Acquisition of T6SS Effector TseL Contributes to the Emerging of Novel Epidemic Strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen with multiple strategies to interact with other microbes and host cells, gaining fitness in complicated infection sites. The contact-dependent type VI secretion system (T6SS) is one critical secretion apparatus involved in both interbacterial competition and pathogenesis. To date, only limited numbers of T6SS-effectors have been clearly characterized in P. aeruginosa laboratory strains, and the importance of T6SS diversity in the evolution of clinical P. aeruginosa remains unclear. Recently, we characterized a P. aeruginosa clinical strain LYSZa7 from a COVID-19 patient, which adopted complex genetic adaptations toward chronic infections. Bioinformatic analysis has revealed a putative type VI secretion system (T6SS) dependent lipase effector in LYSZa7, which is a homologue of TseL in Vibrio cholerae and is widely distributed in pathogens. We experimentally validated that this TseL homologue belongs to the Tle2, a subfamily of T6SS-lipase effectors; thereby, we name this effector TseL (TseLPA in this work). Further, we showed the lipase-dependent bacterial toxicity of TseLPA, which primarily targets bacterial periplasm. The toxicity of TseLPA can be neutralized by two immunity proteins, TsiP1 and TsiP2, which are encoded upstream of tseL. In addition, we proved this TseLPA contributes to bacterial pathogenesis by promoting bacterial internalization into host cells. Our study suggests that clinical bacterial strains employ a diversified group of T6SS effectors for interbacterial competition and might contribute to emerging of new epidemic clonal lineages. IMPORTANCE Pseudomonas aeruginosa is one predominant pathogen that causes hospital-acquired infections and is one of the commonest coinfecting bacteria in immunocompromised patients and chronic wounds. This bacterium harbors a diverse accessory genome with a high frequency of gene recombination, rendering its population highly heterogeneous. Numerous Pa lineages coexist in the biofilm, where successful epidemic clonal lineage or strain-specific type commonly acquires genes to increase its fitness over the other organisms. Current studies of Pa genomic diversity commonly focused on antibiotic resistant genes and novel phages, overlooking the contribution of type VI secretion system (T6SS). We characterized a Pa clinical strain LYSZa7 from a COVID-19 patient, which adopted complex genetic adaptations toward chronic infections. We report, in this study, a novel T6SS-lipase effector that is broadly distributed in Pa clinical isolates and other predominant pathogens. The study suggests that hospital transmission may raise the emergence of new epidemic clonal lineages with specified T6SS effectors.

Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system.

Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections. The strong capacity of P. aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure. The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts. Elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs. In this study, a high-throughput transposon-insertion site sequencing (Tn-seq) approach was employed to identify novel P. aeruginosa biofilm genetic determinants. When analyzing the novel biofilm regulatory genes, we found that the cell division factor ZapE (PA4438) controls the P. aeruginosa pqs quorum sensing system. The ∆zapE mutant lost fitness against the wild-type PAO1 strain in biofilms and its production of 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) had been reduced. Further biochemical analysis showed that ZapE interacts with PqsH, which encodes the synthase that converts 2-heptyl-4-quinolone (HHQ) to PQS. In addition, site-directed mutagenesis of the ATPase active site of ZapE (K72A) abolished the positive regulation of ZapE on PQS signaling. As ZapE is highly conserved among the Pseudomonas group, our study suggests that it is a potential drug target for the control of Pseudomonas infections.