Functional Study Of CapD And Wzy Genes In Bacterial Capsular Polysaccharide Synthesis And Pathogenicity In Elizabethkingia Miricola From Frog

Elizabethkingia miricola(EMi)is a multidrug-resistant opportunistic pathogen that can infect humans and frogs.The frog meningitis-like infectious disease caused by EMi,commonly known as"wryneck disease",is one of the most serious diseases affecting frogs industries in China.However,the pathogenic mechanism and related virulence factors of this bacterium are still unclear,which limits the development of disease prevention and control measures,as well as the development of related drugs and vaccines.Capsular polysaccharide(CPS)is an important virulence factor in bacteria and plays a crucial role in bacterial resistance to host immunity.In previous work,the whole genome of EMi strain FL160902 was determined.In this study,we focused on the capD and wzy genes which are involved in CPS synthesis in EMi,and used homologous recombination to construct capD or wzy gene deletion strain to investigate their biological characteristics and pathogenicity.The main results and conclusions are as follows:1.We analyzed the structure of the EMi CPS synthesis gene cluster and identified the polysaccharide synthesis protein gene capD and the transferases gene wzy in EMi FL160902,and constructed single deletion strain for both genes.The results showed that the EMi CPS synthesis gene clusters were highly conserved at both ends,but the middle region showed high diversity.The conserved region of the gene cluster includes characteristic genes such as wza,wzc and capD,while the variable region includes serotype-specific genes such as wzx and wzy.By homologous recombination,replacing the target gene with the spectinomycin resistance gene,the capD gene deletion strain(ΔcapD)and the wzy gene deletion strain(Δwzy)were constructed,which provided experimental materials for the subsequent study of the biological characteristics and pathogenicity of the strains.2.Deletion of capD or wzy gene affected EMi capsule and biofilm formation,and decreased strain tolerance.To investigate the biological functions of the capD and wzy genes,studies were conducted on the growth characteristics,capsule/biofilm formation,self-aggregation/anti-desiccation/anti-oxidation abilities of the△capD and△wzy strains.The results showed that the growth ability of the mutant strains were not affected,but capsule formation was hindered;biofilm formation was significantly increased by 82.9%and 145.7%in the△capD and△wzy strains,respectively(P<0.001),and their anti-desiccation/anti-oxidation ability were weakened to varying degrees.The self-aggregation ability was enhanced in△capD,whereas it was reduced in△wzy.3.Deletion of capD or wzy gene reduced EMi anti-serum killing activity and weakened pathogenicity to the host.To investigate the effects of the capD and wzy genes on the pathogenicity of EMi,studies were conducted on the anti-serum killing activity,cell adhesion and anti-phagocytosis assays of△capD and△wzy,as well as their pathogenicity to frogs.The results showed that(1)the survival ability of the mutant strains in serum was significantly decreased(P<0.05):at the 90%serum concentration,the survival rate of△capD tended towards 0,while that of△wzy decreased to 19.65%,and that of the wild-type strain was 33.03%.(2)The adhesion ability of the mutant strains to EPC epithelial cells and b End.3 brain microvascular endothelial cells was significantly increased(P<0.05),while their antiphagocytic ability against RAW264.7 macrophage cells was significantly reduced(P<0.001).In the artificial infection experiment in black-spotted frogs showed that,(3)after 5 days of infection,the average bacterial load in the liver,spleen,kidney,and brain tissues of the wild-type strain was significantly higher than that of the two deletion strains(P<0.001).The average bacterial load in these four tissues of the wild-type strain was 1.54×10~4～5.89×10~5 copies/mg which was significantly higher than the average bacterial load in the same tissues of the two deletion strains(2.80×10~2～1.03×10~3 copies/mg,P<0.001).(4)Within 14 days of infection,the mortality rates of theΔcapD andΔwzy in frogs were 30%and 40%,respectively,which were significantly lower than the 75%mortality rate of the wild-type strain(P<0.05).(5)Histopathological observations showed that the degree of tissue damage was milder in frogs infected with the deletion strains.In summary,the deletion of the capD or wzy gene affect the EMi bacterial capsule formation,reduces its tolerance,weakens bacterial immune escape ability,and lowers its pathogenicity to the host.This provides an important reference for further investigate the pathogenic mechanism of EMi and finding effective strategies to treat infections caused by this bacterium.