Novel effector protein EspY3 of Type III Secretion System from enterohemorrhagic Escherichia coli is localized in pedestals

LARZABAL MARIANO,; MARQUES DA SILVA, WANDERSON; A CATALDI

Congreso; VTEC2018; 2018

IntroductionIn a preliminary work we have characterized EspY2 and EspY3 as new translocated effectors of type III secretion system (T3SS) of enterohemorrhagic Escherichia coli (EHEC) O157:H7. The EspY family of effectors present WEX5F motif that is conserved in several well characterized SopD-N Salmonella effectors. In turn, EspY3 presents a LFNEF motif similar with the secretory protein effector PipB2 (PipB family) of Salmonella that target intracellular membrane compartments, and also presents two copies of 8 and 9 tandem of pentapeptide repeats (PPR) in their C-terminal domain. So far, its subcellular localization and its possible function as an effector have not been determined. Methodsenteropathogenic Escherichia coli (EPEC) has greater capacity to produce attaching and effacing lesions, pedestal-type structures and translocate effectors than EHEC O157:H7. Therefore, EspY3 effector was cloned in pLF-3xFLAG vector and recombinantly expressed as fusion product to the FLAG tag in EPEC O127:H6 strain E2348/69. These transformed bacteria were used to perform the epithelial cell infection assays. The subcellular localization of recombinant EspY3 was determined by confocal microscopy using anti-FLAG specific antibodies.Results The capacity of T3SS of EPEC O127:H6 to translocate EspY3 effector was confirmed. Then the subcellular location was determined through epithelial cell infection assays, being able to observe the presence of EspY3 effector associated to the pedestals region, colocalizing with the polymerized actin. Significantly more pronounced pedestals were also observed in relation to EPEC O127:H6 wild type lesions (control without pLF-3xFLAG-EspY3).ConclusionsThe results showed that the EspY3 effector is translocated by the T3SS of EPEC O127:H6, being located subcellularly in the pedestals region where it would be participating in the modulation of the cellular actin, possibly generating the elongation of the pedestals. The 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.