The small RNA McaS as a tool to inhibit curli synthesis and biofilm formation on gastrointestinal pathogenic bacteria.

QUIROGA C; SARNACKI SH; GONZALES MACHUCA AG

Buenos Aires

Congreso; Reunión Conjunta SAIB-SAMIGE 2020; 2020

Reunión Conjunta SAIB-SAMIGE 2020

Salmonella enterica and Escherichia coli serotypes can commonly cause foodborne gastrointestinal (GI) infections, affecting mainly children and elderly individuals. In Argentina, Enterohaemorrhagic E. coli (EHEC) O157:H7 serotype is of serious concern since it causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) reaching the highest levels worlwide. EHEC and S. enterica possess different adhesins that initiate the colonization process. Both bacteria produce curli fibers that are involved in host cell adhesion, dissemination, immune system activation and biofilm formation. Curli adhesin is encoded in two operons csgBAC and csgDEFG, with CsgD as the main transcriptional regulator. Several small regulatory RNAs (sRNA) play a key role in post-transcriptional regulation of curli synthesis by targeting the 5? UTR of the csgDEFG operon. Among them, McaS, an Hfq-dependent sRNA, can effectively decrease the expression of csgD preventing curli production and biofilm formation. In this work, we aimed to generate an McaS expression system capable of inhibit curli synthesis and cellular adherence in order to establish the bases for a possible therapeutic mechanism against GI pathogenic bacteria. We introduced a sequence carrying the McaS sRNA under the control of the Plac promoter into vector pACYC184 (clone pMcaS). This clone was then introduced into different GI pathogenic bacteria and induced McaS expression with 1mM IPTG. We tested two EHEC isolates (one clinical strain, CQ17, and one food-borne strain, CQ146) and a clinical isolate of S. enterica sv. Enteritidis (ArJEG). We also included a uropathogenic isolate (E. coli CQ7) and the reference strain E. coli MG1655. Macrocolony morphology and curli production was determined using LB or LB without NaCl agar plates supplemented with congo red (0,04 mg/mL) and coomassie brilliant blue G (0,02 mg/mL) dyes at different temperatures (28 and 37°C) at two incubation time points (48 and 120 h). Strains were compared against their counterparts carrying the clone pMcaS or the empty vector. A marked reduction in curli production was observed for strains MG1655, CQ146, CQ7 and ArJEG expressing McaS when grown at 28°C after 48 h, becoming increasingly evident at 120 h. No evident phenotype reversion was observed at 37 °C. On the other hand, the adherence to polystyrene plates was measured at 28°C and 37°C after incubation for 48 and 120 h followed by staining with crystal violet (0,01%). A statistically significant reduction was observed on the aforementioned strains at 28°C at both 48 and 120 h (p