Mycoplasma agalactiae Vaccines: Current Status, Hurdles, and Opportunities Due to Advances in Pathogenicity Studies.

Contagious agalactia (CA) is a serious multietiological disease whose classic etiological agent is Mycoplasma agalactiae and which causes high morbidity and mortality rates in infected herds. CA is classified as a notifiable disease by the World Organization for Animal Health due to its significant worldwide economic impact on livestock, primarily involving goat and sheep farms. The emergence of atypical symptoms and strains of M. agalactiae in wildlife ungulates reestablishes its highly plastic genome and is also of great epidemiological significance. Antimicrobial therapy is the main form of control, although several factors, such as intrinsic antibiotic resistance and the selection of resistant strains, must be considered. Available vaccines are few and mostly inefficient. The virulence and pathogenicity mechanisms of M. agalactiae mainly rely on surface molecules that have direct contact with the host. Because of this, they are essential for the development of vaccines. This review highlights the currently available vaccines and their limitations and the development of new vaccine possibilities, especially considering the challenge of antigenic variation and dynamic genome in this microorganism.

Serum Resistance of Mycoplasma agalactiae Strains and Mutants Bearing Different Lipoprotein Profiles.

In order to spread systemically, resistance against complement and other factors present in serum is an important trait in pathogenic bacteria. The variable proteins of Mycoplasma agalactiae (Vpmas) have been shown to affect differential adhesion, invasion and immune evasion, and undergo high-frequency phase-variation in expression. However, nothing is known about their involvement in M. agalactiae's serum susceptibility. To evaluate this, the PG2 strain, the GM139 strain and the six Vpma phase-locked mutants (PLMs, PLMU to PLMZ) were tested for their ability to survive in the presence of non-sensitized and sensitized sheep serum, as well as guinea pig complement. Additionally, the reactivity of the sensitized sheep serum was analysed on the strains via western blotting. Overall data demonstrate PG2 strain to be more susceptible to sheep serum compared to the GM139 strain bearing a different Vpma profile. Significant differences were also observed between the different PLMs, with PLMU and PLMX showing the highest serum susceptibility in serum, while the other PLMs expressing longer Vpma proteins were more resistant. The results are in good correlation with previous studies where shorter lipoprotein variants contributed to a higher susceptibility to complement. Since none of the tested strains and PLMs were susceptible to non-sensitized sheep serum, antibodies seem to play an important role in serum killing.

Sheep Infection Trials with 'Phase-Locked' Vpma Expression Variants of Mycoplasma agalactiae-Towards Elucidating the Role of a Multigene Family Encoding Variable Surface Lipoproteins in Infection and Disease.

The significance of large multigene families causing high-frequency surface variations in mycoplasmas is not well-understood. Previously, VpmaY and VpmaU clonal variants of the Vpma family of lipoproteins of M. agalactiae were compared via experimental sheep infections using the two corresponding 'Phase-Locked Mutants'. However, nothing is known about the infectivity of the remaining four Vpma expression variants VpmaX, VpmaW, VpmaZ and VpmaV as they were never evaluated in vivo. Here, in vivo infection and disease progression of all six Vpma expressers constituting the Vpma family of type strain PG2 were compared using the corresponding xer1-disrupted PLMs expressing single well-characterized Vpmas. Each of the six PLMs were separately evaluated using the intramammary sheep infection model along with the control phase-variable wildtype strain PG2. Thorough bacteriological, pathological and clinical examinations were performed, including assessment of milk quality, quantity and somatic cell counts. Altogether, the results indicated that the inability to vary the Vpma expression phase does not hamper the initiation of infection leading to mastitis for all six PLMs, except for PLMU, which showed a defect in host colonization and multiplication for the first 24 h p.i. and pathological/bacteriological analysis indicated a higher potential for systemic spread for PLMV and PLMX. This is the first study in which all isogenic expression variants of a large mycoplasma multigene family are tested in the natural host.

Host cell interactions of novel antigenic membrane proteins of Mycoplasma agalactiae.

