Evaluating the Genetic Capacity of Mycoplasmas for Coenzyme A Biosynthesis in a Search for New Anti-mycoplasma Targets.

Mycoplasmas are responsible for a wide range of disease states in both humans and animals, in which their parasitic lifestyle has allowed them to reduce their genome sizes and curtail their biosynthetic capabilities. The subsequent dependence on their host offers a unique opportunity to explore pathways for obtaining and producing cofactors - such as coenzyme A (CoA) - as possible targets for the development of new anti-mycoplasma agents. CoA plays an essential role in energy and fatty acid metabolism and is required for membrane synthesis. However, our current lack of knowledge of the relevance and importance of the CoA biosynthesis pathway in mycoplasmas, and whether it could be bypassed within their pathogenic context, prevents further exploration of the potential of this pathway. In the universal, canonical CoA biosynthesis pathway, five enzymes are responsible for the production of CoA. Given the inconsistent presence of the genes that code for these enzymes across Mycoplasma genomes, this study set out to establish the genetic capacity of mycoplasmas to synthesize their own CoA de novo. Existing functional annotations and sequence, family, motif, and domain analysis of protein products were used to determine the existence of relevant genes in Mycoplasma genomes. We found that most Mycoplasma species do have the genetic capacity to synthesize CoA, but there was a differentiated prevalence of these genes across species. Phylogenetic analysis indicated that the phylogenetic position of a species could not be used to predict its enzyme-encoding gene combinations. Despite this, the final enzyme in the biosynthesis pathway - dephospho-coenzyme A kinase (DPCK) - was found to be the most common among the studied species, suggesting that it has the most potential as a target in the search for new broad-spectrum anti-mycoplasma agents.

Mycoplasma nasistruthionis sp. nov. and Mycoplasma struthionis sp. nov. isolated from ostriches with respiratory disease.

Twelve Mycoplasma (M.) strains isolated from the nose, the trachea, and the lung of ostriches (Struthio camelus) displaying respiratory disease were investigated. Analysis of 16S rRNA gene sequences placed five of these strains within the M. synoviae cluster, and seven strains within the M. hominis cluster of genus Mycoplasma, which was further confirmed by analyses of the 16S-23S rRNA intergenic spacer region, and partial rpoB gene and amino acid sequences. Genomic information as well as phenotypic features obtained by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry analysis and serological reactions indicated that the strains examined are representatives of two hitherto unclassified species of genus Mycoplasma, for which the names Mycoplasma nasistruthionis sp. nov., with type strain 2F1AT (= ATCC BAA-1893T = DSM 22456T), and Mycoplasma struthionis sp. nov., with type strain 237IAT (= ATCC BAA-1890T = DSM 22453T), are proposed.

DNA Vaccines Against Mycoplasma Elicit Humoral Immune Responses in Ostriches.

In ostriches, the population densities resulting from intensive rearing increases susceptibility to pathogens such as mycoplasmas. In addition to good management practices, vaccination offers an attractive alternative for controlling mycoplasma infections in food animals, instead of using antibiotics, which often leave unacceptable residues. The use of live attenuated vaccines, however, carry the concern of reversion to virulence or genetic recombination with field strains. Currently there are no commercially available vaccines against ostrich-infecting mycoplasmas and this study therefore set out to develop and evaluate the use of a DNA vaccine against mycoplasma infections in ostriches using an OppA protein as antigen. To this end, the oppA gene of "Mycoplasma nasistruthionis sp. nov." str. Ms03 was cloned into two DNA vaccine expression vectors after codon correction by site-directed mutagenesis. Three-months-old ostriches were then vaccinated intramuscularly at different doses followed by a booster vaccination after 6 weeks. The ability of the DNA vaccines to elicit an anti-OppA antibody response was evaluated by ELISA using the recombinant OppA protein of Ms03 as coating antigen. A statistically significant anti-OppA antibody response could be detected after administration of a booster vaccination indicating that the OppA protein was successfully immunogenic. The responses were also both dose and vector dependent. In conclusion, the DNA vaccines were able to elicit an immune response in ostriches and can therefore be viewed as an option for the development of vaccines against mycoplasma infections.

The identification of oppA gene homologues as part of the oligopeptide transport system in mycoplasmas.

The lack of an annotated oppA gene as part of many oligopeptide permease (opp) operons has questioned the necessity of the oligopeptide-binding domain (OppA) as a part of the Opp transport system in mycoplasmas. This study investigated the occurrence of an oppA gene as part of the oppBCDF operon in 42 mycoplasma genomes. Except for hemoplasma, all mycoplasmas were found to possess one or more copies of the oppBCDF operon and with the help of similarity searches their oppA genes could be identified. Phylogenetic analysis of the combined OppABCDF amino acid sequences allowed them to be grouped into three types. Each type has a unique set of conserved motifs, which are likely to reflect substrate preference and adaption strategies. Our approach allowed the identification of oppA gene homologues for all mycoplasma opp operons and thereby provides a method for re-evaluating the current annotation of oppA genes in mycoplasma genomes.