The oppD Gene and Putative Peptidase Genes May Be Required for Virulence in Mycoplasma gallisepticum

Abstract

Relatively few virulence genes have been identified in pathogenic mycoplasmas, so we used signature-tagged mutagenesis to identify mutants of the avian pathogen Mycoplasma gallisepticum with a reduced capacity to persist in vivo and compared the levels of virulence of selected mutants in experimentally infected chickens. Four mutants had insertions in one of the two incomplete oppABCDF operons, and a further three had insertions in distinct hypothetical genes, two containing peptidase motifs and one containing a member of a gene family. The three hypothetical gene mutants and the two with insertions in oppD 1 were used to infect chickens, and all five were shown to have a reduced capacity to induce respiratory tract lesions. One oppD 1 mutant and the MGA_1102 and MGA_1079 mutants had a greatly reduced capacity to persist in the respiratory tract and to induce systemic antibody responses against M. gallisepticum. The other oppD 1 mutant and the MGA_0588 mutant had less capacity than the wild type to persist in the respiratory tract but did elicit systemic antibody responses. Although M. gallisepticum carries two incomplete opp operons, one of which has been acquired by horizontal gene transfer, our results suggest that one of the copies of oppD may be required for full expression of virulence. We have also shown that three hypothetical genes, two of which encode putative peptidases, may be required for full expression of virulence in M. gallisepticum. None of these genes has previously been shown to influence virulence in pathogenic mycoplasmas. KEYWORDS Mycoplasma gallisepticum, oppD, virulence Mycoplasma gallisepticum is the most important mycoplasmal pathogen in poultry (1, 2) and a particularly useful model of mycoplasmal pathogenesis, in part as a typical representative of the Pneumoniae phylogenic group, which includes the important human pathogen M. pneumoniae. There have been relatively few studies attempting to identify virulence genes in mycoplasmas, in part because of the limited availability of tools for such studies. Signature-tagged mutagenesis (STM) is a useful technique for identifying genes likely to be involved in virulence, as it enables the rapid comparison of populations of mutants used to inoculate animals with those recovered from these animals during the course of infection, allowing identification of mutants with a reduced capacity to persist in experimentally infected animals (3, 4). However, the examination of only pools of mutants may result in a failure to distinguish between a mutation in a gene that is essential in vivo and a mutation in a gene that reduces the capacity of the mutant to compete with other mutants in vivo. In addition, it is not possible to fully assess the virulence of individual mutants within populations. Therefore, STM needs to be complemented with direct comparisons of isolated mutants for their capacity to infect, persist, and cause disease.

Preliminary comparative analysis of the genomes of selected field reisolates of the Mycoplasma synoviae vaccine strain MS-H reveals both stable and unstable mutations after passage in vivo

“…OppF is essential in establishment of systemic infection by M. bovis and its persistence in lower respiratory tract of calves [10]. Also, oppD was found to be required for full expression of virulence of M. gallisepticum in chickens [11].…”

Genome analysis of Mycoplasma synoviae strain MS-H, the most common M. synoviae strain with a worldwide distribution

“…Experimental verification of this hypothesis is required to establish whether the two OppA transporters can co-operate or display broad substrate specificity, whereas OppF and other components of the Opp system have more exacting interactions in M. synoviae . Alternatively, as has been hypothesized for OppD1 in M. gallisepticum [ 36 ], it is possible that the two OppF proteins form dimers in the transport complex and the product of the partial copy of oppF -I in MS-H may retain the capacity to form a heterodimer with the product of full-length copy of oppF -II, resulting in a functional reduction thus a decrease in virulence.

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“…A remarkable example of pseudogenisation was found in the oligopeptide permease (Opp) system. Multiple copies of opp operons are commonly seen in both Gram-negative and Gram-positive bacteria, including M. gallisepticum , and can play a role in virulence [ 33 – 36 ]. The Opp systems comprise an extracellular substrate binding protein OppA, two transmembrane proteins OppB and OppC, which form the pore, and two cytoplasmic ATPases OppD and OppF, which provide the energy for peptide translocation [ 37 , 38 ].…”

“…It has been suggested that in the Hominis group the opp operons underwent duplication and divergence. In M. gallisepticum , one of the opp operons was proposed to be horizontally acquired from a member of the Hominis group [ 36 ]. The correct annotation of oppA sequences in Mycoplasma spp.…”

Deletion of the oligopeptide transporter Lmo2193 decreases the virulence of Listeria monocytogenes

“…To study the role of Opp in C. pseudotuberculosis , the ΔOppD strain showed impaired growth when exposed to the toxic glutathione peptide, and reduced ability to adhere to and infect macrophages compared to the wild-type [ 19 ]. The OppD gene in M. gallisepticum has the continuous ability to reduce the level of virulence in the body, and suggests that the OppD gene may be involved in the regulation of the level of bacterial virulence [ 20 ]. In the study of L. monocytogenes Opp, the lmo2196 gene was described as the substrate binding protein OppA in the ABC transporter [ 21 ], which is a very important factor that L. monocytogenes depends on for survival in low temperature environments, assisting the organism under these conditions to obtain nitrogen resources, regulate the expression of related virulence factors, and help L. monocytogenes survive in macrophages [ 9 ].…”