BACKGROUND: Mycoplasma agalactiae is the main etiological agent of Contagious Agalactia syndrome of small ruminants notifiable to the World Organization for Animal Health. Despite serious economic losses, successful vaccines are unavailable, largely because its colonization and invasion factors are not well understood. This study evaluates the role of two recently identified antigenic proteins (MAG_1560, MAG_6130) and the cytadhesin P40 in pathogenicity related phenotypes. RESULTS: Adhesion to HeLa and sheep primary mammary stromal cells (MSC) was evaluated using ELISA, as well as in vitro adhesion assays on monolayer cell cultures. The results demonstrated MAG_6130 as a novel adhesin of M. agalactiae whose capacity to adhere to eukaryotic cells was significantly reduced by specific antiserum. Additionally, these proteins exhibited significant binding to plasminogen and extracellular matrix (ECM) proteins like lactoferrin, fibrinogen and fibronectin, a feature that could potentially support the pathogen in host colonization, tissue migration and immune evasion. Furthermore, these proteins played a detrimental role on the host cell proliferation and viability and were observed to activate pro-apoptotic genes indicating their involvement in cell death when eukaryotic cells were infected with M. agalactiae. CONCLUSIONS: To summarize, the hypothetical protein corresponding to MAG_6130 has not only been assigned novel adhesion functions but together with P40 it is demonstrated for the first time to bind to lactoferrin and ECM proteins thereby playing important roles in host colonization and pathogenicity.

Predominant Single Stable VpmaV Expression in Strain GM139 and Major Differences with Mycoplasma agalactiae Type Strain PG2.

Although mycoplasmas have a reduced genome and no cell wall, they have important mechanisms for the antigenic variation in surface lipoproteins that modulate their interactions with the host. Mycoplasma agalactiae, the main etiological agent of contagious agalactia, has a multigene family involved in the high-frequency phase variation in surface lipoproteins called variable proteins of M. agalactiae (Vpmas). The Vpma lipoproteins are involved in the immune evasion, colonization, dissemination, and persistence of M. agalactiae in the host. In this paper, we evaluate the Vpma phenotypic profiles of two different strains of M. agalactiae, namely, GM139 and the type strain PG2, to assess possible correlations between Vpma phase variability and the geographic localization, animal origin, and pathogenicity of these two strains. Using monospecific Vpma antibodies against individual Vpmas in immunoblots, we demonstrate that, unlike PG2, which expresses six Vpma proteins with high-frequency phase variation, colonies of GM139 predominantly express VpmaV and do not exhibit any sectoring phenotype for any Vpma. Since VpmaV is one of the most important Vpmas for cell adhesion and invasion, its predominant sole expression in GM139 without high-frequency variation may be the basis of the differential pathogenicity of GM139 and PG2. Additionally, MALDI-ToF MS analysis also demonstrates significant differences between these two strains and their relatedness with other M. agalactiae strains.

Mycoplasma tauri sp. nov. isolated from the bovine genital tract.

Since 2006, a Mycoplasma species unidentifiable to the species level has been regularly isolated from the semen and prepuce of apparently healthy bulls, and occasionally from cattle displaying inflammatory disease of the genital tract. Seven of these Mycoplasma isolates were subjected to a comprehensive taxonomic study. The strains investigated grew well in modified Hayflick's medium and colonies on agar exhibited typical fried egg morphology and produced 'film and spots'. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas with spherically shaped cells bounded by a bi-layered cell membrane. The strains studied neither produced acid from sugar carbon sources nor did hydrolyse arginine or urea, and genome annotation indicated that organic acids (pyruvate, lactate) are used as energy sources. Phylogenetic analyses of 16S rRNA gene sequences, the 16S-23S intergenic spacer region, and partial rpoB gene and protein sequences placed the strains within the Mycoplasma (M.) bovis cluster of the Hominis group with M. primatum, M. agalactiae, and M. bovis being their closest relatives. Genomic information including whole-genome similarity metrics (ANIb, ANIm, TETRA, dDDH, AAI) and phylogenomics, proteomic features revealed by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry as well as serological reactions and polar lipid profiling strongly indicated that the strains examined were representatives of a hitherto unclassified species of genus Mycoplasma, for which the name Mycoplasma tauri sp. nov. with type strain Zaradi2T (=ATCC BAA-1891T = DSM 22451T) is proposed.

Novel antigenic proteins of Mycoplasma agalactiae as potential vaccine and serodiagnostic candidates.

