| Summary: | 碩士 === 國防醫學院 === 微生物及免疫學研究所 === 102 === Capsular polysaccharide is one of the most important virulent factors in K. pneumoniae. Previous studies indicate that CPS gene clusters of diverse K-serotype K. pneumoniae strains contain six conserved genes with the order of galF, acidPPc, wzi, wza, wzb and wzc at 5’ end. Other study indicated that Wzi involved in attachment of the repeat unit of capsular polysaccharide to the outer membrane. As the uniqueness of wzi to their capsular serotypes in K. pneumoniae, serotype-specific primer from wzi could be designed according to their K serotypes. Further analysis showed that the amino acid sequence of Wzi in several K-serotypes, varies with each K-serotype. The interplay between these proteins and capsular polysaccharide as well as its virulence could be further explored.
Reference to the capsule (CPS) gene clusters in group 1 E. coli K30 serotype, whose infrastructure is similar to K20 serotype of K. pneumoniae, give us some hints about gene function of individual genes in K20 CPS cluster. Its actual virulent effect in K. pneumoniae caused by these individual genes is not still familiar.
In this study, individual knockout of the above 6 genes from a highly virulent K. pneumonia K20 serotype strain were performed. wzi gene exchanged from K20 to K1 would also be attempted. Serotype change of each successful clones would be carried out by serum agglutination. Virulence of each mutant would be assessed through serum killing, neutrophil phagocytosis and mouse lethality assay.
Comparison to the parental strain (mucoid, serum-sensitive, and phagocytosis-resistant, LD50=102 CFU), both LD50 of galF and acidPPc mutants; wzi mutant and wza, wzb and wzc mutants decreased 10 folds, 102 folds and more than 105 folds correspondingly.
For low virulent gene- galF and acidPPc, there were no obvious change in its colony morphology, positive agglutination in K20 serum and sensitive in phagocytosis among galF and acidPPc mutants. Lack of making glucose by galF would not change serum killing. Only acidPPc mutant become serum resistant in serum killing implicating that AcidPPc affect LPS function and decrease efficiency of serum killing.
High virulent genes- wza, wzb or wzc, wza, wzb or wzc mutants showed smaller colonies, negative agglutination in K20 serum, sensitive in serum killing and highly sensitive in phagocytosis were demonstrated. Without capsule repeat unit assembling protein and transporter for synthesis may be account for highly susceptible in phagocytosis.
Moderate virulent gene-wzi, wzi mutant was positive in K20 serum agglutination, more susceptible in serum killing and susceptible in phagocytosis. As its capsule become less firm, a tough pellet in centrifugal test and phagocytosis-resistance were easily obtained. Loosing ability for repeat units to anchor tightly in outer membrane leading a loose capsule was attributed to the above results. After complementation K1 wzi to wzi mutant, weak K20 serum agglutination was examined, anti-serum killing and anti- phagocytosis was comparable to that in parental strain. This may be explained by fixing the repeat unit assembling in capsule resulting to no more defective structure in capsule.
In conclusion, the conserved genes of capsule gene cluster in K. pneumoniae have impact in virulence in mice model. The intensity of change in colony morphology, serum agglutination, anti-serum killing and anti-phagocytosis varies among individual genes in this CPS cluster. Relatively, wzi could cause the least effect in affecting serum specificity.
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