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Genetic and biochemical characterization of glycerol uptake in mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence.

ABSTRACT: Highly virulent strains of Mycoplasma mycoides subsp. mycoides SC belonging to the African cluster contain an operon with the genes gtsA, gtsB, and gtsC, encoding membrane ATP binding cassette transporter proteins GtsA, GtsB, and GtsC, which are involved in glycerol transport. Strain Afadé from the African cluster incorporated [U-(14)C]glycerol with a time-dependent increase. The less virulent strain L2 of the European cluster, which lacks gtsB and gtsC, failed to incorporate glycerol. Antibodies against GtsB noncompetitively inhibited glycerol uptake. L-alpha-Glycerophosphate was not transported by M. mycoides subsp. mycoides SC. It is postulated to be synthesized by phosphorylation of glycerol during transport and subsequently metabolized further to dihydroxyacetone phosphate accompanied by release of H(2)O(2). Peroxide production in glycerol-containing growth medium was high for the African strain Afadé but very low for the European strain L2. Virtually no H(2)O(2) was produced by both strains without glycerol. Hence, the efficient glycerol uptake system found in the virulent strain of the African cluster leads to a strong release of peroxide, a potential virulence factor which is lacking in the less virulent European strains. M. mycoides subsp. mycoides SC might have adopted, as a strategy for virulence, a highly efficient uptake system for glycerol which allows the production of an active metabolic intermediate that damages host cells.

SUBMITTER: Vilei EM

PROVIDER: S-EPMC96015 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Genetic and biochemical characterization of glycerol uptake in mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence.

Vilei E M EM Frey J J

Clinical and diagnostic laboratory immunology 20010101 1

Highly virulent strains of Mycoplasma mycoides subsp. mycoides SC belonging to the African cluster contain an operon with the genes gtsA, gtsB, and gtsC, encoding membrane ATP binding cassette transporter proteins GtsA, GtsB, and GtsC, which are involved in glycerol transport. Strain Afadé from the African cluster incorporated [U-(14)C]glycerol with a time-dependent increase. The less virulent strain L2 of the European cluster, which lacks gtsB and gtsC, failed to incorporate glycerol. Antibodie ...[more]

PMID: 11139200

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Project description:BackgroundContagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204.ResultsStrains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204.ConclusionOur results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia.

Project description:Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical ?(1->6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.

Project description:BACKGROUND: Mycoplasma mycoides subsp. mycoides SC is the pathogenic agent of contagious bovine pleuropneumonia (CBPP), the most important disease of cattle in Africa causing significant economic losses. The re-emergence of CBPP in Europe in the 1980s and 1990s illustrates that it is still a threat also to countries that have successfully eradicated the disease in the past. Nowadays, probe-based real-time PCR techniques are among the most advanced tools for a reliable identification and a sensitive detection of many pathogens, but only few protocols have been published so far for CBPP diagnosis. Therefore we developed a novel TaqMan®-based real-time PCR assay comprising the amplification of two independent targets (MSC_0136 and MSC_1046) and an internal exogenous amplification control in a multiplex reaction and evaluated its diagnostic performance with clinical samples. RESULTS: The assays detected 49 MmmSC strains from diverse temporal and geographical origin, but did not amplify DNA from 82 isolates of 20 non-target species confirming a specificity of 100%. The detection limit was determined to be 10 fg DNA per reaction for the MSC_0136 assay and 100 fg per reaction for the MSC_1046 assay corresponding to 8 and 80 genome equivalents, respectively. The diagnostic performance of the assay was evaluated with clinical samples from 19 experimentally infected cattle and from 20 cattle without CBPP and compared to those of cultivation and a conventional PCR protocol. The two rt-PCR tests proved to be the most sensitive methods and identified all 19 infected animals. The different sample types used were not equally suitable for MmmSC detection. While 94.7% of lung samples from the infected cohort were positively tested in the MSC_0136 assay, only 81% of pulmonal lymph nodes, 31% of mediastinal lymph nodes and 25% of pleural fluid samples gave a positive result. CONCLUSIONS: The developed multiplex rt-PCR assay is recommended as an efficient tool for rapid confirmation of a presumptive CBPP diagnosis in a well-equipped laboratory environment.

Dataset Information

Genetic and biochemical characterization of glycerol uptake in mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence.

Publications

Genetic and biochemical characterization of glycerol uptake in mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence.

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