Contagious agalactia (CA) is a serious disease notifiable to the World Organisation for Animal Health (OIE) causing severe economic losses to sheep and goat producers worldwide. Mycoplasma agalactiae, considered as its main etiological agent, inflicts a variety of symptoms in infected animals, including keratoconjunctivitis, mastitis, arthritis, ankylosis, abortions, stillbirths and granular vulvovaginitis. Despite its significance, developing a successful vaccine remains elusive, mostly due to the lack of knowledge about M. agalactiae's pathogenicity factors and pathogenic mechanisms, including its "core" antigens. The aim of this study was to identify, characterize and express antigenic proteins of M. agalactiae as potential vaccine candidates. Predicted proteins of type strain PG2 were analyzed using bioinformatic algorithms to assess their cellular localization and to identify their linear and conformational epitopes for B cells. Out of a total of 156 predicted membrane proteins, three were shortlisted as potential antigenic surface proteins, namely [MAG_1560 (WP_011949336.1), MAG_6130 (WP_011949770.1) and P40 (WP_011949418.1)]. These proteins were expressed in recombinant Escherichia coli strains. Purified proteins were evaluated for their antigenicity using Western blot and ELISA using sera of M. agalactiae-naturally infected and non-infected sheep and goats. All 3 proteins were specifically recognized by the tested sera of M. agalactiae-infected animals. Also, specific rabbit antisera raised against each of these 3 proteins confirm their membrane localization using TritonX-114 phase partioning, Western and colony immunoblotting. In conclusion, our study successfully identified P40 (as proof of concept and validation) and two novel antigenic M. agalactiae proteins as potential candidates for developing effective CA vaccines.

Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants.

Mycoplasma agalactiae exhibits antigenic variation by switching the expression of multiple surface lipoproteins called Vpmas. Although implicated to have a significant influence on the pathogenicity, their exact role in pathogen-host interactions has not been investigated so far. Initial attachment to host cells is regarded as one of the most important steps for colonization but this pathogen lacks the typical mycoplasma attachment organelle. The aim of this study was to determine the role of Vpmas in adhesion of M. agalactiae to host cells. 'Phase-Locked' Mutants (PLMs) steadily expressing single well-characterized Vpma lipoproteins served as ideal tools to evaluate the role of each of the six Vpmas in cytadhesion, which was otherwise not possible due to the high-frequency switching of Vpmas in the wildtype strain PG2. Using in vitro adhesion assays with HeLa and sheep mammary epithelial (MECs) and stromal (MSCs) cells, we could demonstrate differences in the adhesion capabilities of each of the six PLMs compared to the wildtype strain. The PLMV mutant expressing VpmaV exhibited the highest adhesion rate, whereas PLMU, which expresses VpmaU showed the lowest adhesion values explaining the reduced in vivo fitness of PLMU in sheep during experimental intramammary and conjunctival infections. Furthermore, adhesion inhibition assays using Vpma-specific polyclonal antisera were performed to confirm the role of Vpmas in M. agalactiae cytadhesion. This led to a significant decrease (p<0.05) in the adhesion percentage of each PLM. Immunofluorescence staining of TX-114 phase proteins extracted from each PLM showed binding of the respective Vpma to HeLa cells and MECs proving the direct role of Vpmas in cytadhesion. Furthermore, as adhesion is a prerequisite for cell invasion, the ability of the six PLMs to invade HeLa cells was also evaluated using the gentamicin protection assay. The results showed a strong correlation between the adhesion rates and invasion frequencies of the individual PLMs. This is the first report that describes a novel function of Vpma proteins in cell adhesion and invasion. Besides the variability of these proteins causing surface antigenic variation, the newly identified phenotypes are likely to play critical roles in the pathogenicity potential of this ruminant pathogen.

Xer1-independent mechanisms of Vpma phase variation in Mycoplasma agalactiae are triggered by Vpma-specific antibodies.

Despite their small genomes mycoplasmas maintain large multigene families devoted to surface antigenic variation. Although implicated as important factors for mycoplasma pathogenicity and persistence, the role of these antigenic switches in host immune evasion has never been unequivocally proven in these minimalist microbes. Mycoplasma agalactiae exhibits antigenic variation due to Xer1-mediated site-specific DNA inversions of vpma genes encoding abundant multiple surface lipoproteins. To evaluate the biological significance of Vpma oscillations the xer1 recombinase gene has been disrupted in earlier studies to abolish Vpma switching and to generate stable phase-locked mutants (PLMs) steadily expressing a single Vpma product. However, in previous animal infection studies, surprisingly these PLMs switched to new different Vpma phenotypes. The aim of the current study was to demonstrate the influence of anti-Vpma antibodies on change of Vpma expression in PLMs as well as on the wildtype strain. In in vitro assays it is shown that wild type M. agalactiae escapes the negative effects of Vpma-specific antibodies by high-frequency Xer1-mediated switching to alternative Vpma phenoytpes. Even for Xer1-disrupted PLMs that stably expressed the same Vpma for several in vitro generations, the presence of the corresponding Vpma-specific antibody caused repression of the target Vpma and induction of new Vpma phenotypes by novel complex vpma rearrangements like intragenic deletions and gene chimeras. These Xer1-independent vpma recombinations correlated very well with similar PLM switches observed in vivo in an earlier independent study, clearly demonstrating that Vpma phase variation is necessary to express 'Vpma immune evasion proteins' in order to escape the immune response and to survive in the immunocompetent host. The data clearly demonstrate that although the Xer1 recombinase is the sole factor responsible for Vpma switching of wild type M. agalactiae in vitro, other alternative molecular switches operate in its absence under the selective pressure of the immune response. Furthermore, this evasion from the immune attack of the host involves complex vpma rearrangements, a causal relationship that was so far never demonstrated for M. agalactiae, thereby illustrating novel features of its regulation under immune pressure. The results are anticipated to have a direct impact on understanding the in vivo role of surface antigenic variation systems and the immune evasion tactics of other pathogenic mycoplasma species.

