Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species

doi:10.1016/j.vetimm.2009.04.016

Veterinary Immunology and Immunopathology

Volume 131, Issues 3–4, 15 October 2009, Pages 238-245

https://doi.org/10.1016/j.vetimm.2009.04.016 Get rights and content

Abstract

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) has been eradicated in the developed world, but it is still present in many countries of sub-Saharan Africa. After initially successful control measures in the 1960s it has been spreading due to a lack of money, fragmentation of veterinary services, uncontrolled cattle movement, insufficient vaccine efficacy and sensitivity of current diagnostic tests.

In this study we used two-dimensional polyacrylamide gel electrophoresis followed by immunoblot with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry to identify novel immunogenic proteins as candidate molecules for improved diagnostics and vaccines. We identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease as well as severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Most immunogens showed compelling similarity to protein/gene sequences in the two ruminant pathogens M. capricolum subsp. capricolum and M. mycoides subsp. mycoides large colony type both belonging to the mycoides cluster. Three of these proteins, namely glycerol-3-phosphate oxidase, adenylosuccinate synthase, and glyceraldehyde-3-phosphate dehydrogenase, had no compelling homologue in the other distantly related bovine pathogen M. agalactiae. In addition, translation elongation factor Tu, heat shock protein 70, pyruvate dehydrogenase, and FKBP-type peptidyl-prolyl isomerase, which have been found to mediate adhesion to host tissue in other mycoplasmas were shown to be expressed and recognized by sera. These proteins have potential for the development of improved diagnostic tests and possibly vaccines.

Introduction

Contagious bovine pleuropneumonia (CBPP) is regarded as one of the most important livestock diseases in Africa according to the African Union through its Interafrican Bureau for Animal Resources. It affects many cattle stocks especially in sub-Saharan Africa (FAO, 2003) and is caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC). Recently, CBPP outbreaks have occurred in Portugal, Spain, France, and Italy and caused massive economic impact in affected regions for several years (Regalla et al., 1996). CBPP is characterised by acute pneumonia, high morbidity, and high mortality when introduced into a naïve herd. Some animals, however, survive acute disease and develop a chronic carrier state, which is characterised by the development of sequestered areas of necrotic tissue in the lung harbouring live MmmSC (Nicholas and Bashiruddin, 1995).

The disease was eliminated from developed countries using a ‘stamping-out’ policy, movement restriction of affected herds, and vaccination (Newton, 1992). Since a lack of resources for refunding farmers, fragmented veterinary services and ethical beliefs in most parts of Africa strongly object such a stamping-out policy (Thiaucourt et al., 2004), there is an urgent need to improve current control measures. The OIE-recommended diagnostic tests, namely the complement fixation test (CFT) and the competitive-ELISA (cELISA), are hampered by limited sensitivity (Marobela-Raborokgwe et al., 2003). The currently used live vaccines (T1₄₄, T1₄₄ SR) suffer from a limited duration of protection of only one year and the severe side effects they induce at the inoculation site (Tulasne et al., 1996).

Recently, attempts have been undertaken to elucidate host–pathogen interaction (Bischof et al., 2008, Pilo et al., 2005) and to identify pathogen-specific variable surface proteins (Hamsten et al., 2008), lipoproteins (Abdo et al., 2000, Dedieu et al., 2009, Pilo et al., 2003), and other proteins interacting with the host (March et al., 2006). The Mycoplasma-specific codon usage and the very high A + T content of 76% resulting in premature termination and aberrant internal transcription/translation of genomic libraries cloned in Escherichia coli have complicated the detection of immunogenic Mycoplasma proteins (Minion, 1998). In 2004 the full genome sequence of the M. mycoides subsp. mycoides SC type strain PG1 was published (Westberg et al., 2004) and paved the way for the combination of two-dimensional gel electrophoresis (2-DE), immunoblot, and mass spectrometry (MS) as a means to detect immunogenic proteins.

