David Yogev

Epitope Mapping of Immunogenic and Adhesive Structures in Repetitive Domains of Mycoplasma bovis Variable Surface Lipoproteins

ABSTRACT The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units RA1, RA2, RA4.1, RB2.1, RE1, and RF1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion ofM. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (RA1, RA2, RA4.1, RB2.1, RE1, and RF1) and in RF2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer RF1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.

Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein

ABSTRACT A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed inEscherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.

Juxtaposition of an Active Promoter to vsp Genes via Site-Specific DNA Inversions Generates Antigenic Variation in Mycoplasma bovis

Mycoplasma bovis, the most important etiological agent of bovine mycoplasmosis, undergoes extensive antigenic variation of major and highly immunogenic surface lipoprotein antigens (Vsps). A family of 13 related but divergent vsp genes, which occur as single chromosomal copies, was recently found in the chromosome of M. bovis. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal isolates exhibiting phase transitions of VspA or of VspC (i.e., ON-->OFF-->ON) has shown that DNA inversions occur during Vsp phase variation. The upstream region of each vsp gene contains two sequence cassettes. The first (cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhibits 98% homology among all vsp genes, while the second (cassette no. 2), upstream of cassette no. 1, ranges in size from 50 to 180 bp and is more divergent. Examination of the ends of the inverted fragments during VspA or VspC phase variation revealed that in both cases, a change in the organization of vsp upstream cassettes involving three vsp genes had occurred. Primer extension and Northern blot analysis have shown that a specific cassette no. 2, designated A(2), is an active promoter and that juxtaposition of this regulatory element to a silent vsp gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that phase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present within the conserved cassette no. 1. A model for the control of Vsp phase variation is proposed.

In vivo variation of Mycoplasma gallisepticum antigen expression in experimentally infected chickens

The antigen expression profiles of Mycoplasma gallisepticum isolates obtained from tracheal swabs of chickens after aerosol-inoculation with M. gallisepticum strain R or clonal variant R/E were examined in western immunoblots. A reference anti-M. gallisepticum chicken antiserum and antisera from individual infected chickens as well as monoclonal antibodies (mAbs) specific for surface proteins were used to monitor in vivo antigenic variation. mAbs 1E5 and 12D8, recognizing PvpA and p67a, recently shown to undergo high-frequency in vitro phase variation, were used for consecutive staining of colony and western immunoblots in order to distinguish between the resultant phenotypes with respect to the corresponding epitopes. Marked differences in the expression of major immunogenic proteins, including p67a, were observed between the two inocula as well as among reisolates recovered at different times of infection. Comparative western immunoblot analysis of the rapidly changing chicken serum antibody response and reisolates recovered during the course of an experimental infection with M. gallisepticum R or clonal variant R/E suggest that immune modulation may have a key role in generating surface diversity. In addition, comparison of colony immunoblots of strain R inoculum and of reisolated colonies from tracheas of birds 8 days post infection indicated an in vivo selection of the PvpA+p67a- phenotype. This study established that surface antigens of M. gallisepticum are subjected in vivo to rapid alteration in their expression. This variability may function as a crucial adaptive mechanism, enabling the organism to escape from the host immune defense and to adapt to the changing host environment at different stages of a natural infection.

Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen

ABSTRACT A family of 13 related but divergent vsp genes was recently found in the chromosome of the bovine pathogenMycoplasma bovis. The vsp genomic locus was shown to undergo high-frequency rearrangements and to mediate phenotypic switching of variable lipoprotein antigens (Vsps) on the mycoplasma cell surface. Here we report that the vsp gene repertoire is subject to changes. Genetic analysis of M. bovis clonal isolates displaying distinct Vsp phenotypes showed that an intergenic recombination event between two closely related members of the vsp gene family, the formerly expressedvspA gene and the vspO gene, led to the formation of a new chimeric and functional vsp gene,vspC. The 5′ end of the recombination event was identified within the highly conserved vsp-upstream region, while the 3′ end was localized within the first repetitive domain (RA1) present in both vspA and vspOstructural genes. As a result, the vspC gene is an embodiment of the following domains: an N-terminus-encoding region linked to the highly conserved vsp-upstream region provided by the vspO gene; and a C-terminus-encoding region and the more distal and divergent vsp-upstream region acquired from the vspA gene. The generation of chimeric genes encoding surface antigens may provide an important element of genetic variation and an additional source of antigenic diversification within the mycoplasma population.

