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Effect in virulence of switching conserved homologous capsular polysaccharide genes from Klebsiella pneumoniae serotype K1 into K20.

ABSTRACT: The capsular polysaccharides in different serotypes of Klebsiella pneumoniae (KP) coded by the (CPS) gene cluster are characterized by a conserved and a hyper-variable region. We performed a virulence study by switching genes in the highly conserved region of the CPS cluster between strains. Six genes in the CPS conserved region in serotype K20, including galF, acidPPc, wzi, wza, wzb and wzc, were knocked out and replaced by the homologous genes from serotype K1. Compared to the parental K20 strain, the mutants showed a decline in lethality (LD50) in mice from 10-fold to > 105-fold and were categorized in terms of the effect on virulence as low (L) for galF and acidPPC, moderate (M) for wzi, and high (H) for wza, wzb and wzc. Although substituting the acidPPC gene from K1 for acidPPC in the K20 strain fully restored virulence, substitution with the wzi, wza, wzb or wzc homologs from K1 did not. The restoration with wzi from K1 led to a partial restoration of virulence, with the LD50 in mice changing from 104 to 103 CFU. For the wza, wzb and wzc genes, Complementation of K20 wza, wzb and wzc from K1 resulted in varied degrees of lethality in mice. Variable improvement in serum killing and phagocytosis was observed when the knockout mutants were compared with the gene-switched strains. In conclusion, homologous genes for capsule synthesis failed to exhibit the same functionality when switched between serotypes and virulence was decreased in different degree in according to the genes' homology.

SUBMITTER: Lin CL

PROVIDER: S-EPMC5538342 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Effect in virulence of switching conserved homologous capsular polysaccharide genes from Klebsiella pneumoniae serotype K1 into K20.

Lin Chii-Lan CL Chen Fei-Hsu FH Huang Li-Yueh LY Chang Jen-Chang JC Chen Jiun-Han JH Tsai Yu-Kuo YK Chang Feng-Yee FY Lin Jung-Chung JC Siu L Kristopher LK

Virulence 20160826 5

The capsular polysaccharides in different serotypes of Klebsiella pneumoniae (KP) coded by the (CPS) gene cluster are characterized by a conserved and a hyper-variable region. We performed a virulence study by switching genes in the highly conserved region of the CPS cluster between strains. Six genes in the CPS conserved region in serotype K20, including galF, acidPPc, wzi, wza, wzb and wzc, were knocked out and replaced by the homologous genes from serotype K1. Compared to the parental K20 str ...[more]

PMID: 27565716

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Project description:The genomic organization of the chromosomal cps region that is responsible for capsular polysaccharide synthesis in Klebsiella pneumoniae Chedid (O1:K2) was investigated. Deletion analyses and Southern hybridization studies suggested that the central region of the cloned 29-kb BamHI fragment is indispensable for K2 capsular polysaccharide synthesis. The 24,329-bp nucleotide sequence of the Klebsiella cps region was determined and deposited in the EMBL and GenBank databases through DDBJ and assigned accession number D21242. Nineteen possible open reading frames (ORFs) were identified in the sequenced area. Among them, 13 ORFs are very close to each other. Six of the 19 ORFs show considerable nucleotide sequence similarities to Salmonella typhimurium cpsG, cpsB, rfbP, and orf2.8, Escherichia coli gnd, and Haemophilus influenzae bexD, respectively. Moreover, the deduced amino acid sequence of the ORF10 product demonstrated a highly hydrophobic profile and showed putative membrane topology similarity to Rickettsia prowazekii ATP/ADP translocase. Nucleotide sequence similar to the sigma 54-dependent promoter, as well as the usual -35 and -10 sequences, were identified just upstream of ORF3, which is the first ORF in the polycistronic structure. Furthermore, a sequence (GGGCGGTAGCGT) found just downstream of the sigma 54-dependent promoter-like sequence was generally conserved among gene clusters implicated in cell surface polysaccharide synthesis, such as Salmonella rfb and viaB and E. coli kpsMT and rfaQPG. A possible transcriptional terminator with a hairpin loop structure found just downstream of ORF15 that is a homolog of E. coli gnd. K2 capsular polsaccharide biosynthesis in E. coli K-12 depends on cpsB (mannose-1-phosphate guanyltransferase gene), and Klebsiella cpsB, found in the downstream region of the polycistronic structure, was able to complement cpsB of E. coli. Results of transposon insertion and promoter-cloning analyses were consistent with the results of nucleotide sequence analysis.

Dataset Information

Effect in virulence of switching conserved homologous capsular polysaccharide genes from Klebsiella pneumoniae serotype K1 into K20.

Publications

Effect in virulence of switching conserved homologous capsular polysaccharide genes from Klebsiella pneumoniae serotype K1 into K20.

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