Yersinia enterocolitica outer protein P (YopP) inhibits nitric oxide production through sialic acid interaction by a possible extracellular mechanism.

JUAN AGUSTÍN GARAY; JUAN EDUARDO SILVA; BRENDA JOFRÉ; MARÍA SILVIA DI GENARO; ROBERTO CARLOS DAVICINO

Mendoza

Congreso; XL Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2022

Sociedad de Biología de Cuyo

Previous studies of our laboratory demonstrated that the food-borne pathogen Yersinia enterocolitica (Ye) inhibits nitric oxide production(NO) in murine peritoneal macrophages (Mϕ) through Ye outer protein P (YopP) by a carbohydrate-dependent manner. However, theknowledge about the glycan motifs involved in the YopP- Mϕ interaction is underdeveloped. The aim of this study was to explore YopPactivity as lectin and identify the possible N-glycans used as target. Mϕ were obtained by intraperitoneal lavage in C57BL/6 mice underaseptic conditions, cells were plated and Mϕ were purified by incubation for 2 h at 37°C in supplemented DMEM medium in an atmosphereof 5% CO2. Mϕ (1x106cells/ml) were incubated with 5 IU/mL of PNGase F or 2 IU/mL of α 2,6 Syalidase in DMEM medium withoutfetal bovine serum, washed and cultured overnight in supplemented DMEM medium. Subsequently, Mϕ were infected with Ye serotype0:8 (pYV+, WA-314) (Ye wt) or Ye WA-314 deficient in YopP (pYV+, WA-C pYVNalrKanr) (Ye ∆yopP) at MOI 10:1. To elucidatepossible targets, we performed competition tests with five lectins: PNA, SNA, LEL, ECL or MAL II. After infection, Griess, lactatedeshydrogenase (LDH) and urea tests were performed, and YopP expression was determined by Western Blot. Hemagglutination testswere carried out with Ye wt, Ye ∆yopP, purified Yops or periodate treated Ye. We identified YopP presence in the supernatants of Ye wtinfected Mϕ, while LDH assay showed that experimental conditions did not induce cell lysis. These results suggest a possible YopPsecretion by Ye. Yops hemagglutinin results suggest a lectin property of Yops. Moreover, deglycosylated Mϕ (Md) showed increased ureaproduction and decreased NO secretion. Urea production was not modified after infection, while NO production was modulated by YopP.In this regard, the presence of YopP inhibited NO generation in Mϕ meanwhile stimulated NO in Md. SNA is a vegetable lectin withNeu5Ac (alpha2-6) Gal (GalNAc or syalic acid) affinity. We found that SNA did not affect NO production by Ye wt-infected Mϕ whileinhibited NO production in Ye ∆yopP-infected Mϕ. Interestingly, Ye wt-infected Mϕ treated with syalidase showed a significant increasein NO production, suggesting that syalic acid-containing motifs plays a critical role in YopP-mediated effects. Further studies are requiredto consolidate the importance of a possible extracellular role of YopP collaborating with injected intracellular YopP in regulating the NOproduction.