| Cronobacter sakazakii(C.sakazakii)belongs to the family Ente·obacteriaceae,Cronobacter.C.sakazakii is a gram-negative,peritrichous,motile,non-spore,facultative anaerobic bacteria.It is an opportunistic pathogen that can survive in intestines of human and animal.C.sakazakii is widely found in the environment and food,the main ways of infection are environmental exposure and food intake.C.sakazakii,can infect human,especially for low-birth-weight neonates,infants,immunocompromised adults and old people.However,the pathogenic mechanism and important virulence factors of this bacterium are not clear.Therefore,this study aims to explore the mechanism of virulence of C.sakazakii at the gene and protein levels.In this study comparative proteomics was used to screen virulence-related proteins in C.sakazakii.First,38 strains of C.sakazakii were classified by MLST.The results showed that 38 strains were distributed in 17 ST types.According to the classification and the pre-laboratory toxicity results,two strains of C.sakazakii(high virulent strain SAKA80220 and low virulence strain SAKA80221)belonging to ST327 type but with significant virulence differences were selected for comparison proteomic analysis.A total of 2319 proteins were identified,of which 2203 proteins were accurately quantified.In this study,differential protein screening was performed with abundance ratio≥3,≤0.33 and P<0.05.A total of 210 proteins with significant differential expression were screened,among which 67 proteins with high expression and 143 proteins with low expression in the high virulent strain compared to the low virulent strain.The differential proteins were analyzed by metabolic pathw-ays.It was found that the high-expression proteins were mainly related to the synthesis and assembly of flagella,ribosome synthesis,lipopolysaccharide synthesis and transport,energy metabolism and so on.The low-expression proteins are mainly associated with the synthesis of cell membranes and arginine in C.sakazakii.Real-time quantitative PCR was used to verify whether the transcription level was consistent with the translation level of 11 genes.The results showed that the genes expression basically consistent both at the protein level and mRNA level.In detail,the expression of nine genes was consistent at the transcriptional and translational levels;the other two genes expressed high levels at the translational level but did not show significant up-regulated expression at the transcriptional level.According to the related literatures,virulence-related genes nlpD,adh and the membrane-forming gene deoB of C.sakazakii was selected,and gene knockout techiniques were used to study their functions.The sequences of the three genes were obtained in the NCBI database,primers were designed to amplify the upstream and downstream homology arms,and the upstream and downstream homology arms of each gene were ligated into the deletion vector with the kanamycin gene sequence,ordinary PCR and sequencing was employed to verify deletion vector,and the results show that the deletion vector was successfully constructed.The successfully constructed vector was linearized and transferred into wild-type bacteria containing pKD46,and the knockout mutant was screened.After multiple rounds of screening,no knockout mutant stra:in was obtained,suggesting these three genes may play important roles in C.sakazakii,and their deletion may lead to death of the strain.This study lays a foundation for futher understanding the virulence mechanism of C sakazakii,and provideds a reference for control of the pathogenic bacterium and decrease its pollution of food and oil. |
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