Characterization of P40, a Cytadhesin of Mycoplasma agalactiae

Abstract

An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGA Trp codon to the universal TGG Trp codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.Mycoplasma agalactiae is a major pathogen of goats and sheep. It is found particularly in Mediterranean countries but is also reported from many other areas in the world. M. agalactiae causes contagious agalactia, a syndrome that primarily affects the mammary gland, but it can also affect joints, eyes and, in some cases, the lungs (6, 13). Contagious agalactia rises to a chronic state usually explained by the capacity of mycoplasmas to evade the host immune system. Different strategies are used by mycoplasmas for this purpose, including the phase and/or size variation of the membrane surface proteins, which leads to a constantly changing surface structure and the capacity of some lipoproteins to induce the expression of up-and downmodulating cytokines (32). However, a prerequisite for colonization and infection is adhesion to the host cell (32). Adhesion involves particular proteins named adhesins. In some mycoplasmas, such as Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum, adhesion results from the action of multiple proteins, leading to the movement and concentration of the adhesins at a specialized structure, the attachment tip organelle (7,20,21,25,35). In Mycoplasma hominis, adhesins are found dispersed over the cell surface and are directly involved in the attachment to the host cell (17, 31). The close contact between mycoplasma and the target cell membrane resulting from this adhesion is thought to favor the transfer or exchange of metabolic component...

Identification of a 349-Kilodalton Protein (Gli349) Responsible for Cytadherence and Glass Binding during Gliding of Mycoplasma mobile

“…This might be consistent with the fact that neither gliding motility nor a tapered cell pole has been reported for M. hyopneumoniae thus far. Several cytadherence proteins have been identified from M. pneumoniae (2,8,16) and some other mycoplasma species (4,10,12,15,40), but none have shown any similarities to Gli349 in size or in primary structure. This suggests that adhesion proteins are diversified among mycoplasma species.…”

Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples

“…The assay targets an M. agalactiae species-specific region of the p40 gene (GenBank accession no. AJ344229) encoding an immunodominant adhesin that plays a key role in cytoadhesion of M. agalactiae (8). Bacterial adhesion is a key mechanism of mycoplasma virulence, and the protein P40 displays a strong and persistent signal in response to antibodies; thus, the M. agalactiae p40 gene has been proposed as a good candidate for the development of future diagnostic assays (8).…”

“…AJ344229) encoding an immunodominant adhesin that plays a key role in cytoadhesion of M. agalactiae (8). Bacterial adhesion is a key mechanism of mycoplasma virulence, and the protein P40 displays a strong and persistent signal in response to antibodies; thus, the M. agalactiae p40 gene has been proposed as a good candidate for the development of future diagnostic assays (8). The p40 gene sequence displayed regions that were 100% specific to M. agalactiae by nucleotide sequence comparison using BLAST-N version 2.2.14 (National Centre for Biotechnology Information; www.ncbi.nlm.nih.gov).…”

Molecular Characterization of Mycoplasma agalactiae Reveals the Presence of an Endemic Clone in Spain

“…All the information regarding the sampling and the isolation procedure is detailed in the work of Ariza-Miguel et al (11). The species designation of the isolates was confirmed by real-time PCR targeting the p40 gene (12,13). All isolates were analyzed by PFGE with the restriction enzyme SmaI and by MLVA at 4 highly variable VNTR loci (i.e., MagaI VNTR 5, MagaI VNTR 14, MagaI VNTR 17, and MagaI VNTR 19) as previously described (3).…”