Study Of A Novel Toxin-antitoxin System That Contributes To Persister Formation In Pseudomonas Aeruginosa

Persister cells are dormant cells that are highly tolerant to environmental stresses,such as nutrient starvation,oxidative stress and antibiotics.Formation of persister cells is mainly due to phenotype switch.When the environmental stress is removed the dormant persister cells will resume growth.Persister cells might be a major cause of chronic and recurrent bacterial infections.In bacteria,toxin-antitoxin(TA)systems play important roles in persister formation.A TA system is composed of a stable toxin and a labile antitoxin.The toxin represses bacterial growth by inhibiting important bacterial physiological processes,such as DNA replication,transcription,protein synthesis,cell wall synthesis,cell division or reducing membrane potential.Normally,the function of a toxin is neutralized by its cognate antitoxin.Under environmental stress or stochastically,the antitoxin is degraded,which leads to activation of the toxin.Based on the natures of the antitoxins and their mechanisms of action,the TA systems are classified into 7types(I-VII).The type II TA systems are widespread in bacterial genomes.For example,at least 79 and 12 type II TA systems have been identified in Mycobacterium tuberculosis and E.coli,respectively.A typical type II TA system is composed of two genes in an operon that encode a toxin protein and an antitoxin protein.The activity of a type II toxin is inhibited by direct binding to its cognate antitoxin and the transcription of the TA gene operon is repressed by the antitoxin.Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes acute and chronic infections in immunocompromised patients.In P.aeruginosa,at least four pairs of type II TA systems have been shown to contribute to persister formation,including Par D/Par E,Hic A/Hic B,Rel E/Rel B and Hig B/Hig A.A bioinoformatic analysis revealed seven putative type II TA systems on the bacterial chromosome,namely PA14＿51010/PA14＿51020,PA14＿40220/PA14＿40210,PA14＿21710/PA14＿21720,PA14＿28790/PA14＿28780,PA14＿60050/PA14＿60040,PA14＿71340/PA14＿71330,PA14＿28120/PA14＿28130.Understanding the roles of the TA systems will shed light on the persister formation mechanisms of P.aeruginosa.In this study,we examined the roles of seven predicted type II TA systems in the persister formation of a P.aeruginosa wild type strain PA14.Overexpression of a toxin gene PA14＿51010 or deletion of the cognate antitoxin gene PA14＿51020increased the bacterial tolerance to antibiotics.Co-overexpression PA14＿51010 and PA14＿51020 or simultaneous deletion of the two genes resulted in wild type level survival rate.The two genes are located in the same operon that is repressed by PA14＿51020.We further demonstrated the interaction between PA14＿51010 and PA14＿51020.Sequence analysis revealed that PA14＿51010 contains a conserved RES domain.Overexpression of PA14＿51010 reduced the intracellular level of nicotinamide adenine dinucleotide(NAD~+).Mutation of the RES domain abolished the abilities of PA14＿51010 in reducing NAD~+level and promoting persister formation.In addition,overproduction of NAD~+by mutation in an nrt R gene counteracted the effect of PA14＿51010 overexpression in promoting persister formation.In combination,our results revealed a novel TA system that contributes to persister formation through reducing intracellular NAD~+level in P.aeruginosa.