DIFFERENTIAL ACTIVITY OF TWO SAGS OF THE EGC OPERON ON PHAGOCYTIC CELLS

NOLI TRUANT, SOFIA; SARRATEA, BELÉN; FERNÁNDEZ LYNCH MARÍA JULIETA; ANTONOGLOU, BELÉN; DE MARZI MAURICIO; EMILIO L. MALCHIODI; FERNÁNDEZ MARISA MARIEL

Mar del Plata

Congreso; Reunión SAIC-SAI-SAFE 2016; 2016

SAIC-SAI-SAFE

Bacterial superantigens (SAgs) are enterotoxins that bind to the MHC-II molecules and the TCR, activating as much as 20% of T cells and promoting a cytokine storm which enhances endotoxic shock and produces immunosuppression, hindering the immune response against bacterial infection. The egc operon reported in autochthonous S. aureus strains includes natural variants of the SAgs genes seg, sei, sem, sen and seo. Here, we characterize the complementary activity of SEO and SEM on phagocytic cells in the absence of T cell. SEO and SEM were cloned in pET26 vector and expressed in E coli. To test biological activity, SAgs were exposed to different cell cultures to evaluate proliferation and viability by using [methyl-3H] thymidine and MTT. Macrophage activation was characterized measuring arginine activity in cell lysates and by nitrite determination in culture medium. SEO interaction with MCH-II DR1 was evaluated by Western Blot and their affinity constants were estimated by surface plasmon resonance. We have found:1-PBMCs activation was determined for SEO at 0.001ìg/ìl and SEM at 0,01 ìg/ìl; 2-SAgs cytotoxic activity on THP-1 was triggered by SEO at 10 ìg/ìl, and SEM at 100 ìg/ ìl; 3-Both SAgs are toxic to macrophages-differentiated THP-1 at 100 ìg/ìl at 24/48 h, while SEM showed cytotoxicity at 0.1 ìg/ìl on Raw cells at 24/48 h, though SEO only showed effect after 48 h; 4-Exposure of macrophages-differentiated THP-1 to SEM, but no SEO (neither SEI or SEG) showed up to 7-fold nitrite increase; 5-Only SEO increased nitrite up to 1.6 fold in culture medium of RAW cells; 6-SEO reduced arginase activity in cell lysates; 7-SEO-DR1 specific interaction was determined. SEO and SEM exhibited SAg activity on different phagocytic cells showing a complementary cytotoxic activity, favoring tendency to a very soft M1 response that is triggered at different stages for each SAg. The singularity of this differential response may be a key for bacteria?s survival in the host.