El sistema de señalización intercelular quorum sensing participa durante la formación de biofilm en Yersinia enterocolitica.

LUCERO ESTRADA CECILIA; DI MARCO, NATALIA; WEIRICH, JOHANA; BOHN E; ANGELES ZORREGUIETA; AUTHENRITH, INGO

Córdoba

Congreso; XI Congreso Argentino de Microbiología General. SAMIGE.; 2015

Sociedad Argentina de Microbiología General

Yersinia enterocolitica (Ye) causes acute gastroenteritis which may result in severe post-infectious complications as reactive arthritis. It is able to form biofilms onto biotic and abiotic surfaces but till the moment it is not clear the function of biofilm formation in its pathogenesis. Every species of the genera Yersinia is able to produce and release N-acyl-homoserin lactones (AHLs), diffusible molecules that are part of a complex intercellular system known as Quorum Sensing (QS). In Ye the YenI/R system, which presents significant analogy to the LuxI/R family, participates in synthesis and recognition of AHLs. The aim of this work was to obtain mutant strains with yenI/R deletion in order to know if QS participate in Ye biofilm formation. The reference strain Y. enterocolitica WA 1B/O:8 (bio/serotype), was used to obtain mutant strains. The Gibson assembling reaction was used to obtain a suicide plasmid that was cloned into Escherichia coli pir+ by electroporation. After obtaining multiples copies of the plasmid, the E. coli BETA-2168 delta nic35 strain was transformed. The latter strain mated with Ye and then suicide plasmid was integrated into Yersinia chromosome via homologous recombination creating partial diploids (merodiploid) with tetracycline resistance. The suicide plasmid also contains the counter selection marker sacB. The SacB enzyme metabolites yield the cytotoxic product sucrose. Ye merodiploids were cultured on sucrose plates therefore clones that lost the suicide plasmid were selected in this medium. The looping out of the suicide plasmid can either restore the original wild type allele or yield the desired mutant allele. The result of the looping out reaction was tested by colony PCR and also, mutants were validated by sequencing the mutated genes and analyzing the mRNA expression. The crystal violet technique in a 96-well polystyrene plate (PE) was used to observe the biofilm formed after 24 h of incubation at 25°C without shaking in trypticase soy broth added with 0.25% glucose (TSBG). Laser Scanning Confocal Microscopy (LSCM) was used to observe biofilms fixed on glass and stained with DAPI. With the Gibson cloning technique it was possible to obtain the yenI/R mutants without antibiotic resistance. The biofilm observed with mutant strains was significantly thinner than with wild type (WT) strain; the mean values were Ye ΔyenI 0.54±0.09, Ye ΔyenR 0.64±0.08 and Ye WT 1.70±0.23 (p