The Pseudomonas Aeruginosa Citrate Synthase GltA Influences Antibiotic Tolerance And The Type Ⅲ Secretion System Through The Stringent Response

Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen.It is widely distributed in environment and mainly causes acute or chronic infections in immunocompromised people.Antibiotics are the main method for clinical treatment of infections diseases caused by bacteria.P.aeruginosa infections are difficult to cure and because of the high-level antibiotic resistance.There are three main resistance mechanisms of P.aeruginosa,namely inherent resistance mechanism,acquired resistance mechanism and adaptive resistance mechanism.When bacteria are treated with lethal doses of antibiotics,most of the bacteria will die and a small population will form persistent cells and survive.In our experiments,we found that mutation in the citrate synthase gene gltA can cause bacteria to develop tolerance to a variety of antibiotics.At the same time,we found that the increase of antibiotic tolerance in the gltA mutant may be related to the decrease in antibiotic entry efficiency and membrane permeability.It has been reported that defect in the gltA gene results in the defecienty of carbon skeleton for the systhesis of glutamate.Supplementation of glutamate in the culture medium restored the growth and antibiotic tolerance of the gltA mutant.We speculated that the deficiency in glutamate might trigger stringent response that increases antibiotic tolerance in the gltA mutant..In P.aeruginosa,RelA and SpoT control the stringent response through regulating the intracellular homeosdtasis of ppGpp.By detecting the expression levels of sodB,rpoS,relA and H₂O₂ tolerance,we determined that the stringent response was activated in mutation in the gltA mutant.Deletion of relA in the gltA mutant could restore the antibiotic susceptibility.P.aeruginosa secrets a variety of virulence factors.The type Ⅲ secretion system（T3SS）is one of the most important virulence factors that contriobutes to bacterial cytotoxicity.The expression of T3SS gene is regulated by the master regulator ExsA.The exsA gene is located at the end of the exsC-exsE-exsB-exsA operon.The transcription of exsA is driven by the adjacent exsA promoter(P_exsA)and the exsC promoter(P_exsC),which are regulated by cAMP-Vfr and ExsA,respectively.We found that mutation in gltA increased expression of the T3SS genes and bacterial cytotoxicity.Supplementation of glutamate restored the expression of the T3SS genes and the bacterial cytotoxicity of the gltA mutant.We then found that the upregulation of the T3SS genes was due to an increase in the intracellular cAMP level,which upregulated exsA through Vfr.We futher demonstated that the increase of cAMP level was due to the activation of the stringent response in the gltA mutant.In summary,our results demonstrated that mutation in the citrate synthase gene gltA can increase the expression of the T3SS genes and bacterial tolerance to antibiotics by activating the stringent response.