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

LARZABAL MARIANO; BRENDA BUSSACA; AMIGO NATALIA; CATALDI ANGEL

Boston

Congreso; VTEC; 2015

IntroductionVirulence factors translocated to the cytoplasm of host cells by type III secretion system (T3SS) are known as effectors and each pathogen itself has a diverse repertoire of specific infection process. These effectors can be grouped into families with similar biochemical functions and have consensus domains found in eukaryotes that predict their activity. Studies have been conducted to establish the proteomic repertoire T3SS effectors secreted by EHEC O157:H7 strains and may identify 60 genes predicted effectors, some being confirmed as such by protein translocation assays. So far, it has been observed that these effectors play various roles in the pathogenesis and modulation of Rho GTPases, inhibition of apoptosis, interference inflammatory signaling pathway and inhibition of phagocytosis. Thus, these effectors mimic or replace eukaryotic protein activities subverting cellular functions for the benefit of the infection. The family of effectors Espy (1-5) has the WEX5F motif and is conserved in several well 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 present in EHEC and absent in EPEC and Citrobacter rodentium. Other authors have confirmed EspY1 and EspY4 as genuine translocated T3SS effectors of EHEC O157:H7 and EspY5 was characterized as a pseudogene. AimOurs studies want to demonstrate that putative effectors proteins EspY2 and EspY3, predicted as potential virulence factors, are translocated by the T3SS EHEC O157:H7.MethodsespY2 (570 bp) and espY3 (1572 bp) genes were amplified by PCR of EHEC O157:H7 EDL933 strain and the primers were designed from bioinformatic studies of EDL 933 strain sequenced genome. The coding segments for espY2 and espY3 were cloned into pGEM-T Easy vector in E. coli XL-1. The pGEM-espY2 and espY3 constructions were digested with restriction enzymes SpeI and BamHI, and the fragments were subcloned into pLF-3xFLAG. In the same way, the constructions were digested with restriction enzymes HindIII and BamHI, and the fragments were subcloned into pRSET-A expression vector. Were recombinantly expressed EspY2 and EspY3 as fusion product to the FLAG tag in EDL 933 O157:H7 ΔsepL and 6xHis tag in BL-21 AI respectably. EDL 933 O157:H7 ΔsepL pLF-espY2 and espY3 were realized in secretion assays to detect recombinants expressing effectors in the culture supernatant. pRSET-A-espY2 and espY3 BL-21 AI were realized to express recombinant protein to inoculate in mice for obtaining polyclonal antibodies.Results and DiscussionWe have analyzed the genes by bioinformatic studies through Effective T3 software providing pre calculated predictions for bacterial TTSS effectors of all genomes of pathogenic symbionts and publicly available and predictability effectors using sequence data own proteins. We could determine the high probability of EspY2 (score 1) to be an effector secreted by the T3SS. On the other hand, EspY3 ( 0.01683 ) got a low score indicating that no homology with other effectors as predicted. 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.Specific polyclonal antibodies of EspY2 and EsY3 were generated in mice to corroborate the results obtained with the anti FLAG antibody. EspY2 and EspY3 recombinant were expressed and purified from BL-21 bacteria transformed with vectors pRSET-A-espY2 and espY3. These antibodies enabled us to detect by western blot the presence of effectors in the pellet of EDL933 strains O157:H7 ΔsepL pLF-espY2 or espY3 as well as effectors were detected in the culture supernatant. The presence of effectors EspY3 EspY2 in culture supernatants indicate that are being recognized and secreted by the T3SS of EDL933 O157:H7. Infection assays allow us to confirm whether these effectors are translocated to epithelial cells, and we can prove their subcellular localization by confocal microscopy.The characterization of new virulence factors as EspY2 an EspY3 will allow us further clarification of the mechanisms of virulence during the pathogenesis of EHEC O157:H7.