TRANSFORMATION AND CHARACTERIZACION OF YopP DEFICIENT Yersinia enterocolitica MUTANT STRAIN WITH A PLASMID ENCODING THE GREEN FLUORESCENT PROTEIN

MEDINA, AGUSTINA; MANZUR, JIMENA; SILVA, PATRICIA; DI GENARO, MARÍA SILVIA; SILVA, JUAN EDUARDO

San Luis

Congreso; Congreso Nacional de Microbiología General - SAMIGE 2018; 2018

Sociedad Argentina de Microbiología General

Yersinia enterocolitica (Ye) is a Gram-negative bacterium that causes gastrointestinal and genitourinary infection. Ye translocates, through a type III secretion system, bacterial effector proteins into the host cells, interfering with different cellular functions. The machinery of secretion and a set of six effector Yersinia outer proteins (Yops) (YopE, YopH, YopM, YopO, YopP, YopT) are encoded in a 70-kb virulence plasmid (pYV). YopP induces apoptosis in macrophages and dendritic cells by inhibition of NF-kB and MAPK pathways. The green fluorescent protein (GFP), isolated from the jellyfish Aequorea victoria, is frequently used as a reporter for the in vivo tracking of GFP transformed-pathogens. The purpose was to transform a YopP-deficient Ye mutant strain (Ye DyopP) with a GFP-containing plasmid. Moreover, we evaluated the effect of GFP- transformation on the bacterial growth at 27ºC, and on the virulence and invasiveness of this transformed-mutant strain afterin vivo infection of mice. Therefore, the pACYC_EGFP 1000 plasmid (pGFP) of 4569 bps, carrying a cassette of Clorhanphenicol (Cam r) resistance was electroporated into competent Kanamicin-resistant Ye DyopP (Ye DyopP Kan r) in 0.2 mm cuvette (2,5KV, 25µF, LR200ohm, HR 600ohm), and recovered in SOC medium. Transformed bacteria were selected by plating on Kan (50 µg/ml) and Cam (35µg/ml) supplemented Luria Bertani (LB) agar, and green fluorescence was evaluated by UV illumination. Then, several fluorescent clones were picked at random and stored at -80ºC in 10% glycerol-LB broth. Final cell density and specific growth rate (h -1) of GFP-transformed Ye DyopP(GFP-Ye DyopP) was compared with its corresponding parental strain. Therefore, both Ye DyopP and GFP-Ye DyopP were cultured overnight at 27 °C in LB broth in presence of Kan or Kan and Cam, respectively. After 1:1000 dilution in fresh media, bacterial growth was measured hourly for 9 h in aspectrophotometer at 655nm. Moreover, C57BL/6 mice were oragastrically infected with 1-5 x 10 8 Ye DyopP or GFP-Ye DyopP. After 1 and 5 days, Peyer?s patches (PP), mesenteric lymph node (MLN)and spleen (Sp) were obtained and colony forming units (CFU) were recorded. Successful pGFP transformation of Ye DyopP was obtained since green fluorescence was observed in the colonies underUV exposure. GFP-Ye DyopP exhibited similar growth rate to Ye DyopP strain. We did not find significantly differences in the number of CFU/organ in PPs, MLNs and Sp between mice infected with Ye DyopP or GFP-Ye DyopP at 1 or 5 days after infection (Mann Whitney test). Although further studies are necessary, our results indicate reliable expression of GFP-reporter plasmid in Ye DyopP,which does not impact on the bacterial growth nor in vivo virulence and invasiveness.