Function Of Quorum Sensing-I Involved In Escherichia Coli

Pathogenic Escherichia coli are important pathogens for human being and animals, which also cause significant economic losses in animal husbandry industry worldwide. The virulence factors of E. coli pathogens lead to the pathegenesis, and interact with the host cells together. Virulence factors in vivo are strictly regulated, and Quorum Sensing system is actively involved in the regulation process. In this study, the function of Quorum Sensing-I involved in pathogenic E. coli was explored.During bacteria growth, the bacteria can utilize Quorum Sensing system to sense cell population density in surrounding environment, therefore to regulate gene expression. This regulation mechanism is called Quorum Sensing, while the signaling molecules in Quorum Sensing are known as autoinducer. In Quorum Sensing II system, luxS and pfs play significant roles in the synthesis of AI-2. In QS-I, E. coli encodes a single LuxR homolog named SdiA, but does not express the LuxI homo log, the acyl-homoserine lactone (AHL) synthase, which produces AI-1. In the presence of exogenous AHLs, a significant proportion of SdiA, the AHL receptor, is expressed in a folded, soluble form, while it is expressed in nonfunctional, insoluble inclusion bodies in the absence of AI-1. Thus, E. coli can regulate its virulence gene expression in response to the population density of other AHL-positive bacteria. These responses include cell division, antibiotic resistance, motility, and the activity of other QS systems. In previous study, overexpression of SdiA, addition with outsourced AHLs, or construction of endogenous AHLs were used to explore the function of QS-I involved in E. coli, like regulation of cell division, antibiotic resistance, motility, and activity of other QS system. However, isolation of AHL positive strain from animal gastrointestinal tract was tried and always failed, therefore hinder the further study of QS-I function involved in E. coli. In cattle rumen, the AHLs positive bacteria were also failed to screened; however, AHLs signals have been detected in rumen fluids, although the specific bacteria that produce these signaling molecules are unknown. These acyl-homoserine lactones are thought to regulate the gene expression and thus the passage through and survival within the bovine gastrointestinal tract of foodborne pathogens present in cattle, including the human pathogen E. coli O157:H7, which attracts more attention from researchers. However, the specific bacteria that produce these signaling molecules are unknown. Based on the data above and proposition of presence of AHLs positive strains in cattle rumen, we developed methods with which to concentrate rumen fluids and screen the contents for bacteria that produce AHLs. We isolated from cattle rumen fluids a Pseudomonas aeruginosa strain, designated as YZ1, and characterize its AHLs. The interaction between YZ1and E. coli through QS was also examined. JZA1reportor strain contains lacZ report gene, which could activate β-galactosidase actitity under AHLs; therefore JZA1was chosen to quantify YZ1AHLs production, while the maximal production was reached after6h growth.16srDNA method was used to identify YZlas P. aeruginosa. CV026was used to avoid false positive results in screening by active purple color in AHLs. Short or long-side chain AHLs in YZ1was also detected by pSB01/pSB1142biosensors. Mass spectrometry of YZ1culture supernatants indicated the presence of at least three specific classes of acyl-homoserine lactones, C4-HSL、 C8-HSL,3-oxo-C12-HSL. Transformation of E. coli O157:H7with the P. aeruginosa YZ1genes, lasl and rhlI, encoding proteins that produce short-and long-side chain acyl-homoserine lactones conferred upon E. coli the ability to synthesize acyl-homoserine lactones and affected E. coli gene expression. AHL receptor SdiA was up-regulated by1.7folds, motility was reduced by25%, while acid tolerance was increased. These phenotypes were consistent with6folds decrease of fliC and2folds up-regulate of gadA. These data represent the first isolation and characterization of an acyl-homoserine lactone synthase-positive bacterium from cattle rumen, and pointed out the interaction between AHLs positive strain and E. coli in cattle rumen.In previous study, we applied the improved screen method to isolate AHLs positive P. aeruginosa strain YZ1in cattle rumen by report strain JZA1, which would be helpful in further study in relationship between E. coli QS system and exogenous AHLs. Isolation of AHL positive strain in pig intestine always failed, which hinder further understanding of QS-I function in regulation E. coli virulence in pig intestine. We isolated from pig intestine a Aeromonas hydrophilia strain, designated as YZ2, by improved method. The interaction between YZ2and E. coli through QS was also examined. JZA1reportor strain contains lacZ report gene, which could activate P-galactosidase actitity under AHLs; therefore JZA1was chosen to quantify YZ2AHLs production.16srDNA method was used to identify YZ2as A. hydrophilia. CV026was used to avoid false positive results in screening by active purple color in AHLs. Short or long-side chain AHLs in YZ2was also detected by pSB01/pSB1142biosensors. Mass spectrometry of YZ2culture supernatants indicated the presence of C4-HSL. Transformation of E. coli107/86with the A. hydrophilia YZ2genes, ahyl, encoding proteins that produce short-side chain AHLs conferred upon E. coli the ability to synthesize AHLs and affected E. coli gene expression. AHL receptor SdiA was up-regulated by1.8folds, motility was reduced by25%, while acid tolerance was increased by3folds. These phenotypes were consistent with5folds decrease of fliC and4folds up-regulate of gadA. These data represent the first isolation and characterization of an AHLs synthase-positive bacterium from pig, and pointed out the interaction between AHLs positive strain and E. coli in pig intestine.As we confirmed that AHL positive strain was present in pig intestine and regulates E. coli virulence through AHLs, to investigate the effect of the QS-I system on the expression of virulence factors in E. coli, Shiga toxin producing and verotoxin-producing E. coli (STEC and VTEC) was picked up as reference strain, and the yenl gene from Yersinia enterocolitica was cloned into F18ab107/86. Recombinant E.coli transformed with yenl produced AHL, as measured using cross-streak ing assays with the reporter biosensor strain C. violaceum CV026. The AI-1positive recombinant F18ab E. coli exhibited impaired expression of flagella, decreased motility (by33%), reduced biofilm formation (by32%) and AI-2production (by30%), as well as attenuated adherence and invasion on IPEC-J2cells (decreased by25%and30%respectively). This study provides new insights to the crucial function of AI-1in regulating E. coli virulence.In previous study, we found QS-I and-II systems participate in regulation of E. coli flagellin expression together. To elucidate the regulation mechanism, E. coli F18ab strain107/86was modified to either express acyl-homoserine lactone (AHL) synthase (QS-Ⅰ) or deleted for autoinducer2(AI-2) expression (QS-Ⅱ). AHL expression and deletion of luxS (AI-2) both inhibited flagellin expression, as measured by motility assays, bacterial gene expression, and host responses to infection. The QS systems and flagellin were coordinately regulated, as deleting fliC caused decreased QS-II activity.