Preliminary Investigation Of The Modulation Of T3SS By Spectinomycin In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a non-fermenting opportunistic pathogen causing numerous acute and chronic infections.As a human pathogen,it is both invasive and toxigenic,producing a series of virulence factors and colonising diverse host tissues where it may inflict localised tissue damage or disse minate,causing life-threatening systemic infections.The World Health Organization list of global pathogens that are classified as the greatest threat to the human health,lists P.aeruginosa as one of the most critical threats.Previous studies in the laboratory have found that a certain concentration of Spectinomycin(Spe)will affect the expression of type III secretion system(T3SS)in Pseudomonas aeruginosa.In order to explore the mechanism of Spe on T3 SS,we measured the growth of PAO1 and the transcription of exo S under different concentrations of Spe.It was found that when the concentrations of Spe were 15 μg/m L and 30 μg/m L,the effect on the growth of PAO1 was little and the activation of exo S was obvious.Therefore,these two concentrations were selected for follow-up experiment.In addition,this study found that the same concentration of Spe also activates the transcription of exo T and the secretion of Exo T and Exo S proteins.The study found that the activation effect of Spe on T3 SS is unrelated to the mode of action of antibiotics,the two-component system(TCS)gtr S-glt R,genes hpt B,PA1611,las I,rhl I,lads,gac A,tru A,PA4664.However,when the gene vfr is mutated,the activation of Spe disappears at the level of translation and we found Spe can activate the transcription of vfr.In CTX-vfr-flag,Spe also enhances the protein expression of Exo S and Vfr.In addition,when vfr is overexpressed in PAO1,the secretion of Exo S protein is also enhanced.The above experimental results all prove that Spe activates T3 SS expression through Vfr.But at the level of transcription,Spe still activate the transcription of exo S in Δvfr.FimV can regulate the expression of Vfr,but Spe can activate the expression of Exo S at both the transcription level and translation level in ΔfimV,which indicates that FimV has no concern with the activation of Spe.In order to further explore how Spe regulate T3 SS through Vfr,this study used CTX-vfr as a receptor to construct a transposable mutant library,from which 278 strains that regulate vfr expression were screened.The expression of vfr did not enhance after adding Spe in 37 strains.By detecting the secretion of Exo S protein of these 37 strains,as well as random PCR,sequencing,and comparison,were finally deter mined two gene PA4385 and PA5567.Whe we add Spe into medium,the expression of Vfr and Exo S unchanged in Tn PA4385 and Tn PA5567,but increased in the complementation of these two strains.The above experiments show that Spe may activates the expression of Vfr through PA4385 and PA5567 to enhance T3 SS secretion.This study deeply explored the mechanism of Spe effect and provided new ideas for exploring the antibiotics action on expression of virulence factors in Pseudomonas aeruginosa.