EspY2 and EspY3 are novel T3SS translocated effectors of E. coli O157:H7

MARIANO LARZABAL; BRENDA BUSSACA; NATALIA AMIGO; ANGEL A. CATALDI

boston

Congreso; congreso internacional VTEC2015; 2015

IntroductionMore than 60 genes have been predicted by bioinformatic to encode for putative effectors secreted by the type three-secretion system (T3SS) of enterohaemorrhagic Escherichia coli (EHEC) O157:H7. These putative effectors are grouped into 20 different families. The family of effectors EspY (1-5) has the WEX5F motif and is conserved in several well characterized SopD-N Salmonella effectors. SopD-N is secreted by T3SS 1 and 2 of Salmonella, and delivered into the host cell. Genomic analysis demonstrated that EspY family is absent in EPEC and Citrobacter rodentium. Other authors have confirmed EspY1 and EspY4 as genuine T3SS translocated effectors. We propose EspY2 and EspY3 as new translocated effectors by T3SS of EHEC O157:H7.MethodsThe putative effectors EspY2 and EspY3 were cloned into pLF vector and recombinantly expressed as fusion product to the FLAG tag in EHEC O157:H7. The vector has a lacZ promoter sequence inducible by IPTG. Secretion assays were realized to detect recombinants expressing effectors in the culture supernatant.Results and DiscussionFirstly, EspY2 and EspY3 were analyzed by bioinformatic servers specialized in the prediction of secreted effectors by the T3SS. Competent EHEC O157:H7 bacteria were transformed with pLF-3xFLAG- EspY2 and EspY3 vectors, being its expression inducible by IPTG. Through western blot assays the cytoplasmic expression of recombinant effectors was evaluated in the induced bacterial pellet samples, while precipitated supernatant samples allowed us to observe the secretion of T3SS effectors. We have observed cytoplasmic expression of EspY2 and EspY3 as well as their secretion into the medium via the T3SS. This would indicate that the proteins EspY2 and EspY3 would be secreted by T3SS being therefore likely O157:H7 effectors.ImplicationsThe characterization of new virulence factors translocated by T3SS of EHEC O157:H7 will allow us further clarification of the mechanisms of virulence during the pathogenesis.