Mycoplasma gallisepticum inactivated by targeting the hydrophobic domain of the membrane preserves surface lipoproteins and induces a strong immune response.

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.

Mycoplasma gallisepticum produces a histone-like protein that recognizes base mismatches in DNA.

Mycoplasmas are the smallest known microorganisms, with drastically reduced genome sizes. One of the essential biochemical pathways lost in mycoplasmas is methylation-mediated DNA repair (MMR), which is responsible for correction of base substitutions, insertions, and deletions in both bacteria and higher organisms. We found that the histone-like protein encoded by the himA/hup_2 gene of Mycoplasma gallisepticum (mgHU) recognizes typical MMR substrates, in contrast to homologues from other species. The recognition of substitution mismatches is sequence-dependent, with affinities decreasing in the following order: CC > CT = TT > AA = AC. Insertions or deletions of one nucleotide are also specifically recognized with the following sequence-dependent preference: A = T > C. One-nucleotide lesions involving guanine are bound only weakly, and this binding is indistinguishable from binding to intact DNA. Although mgHU is dissimilar to Escherichia coli HU, expression in a slow-growing hupAB E. coli strain restores wild-type growth. The results indicate that mgHU executes all essential functions of bacterial architectural proteins. The origin and the possible role of enhanced specificity for typical MMR substrates are discussed.

Atomic force microscopy investigation of DNA extracted from the vegetative forms and the viable but nonculturable forms of Mycoplasma gallisepticum S6.

Recent studies show that mycoplasmas have various programs of life. This means that changes in morphology and genome expression may occur once the environment of these microorganisms becomes extremely altered. In this article, we report on changes in the DNA molecule obtained from the vegetative forms and the viable but nonculturable (VBNC) forms of Mycoplasma gallisepticum S6. Atomic force microscopy studies show that the above-mentioned forms of the mycoplasma have different values of DNA parameters (height: 0.461 +/- 0.141 and 0.236 +/- 0.069 nm; width: 2.221 +/- 0.286 and 1.291 +/- 0.705 nm for the vegetative and the VBNC forms, respectively). We suppose that the observed phenomenon may be connected with the process of adaptation of these bacteria to severe environments.

The Mycoplasma gallisepticum OsmC-like protein MG1142 resides on the cell surface and binds heparin.

Mycoplasma gallisepticum is an avian pathogen that causes a chronic respiratory disease of chickens and results in significant economic losses to the poultry industry worldwide. Colonization of the host and the establishment of chronic disease are initiated by the cytadherence of M. gallisepticum to the host respiratory epithelium. While several proteins involved in cytadhesion have been characterized, molecules that interact with components of the host extracellular matrix, a process that is central to pathogenesis, are only now being identified. In this study, M. gallisepticum whole cells were shown to bind heparin in a specific and saturable manner. Heparin also significantly inhibited the binding of M. gallisepticum to the human lung fibroblast cell line MRC-5, suggesting a potential role for glycosaminoglycans (GAGs) in cytadherence. M. gallisepticum protein MG1142 (encoded by mga 1142), which displays homology to the osmotically induced (OsmC) family of proteins, binds strongly to heparin, is highly expressed during in vitro culture, and is surface accessible. Recombinant MG1142 bound heparin in a dose-dependent and saturable manner with a dissociation constant (K(d)) of 10+/-1.8 nM, which is within a physiologically significant range, compared to that of other heparin-binding proteins. Binding to heparin was inhibited by the heavily sulfated polysaccharide fucoidan, but not by mucin or chondroitin sulfate A or B, suggesting that electrostatic interactions between the sulfate groups of heparin and the positively charged basic residues of the MG1142 protein are important in binding. The ability of M. gallisepticum to bind GAGs may contribute to host adherence and colonization.

Identification of fibronectin-binding proteins in Mycoplasma gallisepticum strain R.

We have determined that virulent Mycoplasma gallisepticum strain Rlow is capable of binding the extracellular matrix protein fibronectin. Fibronectin was found to be present in M. gallisepticum Rlow protein extracts by Western blotting and peptide sequencing. Mycoplasma gallisepticum Rhigh, the attenuated, high-passage derivative of Rlow, is deficient in this ability. MGA_1199, the M. gallisepticum homologue of the cytadherence-associated protein P65 from Mycoplasma pneumoniae, and MGA_0928, the M. gallisepticum homologue of the M. pneumoniae cytoskeletal protein HMW3, were identified as fibronectin-binding proteins. Peptides from the regions of MGA_1199 and MGA_0928 exhibiting the highest degree of homology with known fibronectin-binding proteins were shown to bind the gelatin/heparin-binding domain of fibronectin. MGA_1199 and MGA_0928 were shown to be absent and aberrant, respectively, in Rhigh, explaining its lack of fibronectin-binding capability. Consistent with its M. pneumoniae counterpart, MGA_1199 (renamed PlpA) was demonstrated to be surface exposed, despite a lack of classical membrane-spanning domains. Due to its demonstrated topology and the strength of interaction between its binding peptide and fibronectin, we propose that PlpA functions as a fibronectin-binding protein in vivo and may possess atypical transmembrane domains.