Comparative Gene Sequence Of Wla Gene Cluster In Campylobacter Jejuni Strains Leading Axonal Guillain-Barré Syndrome

Objective : Campylobacter jejuni (C.jejuni) is the predominant cause of bacterial gastroenteritis in the world and Guillain-Barre syndrome (GBS) is a peripheral neuropathy happened after C.jejuni infection. According to different pathological and physiological representation, GBS can be classified into acute demyelinating pattern (AIDP), acute motor axonal neuropathy (AMAN) and Miller-Fisher syndrome (MFS). Many researches indicate that molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissue is considered to be involved in the pathogenesis of C.jejuni-induced GBS.In this reseach, the wla gene clusters of three C.jejuni isolates from north China which have been proved can cause AMAN type GBS were cloned and sequenced. The nucleotide sequences were analyzed and compaired with the known sequences of C.jejuni strains in Genbank to find out the relevance between the sequence character and ability to cause GBS as also as the markers for local GBS associated C.jejuni. Methods:1 Bacteria culture: The C,jejuni strains used were isolated from stools of AMAN patients from North China.The serotypes are respectively Penner O:19, Penner O:2, Penner O:5. Growth condition was following the procedure of Li CY et al.2 DNA isolation: DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega).3 PCR: PCR reactions were performed as described previously. All PCR products were analyzed by agarose gel electrophoresis.4 Sequence and assemblte: All the PCR products sequenced by Shanghai Sangon Biological Engineering Technology Corporation. Sequencing results were spliced and assembled into a complete sequence by the terminals overlapped each other with the known genome sequence as template.5 Evolutionary analysis: The evolutionary distances from nucleotide sequences were estimated for the wla clusters sequences of the three C.jejuni strains and the corresponding sequence of 6 C.jejuni stains whose genome sequence have published in GeneBank and the phylogenetic tree was constructed using Mega version 4 software.6 Sequence alignment: DNA sequences of wla cluster were compared with C.jejuni NCTC 11168 by BLAST of NCBI database to find out the mutations. Result:1 The wla cluster total length of the three strains spanned about 13kb and lulei strain contained 11 complete ORFs just as NCTC11168, while qiaoyuntao and zhanxing strains contained 12 ORFs, having one more ORF named wlaJ in the cluster between wlaK and wlaI. All the wla clusters of three C.jejuni possessed a G+C content around 29.7%.2 Lulei strain, qiaoyuntao strain and zhanxing strain separately has the highest similarity with RM1221, NCTC11168 and 81-176 strains. In the 12 ORF of zhanxing strain wla cluster, four adjacent genes spanning 4.4kb, wlaM, walL, wlaK and wlaJ, have the highest similarity with NCTC11168. And six adjacent genes from wlaC to wlaI except wlaF are most similar with 81-176 strain. When single gene is compared, the three local strains show homogeneous in some genes.3 The sequence comparison of NCTC11168 and our strains displayed more than 98% identity. The Alignment with the related sequence of NCTC 11168 showed that there were some mutations in the wla gene clusters of the three C.jejuni stains. Lulei strain contained 134 nucleotide mutations, 30 amino acid mutations and the express of wlaB, wlaE, walL, wlaM stopped midway; qiaoyuntao strain contained 24 nucleotide mutations, 3 amino acid mutations and the express of wlaG, wlaK, walM stopped midway; zhanxing strain contained 221 nucleotide mutations, 37 amino acid mutations and the express of wlaB, wlaG, walK stopped midway. In these mutations, lulei strain and zhanxing strain had 7 identical amino acid mutation.Conclusion : The character of gene structure and similarity can be revealed from complete wla gene cluster sequencing of the three stains of C.jejuni and the results of phylogenetic analysis with other C.jejuni stains. When single gene was compared, local strains showed homogeneous in the wlaK, wlaM and some other genes. The nucleotides mutations found in the three C.jejuni stains lead to some amino acid mutation and truncated expression products, and there were also 6 same amino acid mutation in lulei strain and zhanxing strain which may affect the secondary and tertiary structure to change the function of those proteins. And the changes may result in the ability to causing GBS. Our study established the foundation for exploring the biologically characteristic of local GBS-associated C.jejuni stains.