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Identification of Mycoplasma mycoides subsp. mycoides small colony genes coding for T-cell antigens.

ABSTRACT: Genes of the Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) coding for proteins capable of eliciting protective T-cell memory responses have potential for incorporation into a recombinant subunit vaccine against contagious bovine pleuropneumonia (CBPP). Here we used lymphocytes from cattle that had completely recovered from infection to screen products of MmmSC genes for recognition by CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes. Six MmmSC genes (abc, gapN, glpO, lppA, lppB, and ptsG) were expressed as histidine-tagged recombinant polypeptides, or synthetic overlapping peptides, before inclusion in proliferation and gamma interferon (IFN-gamma) assays. Only two MmmSC antigens, LppA and PtsG, consistently induced recall proliferation from immune CD4(+) T cells and IFN-gamma production in all animals tested. Moreover, LppA and PtsG were shown to possess epitopes recognized by both short-lived CD4(+) Tem and long-lived CD4(+) Tcm cells.

SUBMITTER: Totte P

PROVIDER: S-EPMC2916241 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Identification of Mycoplasma mycoides subsp. mycoides small colony genes coding for T-cell antigens.

Totté Philippe P Mather Arshad A Reslan Lina L Boublik Yvan Y Niang Mamadou M Du Plessis Dion D Dedieu Laurence L

Clinical and vaccine immunology : CVI 20100609 8

Genes of the Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) coding for proteins capable of eliciting protective T-cell memory responses have potential for incorporation into a recombinant subunit vaccine against contagious bovine pleuropneumonia (CBPP). Here we used lymphocytes from cattle that had completely recovered from infection to screen products of MmmSC genes for recognition by CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes. Six MmmSC genes (abc, ga ...[more]

PMID: 20534794

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Project description:BackgroundContagious bovine pleuropneumonia (CBPP) is a mycoplasmal disease caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC). Since the disease is a serious problem that can affect cattle production in parts of Africa, there is a need for an effective and economical vaccine. Identifying which of the causative agent's proteins trigger potentially protective immune responses is an important step towards developing a subunit vaccine. Accordingly, the purpose of this study was to determine whether phage display combined with bioinformatics could be used to narrow the search for genes that code for potentially immunogenic proteins of MmmSC. Since the production of IgG2 and IgA are associated with a Th1 cellular immune response which is implicated in protection against CBPP, antigens which elicit these immunoglobulin subclasses may be useful in developing a subunit vaccine.ResultsA filamentous phage library displaying a repertoire of peptides expressed by fragments of the genome of MmmSC was constructed. It was subjected to selection using antibodies from naturally- and experimentally-infected cattle. Mycoplasmal genes were identified by matching the nucleotide sequences of DNA from immunoselected phage particles with the mycoplasmal genome. This allowed a catalogue of genes coding for the proteins that elicited an immune response to be compiled. Using this method together with computer algorithms designed to score parameters that influence surface accessibility and hence potential antigenicity, five genes (abc, gapN, glpO, lppB and ptsG) were chosen to be expressed in Escherichia coli. After appropriate site-directed mutagenesis, polypeptides representing portions of each of these proteins were tested for immunoreactivity. Of these five, polypeptides representing expression products of abc and lppB were recognised on immunoblots by sera obtained from cattle during a natural outbreak of the disease.ConclusionSince phage display physically couples phenotype with genotype, it was used to compile a list of sequences that code for MmmSC proteins bearing epitopes which were recognised by antibodies in the serum of infected animals. Together with the appropriate bioinformatic analyses, this approach provided several potentially useful vaccine or diagnostic leads. The phage display step empirically identified sequences by their interaction with antibodies which accordingly reduced the number of ORFs that had to be expressed for testing. This is a particular advantage when working with MmmSC since the mycoplasmal codon for tryptophan needs to be mutated to prevent it from being translated as a stop in E. coli.

Project description:BACKGROUND: The Mycoplasma mycoides cluster consists of five species or subspecies that are ruminant pathogens. One subspecies, Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC), is the causative agent of contagious bovine pleuropneumonia. Its very close relative, Mycoplasma mycoides subsp. capri (Mmc), is a more ubiquitous pathogen in small ruminants causing mastitis, arthritis, keratitis, pneumonia and septicaemia and is also found as saprophyte in the ear canal. To understand the genetics underlying these phenotypic differences, we compared the MmmSC PG1 type strain genome, which was already available, with the genome of an Mmc field strain (95010) that was sequenced in this study. We also compared the 95010 genome with the recently published genome of another Mmc strain (GM12) to evaluate Mmc strain diversity. RESULTS: The MmmSC PG1 genome is 1,212 kbp and that of Mmc 95010 is ca. 58 kbp shorter. Most of the sequences present in PG1 but not 95010 are highly repeated Insertion Sequences (three types of IS) and large duplicated DNA fragments. The 95010 genome contains five types of IS, present in fewer copies than in PG1, and two copies of an integrative conjugative element. These mobile genetic elements have played a key role in genome plasticity, leading to inversions of large DNA fragments. Comparison of the two genomes suggested a marked decay of the PG1 genome that seems to be correlated with a greater number of IS. The repertoire of gene families encoding surface proteins is smaller in PG1. Several genes involved in polysaccharide metabolism and protein degradation are also absent from, or degraded in, PG1. CONCLUSIONS: The genome of MmmSC PG1 is larger than that of Mmc 95010, its very close relative, but has less coding capacity. This is the result of large genetic rearrangements due to mobile elements that have also led to marked gene decay. This is consistent with a non-adaptative genomic complexity theory, allowing duplications or pseudogenes to be maintained in the absence of adaptive selection that would lead to purifying selection and genome streamlining over longer evolutionary times. These findings also suggest that MmmSC only recently adapted to its bovine host.

Dataset Information

Identification of Mycoplasma mycoides subsp. mycoides small colony genes coding for T-cell antigens.

Publications

Identification of Mycoplasma mycoides subsp. mycoides small colony genes coding for T-cell antigens.

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