Functionality And Regulatory Mechanisms Of Type Ⅳ Secretion System Of Acinetobacter Baumannii

Background and Objective:Acinetobacter baumannii is a Gram-negative bacterial pathogen that primarily causes infections in immunocompromised individuals,affecting the lungs,urinary tract,central nervous system,thoracic and abdominal cavities,thus posing a significant threat to critically ill patients.These bacteria normally are found in surgical rooms,and infections often result from contaminated medical devices.New isolates of A.baumannii are resistant to a wide range of antibiotics,including aminoglycosides,fluoroquinolones,and carbapenems.Multidrugresistant or even pan-drug-resistant strains are more frequently encountered in clinical settings,which made A.baumannii become a major nosocomial pathogen.A.baumannii acquires antibiotic resistance traits through mobile genetic elements such as plasmids,transposons,or integrons,among which conjugative plasmids play a major role.Given that the development cycle of new antibiotics over the past five decades has been far slower than the rate at which bacteria develop resistance to antibiotics,rational design of small molecule inhibitors targeting key components of the relatively conserved conjugative transfer machineries may offer an effective route to address the challenge in combating bacterial multidrug resistance.The large plasmid of A.baumannii has been predicted to encode several proteins homologous to the Dot/Icm proteins of Legionella and Coxiella species.Yet,the role of these Dot/Icm-like proteins in the biology of A.baumannii remains poorly understood.The findings in this project have laid the foundation for the development of small molecules that target crucial components of the conjugative machinery,which holds great promises in addressing the challenge posed by multidrug-resistant A.baumannii.Methods and Results:By using the HHpred homology detection and structure prediction algorithm,we identified 11 potential Dot/Icm homologs in the pAB3 plasmid of A.baumannii.Similar to dot/icm genes in L.pneumophila,these genes are clustered in two regions on the plasmid and their organization in each case is almost identical to the respective dot/icm gene sets of L.pneumophila.Analysis of mutants lacking each of these genes revealed that with the exception of dot C,deletion of which reduced the transfer efficiency,mutations in all other genes completely abolished the mobilization of pAB3 between Ab strains.This T4 SS also promotes the transfer SMPs between A.baumanni and other bacterial species.These results indicate that these Dot/Icm homologs are essential components of the conjugative machinery of A.baumannii.Plasmid dissemination via type Ⅳ secretion systems among bacteria is an energyexpensive process.We thus hypothesized that expression of genes coding for components of conjugation machineries is regulated in response to certain nutrients.To test this hypothesis,we constructed pdot D-gfp fusions to measure the potential inducing effects of various metabolites and found that a selection of metabolites,including succinate and fumarate induce the expression of the dot-like genes thereby improving the conjugation efficiency of the large plasmid pAB3.Results from experiments using fluorescence protein reporter,quantitative PCR indicated that several metabolic intermediates regulate the expression of not only dot/icm genes,also genes of the central carbon metabolism,such as phosphoenolpyruvate carboxykinase(pck G).Furthermore,based on a transcriptome-based investigation of A.baumannii,we found that the GacSA two-component system(TCS)controls the expression of both the dot/icm-like genes and genes involved energy metabolism.Using the XSTREME database,we identified several DNA elements shared by the promoter regions of the dot/icm-like genes and of several central carbon metabolism genes.Further experiments of electrophoretic mobility shift assay(EMSA)revealed that the response regulator Gac A binds to these promoters,establishing that these genes are regulated by the GacSA two-component system.Conclusion:In sum,this study reveals that the 11 Dot/Icm homologs encoded by the large plasmid pAB3 in A.baumannii are essential components of a T4 SS critical for plasmid dissemination.Furthermore,the TCS of GacSA coordinates the energy-consuming conjugation and provides energy for central carbon metabolism by positively regulating the transcription and expression of certain dot/icm genes and meatabolic genes.These findings suggest that targeting key regulatory knots or conjugation system may be a novel strategy to control multidrug-resistant A.baumannii infection.