GREEN FLUORESCENT PROTEIN TRANSFORMATION OF YERSINIA ENTEROCOLITICA SEROTYPE O:3

VELAZQUEZ M ; MANZUR J; SILVA JE; ELIÇABE RJ; DI GENARO MS

San Luis

Congreso; XXXVII Reunión Anual de la Sociedad Argentina de Biología de Cuyo; 2019

Sociedad de Biología de Cuyo

Yersinia enterocolitica (Ye) are Gram-negative bacteria that cause food borne acute or chronic gastrointestinal and systemic diseases. Ye serotype O:3 are the most frequent cause of human yersiniosis and the most associated to postinfectious extraintestinal sequelae such as reactive arthritis. To analyze pathogenic mechanism of these sequelae, identification of the Ye persistence requires the tracking of the bacteria by an easily detectable marker. Although Green fluorescent protein (GFP) transformation systems are well established for many enterobacteria, there are difficulties in establishing such transformation in the genus Yersinia, particularly in the serotype O:3. In this study, we labeled Ye O:3 with a GFP vector, containing chloramphenicol as selection marker, using an electroporation protocol. Competent bacteria were prepared and different electroporation parameters were tested: voltage (2 or 2.5 kv) and plasmid concentration (0.5 or 2 ug). Bacterial growth rate was studied in the presence of chloramphenicol at 25 or 37 ºC. The in vitro GFP expression was analyzed in UV-transilluminator and by flow cytometry. In addition, the stability of the resident Yersinia virulence plasmid (pYV) was checked by autoaglutination and calcium-dependence assays. Furthermore, in vivo GFP expression was investigated by oral infection in C57BL/6 wild-type and TNFRp55-/- mice. We found that the highest electric-field strength (2.5 kv) and the lowest plasmid concentration (0.5 ug) resulted in greater yields of transformants. No adverse effects were observed in growth behavior of the labeled strain compared to wild type (parental) strain. Transformed bacteria expressed GFP at both 25 and 37 ºC. The foreign GFP plasmid did not alter the expression of pYV-encoded virulence factors. At day 5 after oral infection with GFP-Ye O:3, fluorescent colony forming units were detected in Peyer patches, mesenteric lymph nodes and spleen of mice. We conclude that GFP-Ye O:3 maintain the characteristics of the parental strain required for its use in in vivo tracking.