Comparative analysis of the lppA locus in Mycoplasma capricolum subsp. capricolum and Mycoplasma capricolum subsp. capripneumoniae

Monnerat, Marie-Pierre; Thiaucourt, François; Nicolet, Jacques; Frey, Joachim

doi:10.1016/s0378-1135(99)00105-4

Cited by 25 publications

(24 citation statements)

References 38 publications

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“…capricolum and M. mycoides subsp. mycoides biotype Large Colony that both target a gene encoding the LppA lipoprotein, MCCPL PCR (16) and MMMLC PCR (17), respectively. However, they were both shown to be inappropriate for field isolates, with a relative intrataxon sensitivity of only 48 and 26% for M. capricolum subsp.…”

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confidence: 99%

Suppression-Subtractive Hybridization as a Strategy To Identify Taxon-Specific Sequences within the Mycoplasma mycoides Cluster: Design and Validation of an M. capricolum subsp. capricolum -Specific PCR Assay

Maigre¹,

Citti

Marenda

et al. 2008

J Clin Microbiol

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The phylogenetically related Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony are two small-ruminant pathogens involved in contagious agalactia. Their respective contributions to clinical outbreaks are not well documented, because they are difficult to differentiate with the current diagnostic techniques. In order to identify DNA sequences specific to one taxon or the other, a suppression-subtractive hybridization approach was developed. DNA fragments resulting from the reciprocal subtraction of the type strains were used as probes on a panel of M. capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony strains to assess their intrataxon specificity. Due to a high intrataxon polymorphism and important cross-reactions between taxa, a single DNA fragment was shown to be specific for M. capricolum subsp. capricolum and to be present in all M. capricolum subsp. capricolum field isolates tested in this study. A PCR assay targeting the corresponding gene (simpA51) was designed that resulted in a 560-bp amplification only in M. capricolum subsp. capricolum and in M. capricolum subsp. capripneumoniae (the etiological agent of contagious caprine pleuropneumonia). simpA51 was further improved to generate a multiplex PCR (multA51) that allows the differentiation of M. capricolum subsp. capripneumoniae from M. capricolum subsp. capricolum. Both the simpA51 and multA51 assays accurately identify M. capricolum subsp. capricolum among other mycoplasmas, including all members of the M. mycoides cluster. simpA51 and multA51 PCRs are proposed as sensitive and robust tools for the specific identification of M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae.The Mycoplasma genus is composed of wall-less bacteria with small genomes (0.58 to 1.35 Mb) and includes several species known to cause important diseases in humans and animals (24). Most of these species can be grown under laboratory conditions, although they require complex, undefined media and several days to several weeks of incubation because of their limited biosynthetic capacities (24). So far, mycoplasma identification and diagnosis have been based mainly on serological assays after cultivation, but recent ongoing efforts have been directed toward replacing these assays with more rapid and accurate molecular approaches based on the detection of specific DNA sequences.For the veterinary field, members of the so-called Mycoplasma mycoides cluster are of particular concern, because they are important ruminant pathogens that are responsible for economic losses worldwide (1, 28). This cluster includes six taxa, which are phylogenetically closely related based on their 16S rRNA gene sequences (21); present similar serological patterns associated with important intertaxon cross-reactivity (12); and, consequently, are difficult to differentiate. Four taxa contain pathogens for small ruminants, with three, namely, M. mycoides subsp. mycoides biotype Large Colony, M. mycoides subsp. capri,...

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confidence: 99%

Suppression-Subtractive Hybridization as a Strategy To Identify Taxon-Specific Sequences within the Mycoplasma mycoides Cluster: Design and Validation of an M. capricolum subsp. capricolum -Specific PCR Assay

Maigre¹,

Citti

Marenda

et al. 2008

J Clin Microbiol

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“…Moreover, antisera raised against recombinant LppA from M. capricolum subsp. capricolum (California kid) did not cross-react with LppA homologues in any other member of the M. mycoides cluster (Monnerat et al, 1999). LppB sequences from M. mycoides subsp.…”

Section: Discussionmentioning

confidence: 96%

“…capri and Mycoplasma sp. bovine group 7 (Monnerat et al, 1999) and unlike lppB, is located at a site unrelated to the glycerol uptake locus. Comparisons of the predicted amino acid sequences of LppA from type strains of Mycoplasma sp.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of a chromosomal region of Mycoplasma sp. bovine group 7 strain PG50 encoding a glycerol transport locus (gtsABC)

