title: Mycoplasma gallisepticum inactivated by targeting the hydrophobic domain of the membrane preserves surface lipoproteins and induces a strong immune response.
abstract: An innovative approach for inactivation of Mycoplasma gallisepticum using the hydrophobic photoinduced alkylating probe 1, 5-iodonaphthylazide (INA) is described. Treatment of washed M. gallisepticum mid-exponential culture (0.2 mg cell protein /mL) with INA followed by irradiation with far-ultraviolet light (310-380 nm) completely abolished viability. Transmission electron microscopy showed that the majority of the inactivated M. gallisepticum were comparable in size to intact cells, but that part of the INA-treated M. gallisepticum preparation also contained low density cells and membrane vesicles. Confocal microscopy revealed that untreated M. gallisepticum cells were internalized by chicken red blood cells (c-RBCs), whereas the INA-inactivated cells remained attached to the outer surface of the c-RBCs. INA treatment of M. gallisepticum resulted in a complete inactivation of F0F1 -ATPase and of the L-arginine uptake system, but the cytoplasmatic soluble NADH2 dehydrogenase was only partially affected. Western blot analysis of the lipoprotein fraction showed that the INA-treated M. gallisepticum retained their lipoproteins. Following subcutaneous injection of M. gallisepticum INA-bacterin, 100% and 68.8% of chickens were positive by the rapid serum agglutination test and enzyme-linked immunosorbent assay respectively, 2 weeks post-injection. These data suggest that the photoinducible alkylating agent INA inactivates M. gallisepticum but preserves its surface lipoproteins and thus has the potential to be used as a general approach for the inactivation of mycoplasmas for vaccine development.
authors.affiliation: Department of Microbiology and Molecular Genetics, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Hazem
authors.initials: H
authors.lastname: Atalla
authors.affiliation: Division of Avian and Aquatic Diseases, Kimron Veterinary Institute, Beit Dagan, Israel.
authors.forename: Inna
authors.initials: I
authors.lastname: Lysnyansky
authors.affiliation: SAIC-Frederick Inc, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Yossef
authors.initials: Y
authors.lastname: Raviv
authors.affiliation: Department of Microbiology and Molecular Genetics, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Shlomo
authors.initials: S
authors.lastname: Rottem
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology*; D001428:Bacterial Vaccines / Q000737:chemistry / Q000276:immunology; D002645:Chickens / Q000276:immunology*; D057927:Hydrophobic and Hydrophilic Interactions; D008074:Lipoproteins / Q000737:chemistry / Q000276:immunology*; D008297:Male; D045686:Mycoplasma gallisepticum / Q000737:chemistry / Q000276:immunology*; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D008074:Lipoproteins