Biological Characteristics And Virulence Changes Of SlyA Gene Knockout Strain Of Serratia Marcescens

Serratia marcescens is an opportunistic pathogen with a wide range of hosts,causing septicemia,urinary tract infections,wound infections,and other diseases.In the silkworm breeding industry,S.marcescens can infect silkworms which leads to deadly septicemia,causing great losses to the production and economy.The slyA transcriptional regulator is a member of the Mar R family,and it has been reported to regulate virulence-related genes in different bacteria.However,whether the slyA is associated with the pathogenicity of S.marcescens has rarely been reported.In this study,the slyA gene knocked out mutant and its complemented strains were constructed using homologous recombinant technology and the virulence-related biological characteristics were detected.In addition,the molecular basis of slyA gene regulation of pathogenicity changes of S.marcescens was further explored.The following are the key research findings:1.The slyA gene was obtained using S.marcescens SCQ1 genomic DNA as a template.Bioinformatics analysis showed that the slyA gene is 354 bp long and encodes117 amino acids.The SlyA protein is water-soluble,has no transmembrane structure,contains no signal peptide,and is not glycosylated.The SlyA protein is predicted to contain a Mar R-type HTH structural domain.2.The deletion mutantΔslyA and the complementary strain CΔslyA were successfully constructed by homologous recombination technology.3.The biological characteristics of the SCQ1,ΔslyA,and CΔslyA were examined and analyzed.Compared to the wild-type strain SCQ1,theΔslyA showed no significant differences in protease production capacity and antibiotic susceptibility,while it showed significant differences in colony morphology,growth rate,lecithin production capacity,motility,biofilm formation capacity,hemolytic activity,and H₂O₂ stress tolerance.In terms of colony morphology,the prodigiosin production of theΔslyA was significantly weaker than the wild-type strain,since the colonies of theΔslyA and CΔslyA were light pink and orange,respectively.The growth rate of theΔslyA was significantly lower than the wild-type strain SCQ1 during the logarithmic growth period（4-22 h）while exceeding the SCQ1 during the stabilization period（34 h-96 h）,and reached the maximum OD₆₀₀ value of 1.097±0.021 at 58 h.However,the final cell density of theΔslyA was consistent with the wild-type strain SCQ1.The lecithin production ability of theΔslyA and the CΔslyA was stronger than the SCQ1,and the hydrolysis circles were increased by 1.5 and 1.25-fold.The motility of theΔslyA was almost lost and the swimming and swarming diameters of the SCQ1were 4.1 and 3.9-fold and 4.5 and3.7-fold of theΔslyA and the CΔslyA,respectively.The biofilm-forming ability of theΔslyA was significantly reduced compared with the wild-type strain SCQ1.The hemolytic activity assay revealed that theΔslyA completely lost the hemolytic activity,while the areas of hemolytic circles of the SCQ1 and CΔslyA were 0.37±0.07 and0.1227±0.0697.In the stress tolerance assay,the tolerances of high osmolarity,acidic and SDS stress conditions of theΔslyA and the CΔslyA were similar to the SCQ1,while theΔslyA showed higher H₂O₂ tolerance.4.TheΔslyA,CΔslyA,and the wild-type strain SCQ1 were used to infect healthy fifth instar silkworms by injection.The virulence test showed that the wild-type strain SCQ1,theΔslyA,and the CΔslyA were cause silkworms’death,and they caused red,gray,and brown symptoms.The LD₅₀ of theΔslyA and CΔslyA were reduced by 4.05and 1.32-fold,respectively,compared with the wild-type strain.The LT₅₀ of theΔslyA and the SCQ1 was 33.66±0.36 and 69.33±14.147 at 50 cfu/head and 30.68±0.058and 36.34±0.622 at 100 cfu/head,respectively.The survival rate of infected silkworms of theΔslyA was significantly lower than the wild-type strain and the survival rates ofΔslyA-infected silkworms were 20%and 3.33%at 100 cfu/head dose.The bacterial concentration in the hemolymph of freshly dead silkworms of the SCQ1 was twice higher than theΔslyA.In conclusion,the pathogenicity of theΔslyA was stronger than the wild-type strain SCQ1.According to the results of biological characteristics,several virulence-related genes were selected for q RT-PCR.The results showed that the expression of phospholipase A gene plh A,phospholipase A1 gene plh A1,and phospholipase C gene plh C were significantly up-regulated,while the expression of hemolysin-related gene shl A,major pili protein gene fim A,flagellum-related genes flh C and flh D,and biofilm maturation-related gene bsm B were significantly down-regulated in theΔslyA compared with the wild-type strain SCQ1.The expression of the major pili protein gene fim C in theΔslyA was down-regulated but not significantly.Combined with the test results of biological characteristics and q RT-PCR,the enhanced virulence of the mutantΔslyA may be caused by the high expression of lecithinase.However,how slyA regulates the expression of lecithinase in S.marcescens still needs further study.These results showed that the slyA was involved in regulating the growth,motility,biofilm formation,H₂O₂ stress tolerance,prodigiosin,hemolytic activity,and lecithinase production of the S.marcescens SCQ1.Meanwhile,theΔslyA showed enhanced pathogenicity which may caused by the increase in lecithinase production.Therefore,we speculate that the slyA gene not only regulates the synthesis of prodigiosin,but also plays important role in regulating the virulence of the S.marcescens SCQ1,and the regulatory mechanisms need to be further investigated.