Genetic Analyses Of Guillian-Barré Syndrom Associated Campylobacter Jejuni

Campylobacter jejuni(C.jejuni) is a Gram negative microaerophilic bacterium.It is a major cause of human gastroenteritis world-wide.In addition to the burden of disease due to gastroenteritis,C.jejuni infection is highly associated with the development of Guillain-Barre syndrome(GBS),an acute motor paralysis that may result from autoimmune antibodies against C.jejuni antigens.Genetic population studies play an important role to investigate the bacterial pathogen and explore the epidemiology of Campylobacter infection.It was reported the GBS associated C.jejuni had specific genetic character.From June to July,2007,36 cases of GBS occurred in one district in Changchun,Jilin,China.Epidemiology and serological study results indicated it was associated with preceding C.jejuni infection. In order to obtain the genetic characteristics of the outbreak associated strains and the relatedness to other Chinese strains,serotyping,PFGE and MLST analyses were preceded on C.jejuni strains from.China.Seven C.jejuni strains and 7 C.coli strains were identified from the GBS outbreak. The isolates from GBS patients and diarrhea neighbors were C.jejuni.Three of four GBS outbreak associated human isolates(ICDC07001,ICDC07002,and ICDC07003) belonged to serotype HS:41 which were different from the serotype of the strains from GBS patients' house feeding poultry.There were totally 9 kinds of serotype among 26 C.jejuni except 9 strains belonged to un-typed ones.PFGE were performed according to the standard PulseNet PFGE protocol for C.jejuni (http://www.cdc.gov/pulsenet/protocols/campy_protocol.pdf) with SmaI and KpnI digestions.All four human C.jejuni isolates had indistinguishable SmaI and KpnI PFGE profiles and they were different from the PFGE profiles of the chicken isolates. The PFGE profiles of the GBS outbreak strains were unique when compared to 5,284 entries in the PulseNet National Campylobacter database.This PFGE profile of the human C.jejuni isolates from the GBS outbreak were most closely similar to C.jejuni isolates associated with an outbreak in Louisiana in 1995 and was similar to 4 of GBS associated strains from Hebei province,China.The entire four human C.jejuni isolates from GBS outbreak had the same sequence type(ST).This ST was new in the public MLST database and was assigned ST-2993.The chicken isolates from GBS outbreak were ST379 and ST2274 which had been seen previously in the database.There was no any founder ST was identified from Chinese strains by eBURST cluster analysis. SplitsTree analysis indicated the STs from GBS associated strains did not form any colonal complex.Four isolates from the MLST database had similar MLST profiles with ST-2993.There strains were found being associated with GSB and severe system infection.The genome size of ICDC07001 was similar with other human C.jejuni isolates such as C.jejuni 81-176,NCTC11168 and 81-116 but smaller than the chichen isolate RM1221 and C.jejuni sub.doylei 269.97.There was one C.jejuni integrated element, a Campylobacter Mu-like phage(also termed CMLP1,encoding several proteins with similarity to bacterio-phage Mu and other Mu-like biosynthesis protein) reversely inserted between CDS01122(Cj0723c,putative integral membrane zinc metalloprotease) and CDS01219(Cj0725c,molybdenum cofactor) in ICDC07001. Similar as other C.jejuni genome character,there were variant genes in the regions of surface structure,such as the lipooligosaccharide(LOS) biosynthesis,Polysaccharide capsular(CPS) biosynthesis,flagella modification(FM),and DNA restriction modification(R/M) loci.From genomic comparative study,ICDC07001 were 98.99% similar with C.jejuni strain 260.94 which was isolated from GBS patient in South Africa and had same serotype with ICDC07001.Twenty five CDSs were found specific on ICDC07001 compared to NCTC11168,81-176,RM1221,81-11,269.97and C.curvus₅25.92,C.fetus₈2-40 and C.hominis_BAA-381,which were located in the CMLP1,CPS,LOS and FM regions.Three specific CDSs were in Oxidative phosphorylation region.ICDC07001 has a specific FspA2 gene and partial of IS606 tnpB located at the upstream of tonB.Comparative analysis was performed by sorting the studied strains into three groups.Twenty-seven genes were specifically common in the C.jejuni infection outbreak associated group.Eighteen genes were common in the gastroenteritis group and thirteen genes were common in the GBS associated group and nine of them belonged to LOS genes.LOS cluster of ICDC07001 was similar with type ALOS,which was previously reported to associate with GBS.Sialyl-transferase gene from ICDC07001 was cst-â…¡(Asn51) which had bothα-2,3 andα-2,8-sialyltransferase activity.It was referred to express the mimic of the GM1 and GD1b gangliosides which was consistent with the serum anti-gangliosides tests results for GBS patients.The CPS of ICDC07001 was only similar with the same serotype strains 260.94.Neighbors-joining phylogenetic analysis of Campylobacter and other bacteria based on the similarities of 16SrRNA,rpoB and flaA-SVR gene sequences were performed. A good congruence result was obtained between these genes.The most closely related strain to ICDC07001 was strain 260.94.C.jejuni 81-176,81-116 and HB93-13 were also closed related to ICDC07001.A primary proteomic database of sequenced C.jejuni NCTC11168 was set up in this study.Comparative proteomic analysis was performed between 37℃and 42℃culture in vitro with different media.There were the subsets of optimal expression proteins for certain temperature environment.The optimum expressed proteins at 37℃were most related to the stress enhancer and bio-film forming.We did not find any significantly different expression of the PEB1, CadF and CDT between 37℃and 42℃both in BHI and agar media.In summary,the present study gave us a comprehensive profile of the genetic character of the GBS associated strains and the strains isolated in China.Pathogen tracking study for the GBS outbreak associated strains proved C.jejuni isolates from human were same but different from the poultry ones.The specific genetic character of ICDC07001 was obtained by the genome comparative analysis which gave us clues for further study.In this study,we found CPS but not LPS genes were related to the serotype of C.jejuni.All the studied GBS associated strains had similar LOS.Optimal expressed proteins under different temperature may contribute to host adaptation or the pathogenic specialty of human disease.