This manuscript describes the application of 2-DE, immunoblot and MS to detect immunogenic proteins, which might be useful for the development of next generation vaccines and diagnostics. Additionally, a sequence comparison of identified immunogens with the two closely related ruminant Mycoplasma species M. mycoides subsp. mycoides large colony type and M. capricolum subsp. capricolum (Manso-Silvan et al., 2007) as well as the bovine pathogen M. agalactiae PG2 was performed to assess their specificity.

Section snippets

Bacterial strains and culture conditions

The MmmSC type strain PG1^T was grown as stationary culture for 3 days at 37 °C in 1000 ml bottles in modified PH media (Kirchhoff and Rosengarten, 1984), supplemented with 20% horse serum (WDT, Garbsen, Germany). The cells were then centrifuged at 20,000 × g for 30 min at 4 °C and washed three times in phosphate-buffered saline (PBS; 150 mM NaCl, 1.5 mM KH₂PO₄, 9 mM Na₂HPO₄×12H₂O, 2.5 mM KCl [pH 7.2]). Washed cell pellets were stored at −20 °C.

Raising defined sera

A group of 15 cattle was infected as described previously (

2D gel electrophoresis and immunoblotting

Since no 2D proteome reference map of MmmSC is currently available, an overview of the total cell lysate protein distribution of MmmSC PG1^T was generated using broad-range 24 cm non-linear pH gradient (3–11 NL) strips. Protein preparations of whole cell lysates were obtained from cultures grown under standard conditions. 2-DE revealed approximately 300 prominent protein spots upon staining with colloidal Coomassie (Fig. 1A), and upon staining with the more sensitive silver stain approximately

Discussion

One way to identify immunogenic proteins potentially suited for the development of improved control measures is 2-DE combined with MS analyses. In mycoplasmas this approach is particularly attractive since the high A + T content of 76% (Westberg et al., 2004) and the use of TGA as a tryptophan codon rather than a stop codon (Minion, 1998) hampers a genetic approach using E. coli expression libraries. With the genome sequence of MmmSC PG1^T now published, the essential prerequisite for this

Acknowledgments

Joerg Jores was partly funded by the Bundesministerium für wirtschaftliche Zusammenarbeit und Entwicklung, BMZ (German Federal Ministry for Economic Cooperation and Development). The work was supported from the Advisory Service on Agricultural Research for Development of GTZ in Germany (project number 04.7860.2–001.00). This is ILRI publication number 200803.

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¹: J.J. and J.M. contributed equally to this work.

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Research paperAnalysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species

Abstract

Introduction

Section snippets

Bacterial strains and culture conditions

Raising defined sera

2D gel electrophoresis and immunoblotting

Discussion

Acknowledgments

Trends Microbiol.

J. Immunol. Methods

Vet. Immunol. Immunopathol.

Vet. Microbiol.

J. Comp. Pathol.

Microb. Pathog.

Vet. J.

Vet. Microbiol.

Vet. Microbiol.

Antigenic and genetic characterization of lipoprotein LppQ from Mycoplasma mycoides subsp. mycoides SC

Clin. Diagn. Lab. Immunol.

Pathogenicity of Mycoplasma capricolum subspecies capricolum for cattle immunosuppressed with Trypanosoma congolense

Isr. J. Vet. Med.

Surface localized glyceraldehyde-3-phosphate dehydrogenase of Mycoplasma genitalium binds mucin

Mol. Microbiol.

MolliGen, a database dedicated to the comparative genomics of Mollicutes

Nucleic Acids Res.

Cytotoxicity of Mycoplasma mycoides subsp. mycoides small colony type to bovine epithelial cells

Infect. Immun.

Members of the 70 kDa heat shock protein family specifically recognize sulfoglycolipids: role in gamete recognition and mycoplasma-related infertility

J. Cell Physiol.

Analysis of the Actinobacillus pleuropneumoniae ArcA regulon identifies fumarate reductase as a determinant of virulence

Infect. Immun.

Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis

Electrophoresis

Taxonomy of the Mycoplasma mycoides cluster

Isr. J. Med. Sci.

Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae

Mol. Microbiol.

Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides small colony identifies LppA as a major T-cell antigen

Comp. Immunol. Microbiol. Infect. Dis.

Contagious bovine pleuropneumonia

EMPRES Transbound. Animal Dis. Bull.

Research paper
Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species