| The research of aba I/aba R quorum sensing system in Acinetobacter baumannii Acinetobacter baumannii is a Gram-negative coccobacillus,clinically important,opportunistic pathogen that causes a wide range of clinical infections.Acinetobacter baumannii can cause pneumonia,wound infection,urinary system infection,bacteremia and meningitis.With the increase of clinically drug-resistant Acinetobacter baumannii strains,novel therapies for controlling infections caused by resistant strains are one of the major challenges we currently face.Bacteria use virulence factors to interact with the host and subsequently adhere to and invade host cells to cause infection.Prevention of bacterial virulence factors constitutes an important alternative strategy for the control of bacterial infectious diseases,namely antiviral therapy.Previous studies have emphasized the importance of iron acquisition,transports,cell-associated pili,lipopolysaccharides,and outer membrane proteins such as omp A,omp33,and sur A1 for virulence,but studies of quorum sensing(QS)effects on pathogenicity in A.baumannii are limited.QS system is a mechanism widely existing in bacteria to coordinate community behavior through sensing bacterial population density,and participates in the regulation of various biological functions,playing an important role in the regulation of bacterial virulence and the pathogenesis process.The quorum sensing mechanism of A.baumannii is mediated by the typical lux I/lux R QS system(aba I/aba R)system,which is similar to the typical lux I/lux R system in other Gramnegative bacteria.Including the sensing protein Aba I(as an AHLs signaling molecule synthase)and the transcription regulatory protein Aba R(as an AHLs recognition receptor).The aba I gene encodes 183 amino acids.The aba R gene encodes 238 amino acids.Previous studies on A.baumannii had focused on the role of QS systems in drug resistance,biofilm formation,and motility,but studies of QS effects on pathogenicity in A.baumannii are poor.In this study,A.baumannii ATCC17978 was taken as the research object to study the virulence of the aba I/aba R quorum sensing system.The modified plasmid was transferred from the donor strain Escherichia coli S17-1 to the recipient strain A.baumannii ATCC 17978 by conjugative transfer method,and after two crossrecombination,a marker-free knockout strain was obtained.A single knockout strain and a double knockout strain of A.baumannii aba I and aba R were constructed by conjugation transfer method.Explore the biological functions of mutant and wild strains through a series of biological experiments.Transcriptome study of A.baumannii wild strains and knockout strains were conducted by using RNA-sequencing technology.The expression network of quorum sensing system was constructed by weighted coexpression network analysis(WGCNA),which combined omics,phenotypic and molecular data.The gene interaction network of A.baumannii QS system was constructed to screen out the gene information related to the quorum-sensing system and bacterial virulence of A.baumannii.Further clarify the global regulation of the QS system of A.baumannii.It laid the foundation for effective control of A.baumannii infection.Part I.A.baumannii quorum sensing aba I/aba R gene knockout The transformed plasmid was transferred from the donor bacterium Escherichia coli S17-1 to the recipient bacterium A.baumannii ATCC 17978 by a conjugation transfer method,and after two cross-recombinations,a label-free knockout strain was obtained.Using this method,single and double knockout strains of aba I and aba R genes of A.baumannii ATCC 17978 strain were successfully constructed.The knockout gene was successfully replenished and overexpressed by shuttle plasmid p ME6032.The receptor gene aba R of A.baumannii is difficult to be knocked out.In this study,this gene was knocked out for the first time by the traceless knockout method,and the success rate of obtaining gene deletion strains by this method was higher and the stability was stronger,laying a foundation for the subsequent study of quorum-sensing system.Part II Biological characteristics of A.baumannii wild and knockout strains Through a series of biological experiments to explore the biological functions of mutants and wild strains,we found that the aba I gene of A.baumannii affects the secretion of signal molecules,cell morphology,growth and reproduction,motility and biofilm formation ability of the bacteria,and the strain without the aba I gene showed no serum resistance and the virulence of the strain decreased significantly.aba R gene affects the secretion of signal molecules,motility and biofilm formation ability of bacteria,but the strain with aba R gene knockout still has serum resistance,showing increased virulence in mouse infection model.After deletion of aba I and aba R genes,signaling molecule secretion and motility were affected,no serum resistance was found,and virulence was significantly reduced.In conclusion,the quorum sensing system of A.baumannii is related to the pathogenicity of Acinetobacter baumannii,but the molecular mechanism of quorum sensing and pathogenicity of Acinetobacter baumannii is still unclear.In the third part,we will further study the molecular function of Acinetobacter baumannii’s quorum sensing system through transcriptomics.Part III Transcriptome study on quorum sensing system of A.baumannii Compared with WT,a total of 159 genes were differentially expressed in the Δaba I mutant,of which 126 genes were up-regulated and 33 genes were down-regulated.In the Δaba R mutant,324 genes were differentially expressed(211 up-regulated,113down-regulated).In the Δaba IR mutant,there are 123 differentially expressed genes(79 up-regulated and 44 down-regulated).The GO function analysis showed that the differential genes in the Δaba I mutant were mainly annotated in biological processes such as heterosexual substance metabolism,toxin metabolism/catabolism,and cellular components such as respiratory chain complex I.Enrichment of KEGG pathway showed that the differential genes of Δaba I mutants were mainly enriched in related pathways such as phenylalanine metabolism and oxidative phosphorylation.In theΔaba R mutant strain,the differential genes are mainly annotated in biological processes such as ribosome assembly and translation,cellular components such as cytoplasmic ribosomes,and and molecular functions such as the structure of ribosomes.Enrichment of KEGG pathway indicated that the differential genes of Δaba R mutants are mainly involved in the ribosomes,degradation of valine,leucine,and isoleucine.In the Δaba IR mutant strain,the differential genes are mainly annotated in biological processes such as metal ions,cellular components such as the periplasmic space surrounded by the outer membrane,and molecular functions such as copper ion binding.Enrichment of the KEGG pathway indicated that the differential genes of the Δaba IR mutant are mainly involved in the ABC transporter and other related pathways.RNA-seq analysis showed that the aba I gene may indirectly affect the virulence of bacteria by affecting the expression of key genes related to NADH dehydrogenase in the respiratory chain.Knockout of this gene leads to significant inhibition of strain energy production and transformation,which may lead to the bacterial virulence is reduced.The aba R gene may indirectly affect the virulence of bacteria by affecting the expression of key genes related to phenylacetic acid(PAA),branched chain amino acids(BCAAs),propionic acid catabolism,and ribosomal protein.After the aba R gene is knocked out,the expression of these related genes is significantly up-regulated,enhancing the virulence and immune escape ability of bacteria.The weighted co-expression network analysis showed that the genes related to the virulence of A.baumannii are mainly involved in lipid transport and metabolism,amino acid transport and metabolism,and energy metabolism.The genes AUO97_RS14360,AUO97_RS14365,AUO97_RS14370,AUO97_RS14375,AUO97_RS14380 enriched in the leucine degradation pathway may be closely related to the virulence of A.baumannii. |
