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.
Highlights
Mycoplasmas are closely related to Gram-positive bacteria from which they developed by genome reduction [1]
To begin to explore the effect of UV-irradiation of M. gallisepticum cells treated with iodoazidonaphtalenes, we tested the effect of UV-irradiation on the viability of M. gallisepticum (Table 1)
Irradiation of up to 2 min had very little effect on M. gallisepticum viability, whereas a decrease of 2–3 logs in colony forming units per mL (CFU/mL) was measured after 4 min of irradiation
Summary
Mycoplasmas are closely related to Gram-positive bacteria from which they developed by genome reduction [1]. These microorganisms are characterized by a small size (0.2–0.3 μm), minute genome (0.58–1.38 Mb) and the lack of a cell wall and many metabolic pathways [2]. Inactivation of M. gallisepticum by a Photoinducible Alkylation. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. Different species of mycoplasma showed decreased susceptibility to many commercial available antimicrobials (reviewed in [3]). In view of the decreasing efficacy of antibiotics in controlling mycoplasma infections, there is increasing interest in immunoprophylaxis. A new approach is needed in the ongoing pursuit of improved mycoplasma vaccines
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