Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein

https://doi.org/10.1016/S0378-1135(99)00164-9Get rights and content

Abstract

The gene encoding the P48 major surface lipoprotein of M. agalactiae has been recently characterised. Since its product plays an important role in the immune response of infected animals, in this study we analysed a recombinant P48 expressed in E. coli. Multiple point mutations were introduced by site directed mutagenesis in order to convert four tryptophan TGA codons, which are a typical feature of the mycoplasma genetic code, into the standard TGG. The mutated p48 gene was subcloned into pGex-2T and expressed in fusion with glutathione-S transferase. Following purification steps, P48 was eluted from carrier protein by thrombin digestion and used in Western blot and indirect ELISA using well-characterised sheep sera. Results demonstrate that specific antibodies against P48 are detected 3 weeks after onset of clinical disease and the recombinant P48 is a diagnostically relevant marker of M. agalactiae infection.

Introduction

Mycoplasma agalactiae is the etiological agent of contagious agalactia (CA) of sheep and goats, a chronic disease occurring in the Mediterranean basin, characterised by mastitis, arthritis and keratoconjunctivitis (Nicolet, 1994, Bergonier et al., 1997). Membrane surface lipoproteins play a fundamental role in the biology and pathogenesis of mycoplasma. Coat proteins, adhesins and transporters are typical mycoplasma membrane proteins, while phase variation and/or dysregulation of cytokine expression are features by which they may evade the host immune response (Razin et al., 1998).

In the last few years, different mycoplasma mechanisms that involve variability of membrane proteins have been studied at the gene level, and the immunomodulatory activities of such lipoproteins have been characterised in detail (Razin et al., 1998). Little is known of surface lipoproteins of M. agalactiae. Variable expression has been demonstrated by monoclonal antibodies in a low kDa range surface proteins (Bergonier et al., 1996), while highly immunogenic surface lipoproteins have been described by different authors in the range 45–55 kDa (Contini et al., 1989, Tola et al., 1997).

In order to characterise virulent factors involved in the pathogenesis of CA and to identify membrane proteins as potential candidate for the diagnosis and control of the infection, we recently cloned and sequenced the entire open reading frame encoding for the P48 lipoprotein of M. agalactiae (Rosati et al., 1999).

P48 is quantitatively the major Triton X-114 phase protein (Pittau et al., 1990); moreover, it is highly hydrophilic and shares conserved motifs with membrane proteins of M. fermentans (Matsumoto et al., 1998, Calcutt et al., 1999), whose biological activities are related to complement activation and cytokine induction. Previous Western blot (WB) analysis has also demonstrated that P48 is one of the antigens first recognised at the beginning of infection (Contini et al., 1989) and a membrane protein-based indirect Elisa has been proposed for diagnosis of CA (Cannas et al., 1992, Romano et al., 1995). This makes the P48 an ideal starting point to clarify pathogenetic mechanisms and immunomodulation in the course of infection.

Cloned genes usually allows the expression of recombinant product or its subunits for rapid antigenic and biological characterisation, since large amounts of protein can be rapidly expressed and purified. Mycoplasma genes are difficult to express in E. coli since the tryptophan codon UGA is used in place of UGG, a non rare feature in low G + C branch of eubacteria. As a consequence, mycoplasma proteins are usually expressed in a truncated form since E. coli will stop translation because UGA is treated as a stop codon (Razin et al., 1998). In order to accomplish a gene function study of P48 we cloned and expressed in E. coli a recombinant variant in which four UGA codons were selectively mutated into UGG. Antigenic characterisation of this recombinant product was also performed.

Section snippets

Plasmids and bacterial host

The plasmid pUC18 has been originally used to clone a 6.6 kb insert of M. agalactiae originated by Pst1 digestion of genomic DNA (pUC/6.6Kb-PstI). The fragment contains the whole open reading frame of p48 gene. The nucleotide sequence has been deposited in EMBL data base under accession number AJ132423. This plasmid was used as a template for site directed mutagenesis. The plasmid pGEX-2T (Pharmacia, Uppsala, Sweden) was used as a prokaryotic expression vector. Escherichia coli strain JM105 was

Results

Site directed mutagenesis performed with the method described is shown in Fig. 1b. After the second PCR run, a single band was detected of the expected length. Subsequently, it was efficiently cloned in the expression vector. Sequence analysis of insert confirm that all mutated nucleotides were correctly introduced and the gene was cloned in the expected frame. The GST/P48 fusion protein was expressed in soluble form in E. coli and migrated as <80 kDa in SDS-PAGE, corresponding to fusion of GST

Discussion

In this study, we cloned and expressed in E. coli the whole P48 gene of M. agalactiae, an immunodominant surface antigen that, in its recombinant form, demonstrated it to be a diagnostically relevant marker of CA infection. Several features seem to support this finding. The r-P48 does not fall in the size range of variable surface antigens and mechanisms of variation, such as inversion, insertion, repetition of polymeric sequences, frameshift mutations, have not been found at the genetic level (

References (24)

  • E. Cannas et al.

    Antibody detection by ELISA using membrane proteins of M. agalactiae

    Proc. Ann. Meet. Ital. Soc. Vet. Sci.

    (1992)
  • Cody and Smith, 1991. Applied statistics and the SAS® programming language, 3rd Edn., Prentice Hall, Englewood Cliff,...
  • Cited by (0)

    View full text