Research On The Regulation Mechinism Of RcsB And BaeR Transcription Factors On The Type ? Secretion System In Extraintestinal Pathogenic Escherichia Coli

Microbe have envovled a remarkable series of specific and sophisticated macromolecular device that can export various substrate molecules across the bacterial cell membrane,including protein,DNA,virulence factors and so on,and facilitate bacteria colonization and growth,survival,infection and development of disease in detrimental milleu.Recently,considerable advance has been made towards revealing the molecular and molecular mechanisms of types I-? secretion systems in Gram-negative bacteria.These studies have strengthened our understanding of the complicated mechanisms.Type ? secretion system is a cell membrane spanning device that translocates effector proteins into eukaryotic and prokaryotic cells,and plays a vital role in pathogenesis.Different functions of Type ? secretion system?T6SS?have been uncovered,including bacterial competition,qurom sensing,cytotoxicity,colonization in host,virulence,survival in host cells and so on.At present,considerable progress has been made in the functions of T6SS,but there is still much to be discovered in the regulation mechnisms of T6SS.In our lab,we identified a functional T6SS in isolate porcine extraintestinal pathogenic E.coli?Ex PEC?PCN033,and found that T6SS of Ex PEC PCN033 were involved in interaction with competing bacteria or host cells,bacterial competition,and pathogenesis.Concrete research are as follows:1. Construction and transcriptome analysis of mutant Rcs FS2DM3QPolymyxin B is an antimicrobial peptide produced by Panibacillus polymyxa,which is highly effective against E.coli.We treated extra-intestinal pathogenic Escherichia coli?Ex PEC?PCN033 and nonpathogenic strain of E.coli K12 strain W3110?devoid of T6SS?with polymyxin B.Growth kinetics curve indicated that Porcine Ex PEC PCN033 is more tolerant than E.coli W3110 to polymyxin.Also we found that intracellular ROS levels were much higher in PMB treated cells.Rcs cascade is one of bacterial envelope stress response systems.The outer membrane lipoprotein Rcs F is an essential element of the Rcs signal transduction system.It is known that most of the stress signals that activate the Rcs cascade require Rcs F protein.The amino acid serine at position 2 determines retention of Rcs F in inner-membrane,and amino acid at position 3 methionine determines the retention efficiency of lipoprotein Rcs F.We constructed mutant Rcs FS2DM3Q by site-directed mutagenesis,Serine at position 2 was mutated to aspartic,methionine at position 3was mutated to glntamine?or Glu,Asp?produce 100%strongest retention efficiency in vitro system,and activate the Rcs signal transduction system.Comparing to wt PCN033,mutant Rcs FS2DM3Q showed increased tolerance when bacteria were exposed to different concentration of polymyxin B.The q RT-PCR assays showed that expression levels of Rcs Cascade were strongly induced.Comparing to wild type PCN033,mutant Rcs FS2DM3Q characterizes in increased adherence and invasion capacity,intracellular replication ability,biofilm formation ability,and reduced motility.We conducted the transcriptome sequencing analysis of wt PCN033 and mutant Rcs FS2DM3Q.RNA-seq results showed that there were 1493 differentially expressed genes?log2[foldchange]>1,P<0.001?.1148 genes upregulated,and 345genes downregulated.Further analysis showed that most of genes in T6SS cluster from PCN033 were upregulated,and the RNA-seq data were verified by q RT-PCR?2. Exploration of Regulation mechanism of T6SS by Rcs CascadeWe identified several potential transcriptional regulators that might directly bind T6SS gene cluster by analysis of RNA-seq data and relevant studies.And we predicted the promoter region of T6SS gene cluster by bioinformatics analysis.An array of protein-DNA binding assay was performed in vitro.We obtained five transcriptional regulators that directly bound to promoter DNA of T6SS.Surface plasma membrane resonance assay further verified the EMSA results.The binding sites of Rcs B and Bae R were identified by DNase I footprinting assay.We constructed?Rcs B mutant strain.And deletion of rcs B gene,genes of T6SS were significantly downregulated.In conclude,response regulator Rcs B and Bae R positively regulated T6SS of Ex PEC PCN033.Mutant strain?rcs B was more sensitive to PMB.Growth characterization assayrevealed that deletion of rcs B/T6SS core structural elements hcp/vgr G remarkably inhibited the growth of bacteria cells under stress.Through a series of phenotypic analysis,including survival rates,intracellular ROSs measurement,inductively coupled plasmon resonance atomic absorption spectrometry?ICP-MS?indicated that activation of T6SS via Rcs B facilitated the scavenge of ROSs and better bacterial survival.Levels of intracellular ROSs in mutant Rcs FS2DM3Q was the lowest among all strains,and activation of T6SS in Ex PEC PCN033 by Rcs system contributes to elimination of ROS induced by polymyxin B,and facilitate bacterial tolerance.Taken together,Rcs system was activated by detecting extracellular polymyxin B,accompanied by production and accumulation of reactive oxygen species which caused damage and cell death,and T6SS of Ex PEC was induced by Rcs system via Rcs B for elimination of ROSs,bacterial survival,adaptability and pathogenicity.3. Exploration of Regulation mechanism of T6SS by TCS Bae SRBased on the known stress signals that can activate the two-component regulation system Bae S/Bae R,we chose stresses indole,ciprofloxacin,zinc ions,and sodium tungstate etc.We treated Ex PEC PCN033 and W3110 with different concentrations of Zn SO₄.There is no difference in PCN033 with or without zinc ions.The W3110 strain showed significantly slower growth rate comparing to untreated cells.The Ex PEC PCN033 is more tolerant than W3110.Moreover,we found that only the stress singal zinc ions can activate the Bae SR signal transduction system and the type ? secretion system at the same time.Deletion of bae R/bae SR decreased zinc resistance of bacteria.We constructed two single gene deletion mutant?Bae R and?Bae S,one double gene deletion mutant?Bae SR.The q RT-PCR and western blot assays desmonstrated that response regulator Bae R positively regulated expression of T6SS in Ex PEC PCN033.The growth rate of?Bae R and?Bae S mutant strains were significant inhibited under stress.Moreover,T6SS mutant?hcp1/hcp2/hcp3 is more sensitive than wild type after exposure to external zinc,and complementation of hcp1/bae R largely restored growth defect.Our study uncovers a new regulation mechanism of Bae SR system in response to metal stress.In conclusion,it reveals that Bae R-regulated T6SS is critical for bacteria survival under toxic zinc condition.T6SS contributes to zinc stress resistance in a Bae SR system-dependent manner.