protein gene. Moreover, each subgroup of FspA group 1 is linked with a specific allele of the integral membrane protein. This may imply that integral membrane proteins relate to virulence of FspA or that the two proteins co-evolved from a common precursor. However, this remains to be determined and more information about the functional association of the two proteins remains lacking.
This study reveals greater heterogeneity of FspA (group 1, 2 and 2p) among Thai C. jejuni isolates than previously reported . This may be because the selected C. jejuni isolates for this study were representative isolates from of PFGE clusters of isolates from multiple diarrhea studies in Thailand . Moreover, these isolates were obtained from various regions of Thailand over time suggesting spatial and temporal distribution among C. jejuni isolates in Thailand, whereas all previously examined Thai isolates were from the 1999 Cobra Gold exercises . Interestingly, the association of only the FspA group 1 gene with the Cj0860 alleles that was reported previously continued to be found in these additional strains. Moreover, subgroups of FspA group 1 were associated with specific groups of Cj0860. However, the significance of the observed differences and the roles of these genes in virulence need further study.
Of three hundred and thirty four C. jejuni isolates, 274 isolates were from US soldiers with diarrhea who participated in the Cobra Gold exercises during 1998-2003 and 60 isolates were from foreigners and Thai adults with diarrhea seen at Bumrungrad Hospital in 2001-2002. The 334 isolates had been previously clustered by PFGE analysis. A dendrogram of PFGE patterns was generated and grouped into 103 clusters. A total of 103 C. jejuni isolates representative of each cluster from the PFGE analysis were selected for this study . Among 103 C. jejuni isolates, 76 isolates were isolated from the US soldiers in the Cobra Gold exercises and 27 isolates were isolated from the foreigners and Thai adults at Bumrungrad Hospital in Bangkok. All C. jejuni isolates were kept at -70 °C in glycerol medium and later were subsequently grown on blood agar plate (BD Diagnostic Systems, Sparks, MD, USA.) and incubated at 37 °C for 48 h under microaerobic condition. All isolates were from stool samples collected with informed consent under human use protocols approved by the appropriate ethical review committees.
PCR of alleles of Cj0859c (fspA) and Cj0860
DNA templates were extracted from fresh, subcultured C. jejuni isolates after 48 h incubation using DNeasy tissue kits (Qiagen GmbH, Hilden, Germany). The samples of DNA were estimated for purity and quantity by gel electrophoresis compared to the known molecular weight marker and these DNA were kept at -20 °C for further use as DNA template for PCR. The fspA gene (Cj0859c) and the adjacent integral membrane protein gene (Cj0860), if present, were amplified using 0.4 μM of primer pg06.14 (5' -CCTATTTATGGATTGCAATTTCACCCCG -3') that bound to pabA gene (Cj0861) and 0.4 μM of primer pg06.15 (5' -CTTGAAACGATCAAGGGTAGGGCAGC -3') that bound to murA (Cj0858c)  in 50-μl reactions containing 1-10 ng DNA template, 1 × PCR buffer II, 3.0 mM MgCl2, 0.4 mM each dNTP and 2.5 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems). DNA amplification was performed using an initial denaturation step at 94 °C for 5 min; followed by 35 cycles of amplification (denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min) and ending with a final extension at 72 °C for 10 min. Amplification products were purified for sequencing by Wizard SV gel and PCR clean-up system (Promega, WI, USA).
Sequencing of PCR products and analysis of sequences
All amplified products were submitted for sequencing commercially (Macrogen, Seoul, South Korea). An approximately 800-bp PCR products were sequenced using the two PCR primers (pg06.14 and pg06.15) as previously described, whereas 1600-bp PCR products were sequenced using the two PCR primers (pg06.14 and pg06.15) and primer pg0553re (5'- GCTATTTAAGGAATTGTTAATTTGCAT-3'). The DNA sequences were edited and assembled by Sequencher software version 4.7 (Gene Codes Corporation, MI, USA). The deduced amino acid sequences were analyzed for their heterogeneity such as alignment, clustering and sequence information using CLC Free Workbench 4 software . C. jejuni 81-176 [GenBank:CP000538] and CG8486 [GenBank:EF058232] were used as reference sequences.
All study projects described here were financially supported by the Military Infectious Diseases Research Program, Walter Reed Army Institute of Research, Washington, DC, USA. We would like to thank Apichai Srijan, Department of Enteric Diseases at the Armed Forces Research Institute of Medical Sciences (AFRIMS), for his kind provision of C. jejuni strains. The views expressed in this article are those of the author and do not reflect the official policy of the Department of the Army, Department of Defense, or the U.S. Government.
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