DNA probes for Mycoplasma gallisepticum and Mycoplasma synoviae: Application in experimentally infected chickens

DNA probes specific for Mycoplasma gallisepticum and M. synoviae were selected from genomic libraries prepared in the pUC13 vector. The probes hybridized with the DNA of a wide spectrum of strains within each homologous species, but did not react with the heterologous species or with DNA from any other avian mycoplasma or bacteria tested. Experimental infection and contact exposure of chickens to M. gallisepticum served as models to test the effectiveness of the DNA probe in diagnosis as compared with serological and culture detection methods carried out in parallel. A correlation was generally found between the level of M. gallisepticum in tracheal swabs and the effectiveness of the probe, although a predictably reactive level of mycoplasmas was not always detected. Treatment of clinical specimens with acetylcysteine to disrupt mucus improved the detection rate. Dot-blot hybridization with probe pMG4 enabled positive identification of M. gallisepticum at an early stage of infection, prior to the development of a serological response in the infected chicken. Results are obtainable within 4 days of sampling, much more rapidly than culture, and also in clinical specimens from which mycoplasma isolation is impossible, such as carcasses. The results indicate that the use of DNA probes for the early and rapid detection of M. gallisepticum infection is feasible; a development which can replace laborious culture techniques and less effective serological methods, and thus reduce the time required for diagnosis.

Genetic Mechanisms of Surface Variation

The mycoplasma cell surface is unique among prokaryotes and is highly evolved to accommodate the lifestyle of these diverse organisms during interaction with their respective vertebrate and plant hosts. Particularly in the absence of a cell wall, several adaptive features must be in place to support the survival of these organisms during transmission and residence in varied niches, both extracellular and in some cases within host cells (22, 24, 68). As with any pathogenic organism, the mycoplasma surface reflects the functional interaction of individual organisms with host compartments, including interactions with the innate and adaptive immune systems. Quite unlike other bacteria, however, all functions are expressed from remarkably limited gene sets that are rapidly becoming delineated through mycoplasma genome sequencing projects (11, 26, 28, 36). Insights from these projects as well as many experimental studies now reveal one striking aspect of mycoplasmal biology: the prevalence of mutation-based systems as an adaptive strategy for survival. In contrast to their eubacterial counterparts, mycoplasmal genomes so far have revealed few of the complex systems for classic gene regulation and environmental sensing that endow adaptive flexibility for individual bacterial cells.

Cytadherence-Deficient Mutants of Mycoplasma gallisepticum Generated by Transposon Mutagenesis

ABSTRACT Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gmr HA-negative (HA−) colonies displaying a stable HA− phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA− mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA− mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.

Intraspecies polymorphism of vsp genes and expression profiles of variable surface protein antigens (Vsps) in field isolates of Mycoplasma bovis

To assess the extent of interstrain variation, 50 isolates of Mycoplasma (M.) bovis including the type strain PG45 were examined for the presence of a family of variable membrane surface lipoproteins (Vsps) and their genes. Southern hybridization using a genomic fragment carrying three distinct vsp genes (vspAEF) revealed a striking heterogeneity, with only 2/50 strains having identical banding patterns. Cluster analysis of the data showed that most isolates from interrelated herds (groups 1, 2 and 3) were combined in a cluster of 50% homology, while isolates from distinct geographical regions (groups 4, 5 and 6) were linked only at 18% homology. Vsp antigen expression was monitored by Western immunoblotting using four specific monoclonal antibodies (MAbs). Resembling the findings at the DNA level, interstrain variation of Vsp expression among groups 1-3 was less pronounced than among non-interrelated isolates from groups 4-6. Ten out of 50 strains did not hybridize with the vspAEF gene probe at high-stringency conditions, 8/50 failed to react with any of the Vsp-related MAbs, and 6/50 proved negative in both assays. Interestingly, most of these isolates produced hybridization signals at low stringency suggesting major distinctions in their vsp gene structure. The extensive evidence obtained on interstrain vsp gene polymorphism and variation in Vsp expression could provide a basis for a future understanding of the pathogenic potential of individual M. bovis strains.

Genetic and antigenic relatedness between Mycoplasma gallisepticum and Mycoplasma synoviae

The two avian pathogens Mycoplasma gallisepticum and Mycoplasma synoviae were found, by Southern blot hybridization of their digested DNAs, to share genomic nucleotide sequences additional to those of the highly conserved ribosomal RNA genes. The assumption that some of the shared sequences encode for antigens or epitopes common to both mycoplasmas was supported by Western immunoblot analysis of cell proteins of one mycoplasma with specific antiserum to the other mycoplasma. Interestingly, the band patterns of reactive antigens were different for some of the M. gallisepticum strains, supporting the concept that the species is genotypically variable. The results of the present study may explain the cross-reactivity of the two mycoplasmas noted previously in a variety of routine serological tests.