Surface glycotopes and epitopes are differentially displayed among Mycobacterium sp. isolates. A flow cytometry approach

SCHIERLOH P; YOKOBORI N; SABIO Y GARCIA C; LOPEZ B; BARRERA L; SASIAIN MC

Simposio; IV resunión de la Sociedad Latinoamericana de tuberculosis y otras micobacteriosis (SLAMTB); 2009

Introduction: Mycobacterial cell wall has unique biochemical composition that exerts many roles during host-pathogen interactions. Particularly, membrane glycoconjungates, which can be recognized by specific lectins, are associated with virulence and immune resistance. Aim: To develop low cost and fast assays to characterize glycosylation and antigenic exposure/availability in mycobacterial cell wall for application in comparative analysis of local Mtb clinical isolates. Methods: Surface glycosylation of Mtb strains (H37Rv, CDC1551 and local clinical isolates), M. bovis-BCG and M. smegmatis was characterized by employing biotin- or FITC-plant lectins (UEA, WGA, PNA, SNA, ConA, PHA, PSA and Pokeweed Mitogen). To seek for differential immunoreactivity, a pool from human serum with anti-PPD/LAM/Mycobacterial Total lipids titer >1/103 combined with a panel of FITC-anti-human Igk, Igl, IgG, IgM, IgA and IgE antibodies were used. Mycobacteria staining were measured by two color flow cytometry using Propidium iodide (PI) as counter stain. Results: Different glycosylation patterns between mycobacteria were detected not only at specie but also at spoligotype family level, that is, different isolates from LAM/Haarlem families bind ConA, PSA and WGA at different extent (p<0.05). Similarly, differential antibody isotype usage was also confirmed at isolate level. Conclusion: Herein we describe a novel flow cytometry application that may serve as starting step to characterize mycobacterial cell wall components that can be recognized by different immune receptors at innate and adaptive level. Acknowledgments: To Colorado State University and INTA-Castelar for kindly provide Mtb fractions and H37Rv, CDC1551 and M.smegmatis strains. This work was supported by ANCyT, CONICET and Roemmers Foundation. Keywords: Mycobacterium sp., cell wall, lectins.