| Burkholderia pseudomallei is an aerobic, Gram-negative, motile bacillus, exhibitingbipolar staining, first described by Alfred Whitmore in Rangoon, Burma in1911. Accordingto differnce of utility capacity of L-Arabinose, as some experts suggested, B. pseudomalleithat cannot use L-Arabinose is considered to be pathogenic strain, vice versa. It was alsoclassified as a category B pathogen by the US CDC in2006and listed as a bio-threat after“9.11”. B. pseudomallei could cause a tropical disease, Melioidosis, which could displaymulti-abscess, tough pneumonia and fatal septicemia, with a mortality rate of up to40%. InChina, Hainan, Guangdong, Guangxi and Taiwan are all disaster areas of Melioidosis. Withthe increasing imported cases in many non-endemic regions, more attention is paid on thisbug overseas, while in China it isn’t enough.A standard B. pseudomallei strain is not available because of the barrier of itsbio-threat characteristics. So we must first establish a Chinese standard strain for furtherstudy on Melioidosis. Genome sequencing is the most prompt and effective approach togain detailed molecular informations and genetic characteristics associated with a certainmicrobe. So we sequenced a complete genome of a B. pseudomallei strain and analyzed itsgenetic characteristics.Besides, B. pseudomallei-infected cell model is necessary for pathogen research.Although many cell models are applied in B. pseudomallei infection studies, none of themhas pointed out a criteria for assessing a reliable infected cell model nor involved a Chineseclinical strain. So we improved previous infection models, describing the process in detailand forming a reliable infection model for our furhter study.Methods1. Whole-genome shotgun sequencing was performed with454Titanium. Newbler2.3was used to perform the assembly of raw sequencing reads. The order of contigs weredetermined by alignment with the published genome sequence of B. pseudomallei strain 1106a. Gaps between contigs were closed by local assembly and sequencing PCR productsusing an ABI3730capillary sequencer, followed by online annotation, error checking,submission and data analysis.2. Antisera of B. pseudomallei BPC006was produced by animal immunization withultrasonic, formaldehyde and heat treated antigen.3. TEM and immunofluorescence were used for observation of intracellular B.pseudomallei. With Giemsa staining and inverted microscope, we can have a step-by-stepimage about cell fusion and MNGC(multinucleated giant cells) formation. we detected theinvasion rate of B. pseudomallei and examined proinflammatory cytokines to assess thereliability of B. pseudomallei BP006-infecting model.Results1. We conducted the B. pseudomallei BPC006whole genome and assembled the twocomplete circle chromosomes with no gap and deposited them under accession numberCP003781and CP003782in GeneBank. There are many phage-related insertions, deletionsand mutantions. Then phylogenetic trees based on pan-genome and NCBI database wereconstructed.2. ELISA and agglutination titers of antisera were determined to be up to1:64000and1:128, respectively. The antisera were found to react with the intracellular B. pseudomalleispecifically.3. B. pseudomallei infected RAW264.7cell at MOI=100, induced cell fusion betweenhost cells and multinucleate giant cell formation, which showed a “doublet” change during1to24hours post infection.Conclusions1. This is the first genome sequence of a B. pseudomallei isolate in China. With theseresults we could establish a standard strain for further study on B. pseudomallei and formthe base of its diagnosis and prevention.2. We also have produced antisera of B. pseudomallei BPC006, an effective tool forimmunostaining, diagnosis and cellular microbiology.3. A stable B. pseudomallei-infected cell model is set up, laying the foundation ofstudy on interaction between the pathogen and its host cell. |
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