2003

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Mycoplasma species bovine group 7, represented by the type strain PG50, is one of six members of the Mycoplasma mycoides cluster and has been implicated in sporadic and outbreak cases of polyarthritis and mastitis in Australian dairy cattle. This study describes cloning and sequencing a 7?9 kb region of the PG50 chromosome and identification of genes involved in glycerol transport (gtsA, gtsB and gtsC) that are followed by a putative lipoprotein gene lppB and a genomic locus containing two ORFs encoding putative membrane proteins. Long range PCR using primers spanning gtsABC and downstream flanking genes, and Southern hybridization analyses using a suite of probes derived from M. mycoides subsp. mycoides small colony type (SC) strain Afadé for gtsA, gtsB and gtsC, lppB and the two downstream genes confirmed that these genes were conserved among Mycoplasma sp. bovine group 7 isolates and mycoplasmas belonging to the M. mycoides subcluster [M. mycoides subsp. mycoides SC, M. mycoides subsp. mycoides large colony type (LC) and M. mycoides subsp. capri ] but were absent in mycoplasmas belonging to the Mycoplasma capricolum subcluster (M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae). M. capricolum subsp. capricolum type strain California kid did not hybridize with the probe for gtsA and gave only weak or no hybridization signals with probes derived from the loci downstream of gtsABC, suggesting that this region has diverged in mycoplasmas belonging to subspecies of M. capricolum. It is shown that PG50, after the addition of a physiological concentration of glycerol to the growth medium, generates H 2 O 2 at levels comparable with strain Afadé , implying that the glycerol transport system is functional in Mycoplasma sp. bovine group 7. This suggests that in PG50, as in M. mycoides subsp. mycoides SC, glycerol uptake is followed by phosphorylation to glycerol 3-phosphate and then conversion to dihydroxyacetone phosphate, catalysed by L-a-glycerophosphate oxidase, resulting in the production of H 2 O 2 . The ability of Mycoplasma sp. bovine group 7 to generate significant amounts of hydrogen peroxide may be important in pathogenesis.

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“…In addition, two other antigenic proteins have been characterised thus far in M. mycoides subsp. mycoides SC: the 72 kDa antigen, also known as LppA [8,16,17] and the 48-kDa protein LppQ [2]. In an attempt to characterise the other major antigenic protein bands, we identified the lppC gene.…”

Section: Discussionmentioning

confidence: 99%

Antigenic and genetic characterisation of lipoprotein lppC from Mycoplasma mycoides subsp. mycoides SC

et al. 2003

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-Lipoprotein LppC, an immunodominant antigen, and its corresponding gene lppC were characterised in Mycoplasma mycoides subspecies mycoides small colony (SC) type, the etiological agent of contagious bovine pleuropneumonia (CBPP). The lppC gene was found in the type strain of M. mycoides subsp. mycoides SC and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. Southern blot analysis indicated the presence of at least four copies of lppC in the genome of M. mycoides subsp. mycoides SC, of which only one seems to be functional. Genes homologous to lppC have also been detected in closely related mycoplasmas such as M. mycoides subsp. mycoides large colony (LC) type and in M. sp. bovine group 7. LppC is encoded as a precursor with a consensus sequence for a prokaryotic signal peptidase II. The amino acid sequence of LppC and its precursor showed similarity to both LppB (at the N-terminal domain) and LppQ (at the C-terminal domain), two lipoproteins described previously in M. mycoides subsp. mycoides SC. The N-terminal domain of the mature LppC seems to be surface exposed. The C-terminal domain presented an integral membrane structure made up of five repeated units, rich in hydrophobic and aromatic amino acids, which may have pore forming potential in the mycoplasmal membrane. A recombinant peptide representing the N-terminal half of LppC was obtained following cloning in vector pETHIS-1 and expression in Escherichia coli hosts. The recombinant protein was used on immunoblots for serological analysis of sera from cattle that were naturally or experimentally infected with M. mycoides subsp. mycoides SC.Mycoplasma mycoides subsp. mycoides SC / contagious bovine pleuropneumonia / multiple copy gene / antigenic protein / lipoprotein

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Comparative analysis of the lppA locus in Mycoplasma capricolum subsp. capricolum and Mycoplasma capricolum subsp. capripneumoniae

Cited by 25 publications

References 38 publications

Suppression-Subtractive Hybridization as a Strategy To Identify Taxon-Specific Sequences within the Mycoplasma mycoides Cluster: Design and Validation of an M. capricolum subsp. capricolum -Specific PCR Assay

Suppression-Subtractive Hybridization as a Strategy To Identify Taxon-Specific Sequences within the Mycoplasma mycoides Cluster: Design and Validation of an M. capricolum subsp. capricolum -Specific PCR Assay

Characterization of a chromosomal region of Mycoplasma sp. bovine group 7 strain PG50 encoding a glycerol transport locus (gtsABC)

Antigenic and genetic characterisation of lipoprotein lppC from Mycoplasma mycoides subsp. mycoides SC

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