It is crucial that the substances used for inactivation cause minimal alteration of the antigenes

It is crucial that the substances used for inactivation cause minimal alteration of the antigenes. complete inactivation of F0F1 CATPase and of the L-arginine uptake system, but the cytoplasmatic soluble NADH2 dehydrogenase was only partially affected. Western blot TD-106 analysis of the lipoprotein fraction showed that the INA-treated retained their lipoproteins. Following subcutaneous injection of 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 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. Introduction Mycoplasmas are closely related to Gram-positive bacteria from which they developed by genome reduction [1]. These microorganisms are characterized by a small size (0.2C0.3 m), minute genome (0.58C1.38 Mb) and the lack of a cell wall and many metabolic pathways [2]. Many species of mycoplasma are known as pathogens and are implicated in a number of serious diseases including atypical pneumonia in man, contagious bovine and caprine pleuropneumonia, contagious agalactia in small ruminants, calf pneumonia, enzootic pneumonia in pigs and chronic respiratory disease in poultry. However, at present there are no effective control measures for many of TD-106 these infections. Indeed, mycoplasmas are intrinsically resistant to several antimicrobial classes including beta-lactams. Moreover, 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. However, there are few effective vaccines against mycoplasma, most of which provide only transient or partial immunity (reviewed in [4]). Therefore, a new approach is needed in the ongoing pursuit of improved mycoplasma vaccines. The selective labeling of proteins in biological systems by photosensitization of alkylating probes was previously described [5]. The studies showed that by targeting the hydrophobic domain of enveloped viruses, such as influenza virus, Ebola virus, and a variety of retroviruses, by 1, 5-iodonaphthylazide (INA) followed by photosensitization by irradiation with ultraviolet (UV) light, a complete inactivation of the viruses was achieved while their structural integrity and immunogenicity remained unaffected [6C8]. In this study, we used and preparation of membranes strain Rlow (p.8) was obtained from the Mycoplasma Unit Strain Depository at the Kimron Veterinary Institute, Beit Dagan, Israel and was used throughout this study. The organism was grown in a modified Hayflicks medium [10] supplemented with 10% heat-inactivated fetal calf serum (Biological Industries, Beit Haemek, Israel) at 37C for 36C48 hrs. Membrane lipids were metabolically labeled by growing the cells in a medium containing 0.3 Ci of [9, 10(N)-3H] palmitic acid (53.0 Ci/mmol; New England Nuclear) per mL. In most experiments, the organisms were harvested at GRB2 the mid-exponential phase of growth (for 20 min, washed once and resuspended in a buffer solution containing 0.25 M NaCl and 10 mM Tris-HCl adjusted to pH 7.5 (referred as TN buffer). Paraformaldeyde (PFA) treated cells were obtained as described before [11]. Cell membranes were obtained from washed cells by ultrasonic treatment in a W-350 Heat Systems sonicator operated at 200 W and 50% duty cycles [12] at 4C for 2 min. The membranes were collected from the cell extracts by centrifugation at 34,000 g for 30 min, washed once, resuspended in TN buffer, and kept at ?70C until used. Treatment of with iodoazidonaphtalenes Treatment of with iodoazidonaphtalenes was performed as described elsewhere [13, 14]. In brief, the iodoazidonaphtalenes (60C200 M), INA, 1-azidonaphtalene (AzNAP), 1,5-diazidonaphtalene (DAN), 1,5-diiodonaphtalene (DINAP), and 1-azido 3,5-diiodonaphtalene (DINA) were incubated with washed mid-exponential cells in TN buffer (0.2 mg cell protein /mL) at 37C for 30 min. Glutathione (20 mmol/L) was then added to neutralize residual iodoazidonaphtalenes in the aqueous phase. The treated samples were irradiated by TD-106 far-UV light using A 100-W ozone-free mercury.