| Pseudomonas aeruginosa is an opportunistic gram-negative bacterium that causes acute-chronic infections in humans.An obvious feature of P.aeruginosa is displaying resistance to various antibiotics.The type III secretion system(T3SS)plays an important role in P.aeruginosa acute infection.In this study,we constructed a transposon mutagenesis library and identified a set of mutants with altered exo S expression.Among them,we focused on studying the role of CysB and its regulatory mechanism.CysB is required for cysteine biosynthesis.Our data showed that the expression of exo S is strongly reduced in the cysB deletion mutant compared to that in wild-type PAO1 strain.On the other hand,CysB affects the activity of other virulence factors.Mutation of cysB reduced bacterial motility,increased exopolysaccharides production and biofilm formation.In the pneumonia model experiment,deletion of cysB decreased P.aeruginosa pathogenicity.To further investigate the function and the regulatory mechanism of CysB,we performed RNA-seq assays to gain the CysB-regulated genes.RNA-seq results showed that CysB regulates 280 genes.Importantly,the expression of T3SS-related genes was drastically reduced in cysB mutant compared to that in wild-type parent.Further experiments demonstrated that CysB impaired T3 SS expression and bacterial pathogenicity through regulating Ret S.Moreover,we solved the crystal structure of the CysB protein,which is similar to other Lys R superfamily proteins.Structural superposition revealed one possible DNA-binding model for CysB.Structural comparison revealed great flexibility of CysB RD domain,which may play important role in bending and transcriptional regulation of target DNA.In conclusion,we firstly found that CysB is closely related to T3 SS and bacterial pathogenicity.These findings extand our understanding of T3 SS complex regulatory network.Moreover,the crystal structure of CysB provides new insights for studying the function of its homologs in other bacterial species. |
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