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

MEDINA AGUSTINA; SILVA JUAN; MANZUR JIMENA; DI GENARO SILVIA

Congreso; XIII Congreso Argentino de Microbiología General; 2018

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 ∆yopP) 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 after in vivo infection of mice. Therefore, the pACYC_EGFP1000 plasmid (pGFP) of 4569 bps, carrying a cassette of Clorhanphenicol (Camr) resistance was electroporated into competent Kanamicin-resistant Ye ∆yopP (Ye ∆yopP Kanr) 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 ∆yopP (GFP-Ye ∆yopP) was compared with its corresponding parental strain. Therefore, both Ye ∆yopP and GFP-Ye ∆yopP 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 a spectrophotometer at 655nm. Moreover, C57BL/6 mice were oragastrically infected with 1-5 x 108 Ye ∆yopP or GFP-Ye ∆yopP. 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∆yopP was obtained since green fluorescence was observed in the colonies under UV exposure. GFP-Ye ∆yopP exhibited similar growth rate to Ye ∆yopP strain. We did not find significantly differences in the number of CFU/organ in PPs, MLNs and Sp between mice infected with Ye ∆yopP or GFP-Ye∆yopP 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 ∆yopP, which does not impact on the bacterial growth nor in vivo virulence and invasiveness.