Vpma phase variation is important for survival and persistence of Mycoplasma agalactiae in the immunocompetent host.

Despite very small genomes, mycoplasmas retain large multigene families encoding variable antigens whose exact role in pathogenesis needs to be proven. To understand their in vivo significance, we used Mycoplasma agalactiae as a model exhibiting high-frequency variations of a family of immunodominant Vpma lipoproteins via Xer1-mediated site-specific recombinations. Phase-Locked Mutants (PLMs) expressing single stable Vpma products served as first breakthrough tools in mycoplasmology to study the role of such sophisticated antigenic variation systems. Comparing the general clinical features of sheep infected with a mixture of phase-invariable PLMs (PLMU and PLMY) and the wild type strain, it was earlier concluded that Vpma phase variation is not necessary for infection. Conversely, the current study demonstrates the in vivo indispensability of Vpma switching as inferred from the Vpma phenotypic and genotypic analyses of reisolates obtained during sheep infection and necropsy. PLMY and PLMU stably expressing VpmaY and VpmaU, respectively, for numerous in vitro generations, switched to new Vpma phenotypes inside the sheep. Molecular genetic analysis of selected 'switchover' clones confirmed xer1 disruption and revealed complex new rearrangements like chimeras, deletions and duplications in the vpma loci that were previously unknown in type strain PG2. Another novel finding is the differential infection potential of Vpma variants, as local infection sites demonstrated an almost complete dominance of PLMY over PLMU especially during early stages of both conjunctival and intramammary co-challenge infections, indicating a comparatively better in vivo fitness of VpmaY expressors. The data suggest that Vpma antigenic variation is imperative for survival and persistence inside the immunocompetent host, and although Xer1 is necessary for causing Vpma variation in vitro, it is not a virulence factor because alternative Xer1-independent mechanisms operate in vivo, likely under the selection pressure of the host-induced immune response. This singular study highlights exciting new aspects of mycoplasma antigenic variation systems, including the regulation of expression by host factors.

Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection.

Mycoplasma agalactiae is a worldwide serious pathogen of small ruminants that usually spreads through the mammary route causing acute to subacute mastitis progressing to chronic persistent disease that is hard to eradicate. Knowledge of mechanisms of its pathogenesis and persistence in the mammary gland are still insufficient, especially the host-pathogen interplay that enables it to reside in a chronic subclinical state. This study reports transcriptome profiling of mammary tissue from udders of sheep experimentally infected with M. agalactiae type strain PG2 in comparison with uninfected control animals using Illumina RNA-sequencing (RNA-Seq). Several differentially expressed genes (DEGs) were observed in the infected udders and RT-qPCR analyses of selected DEGs showed their expression profiles to be in agreement with results from RNA-Seq. Gene Ontology (GO) analysis revealed majority of the DEGs to be associated with mycoplasma defense responses that are directly or indirectly involved in host innate and adaptive immune responses. Similar RNA-Seq analyses were also performed with spleen cells of the same sheep to know the specific systemic transcriptome responses. Spleen cells exhibited a comparatively lower number of DEGs suggesting a less prominent host response in this organ. To our knowledge this is the first study that describes host transcriptomics of M. agalactiae infection and the related immune-inflammatory responses. The data provides useful information to further dissect the molecular genetic mechanisms underlying mycoplasma mastitis, which is a prerequisite for designing effective intervention strategies.

Genetic loci of Mycoplasma agalactiae involved in systemic spreading during experimental intramammary infection of sheep.

Mycoplasmas are amongst the most successful pathogens of both humans and animals yet the molecular basis of mycoplasma pathogenesis is poorly understood. This is partly due to the lack of classical virulence factors and little similarity to common bacterial pathogenic determinants. Using Mycoplasma agalactiae as a model we initiated research in this direction by screening a transposon mutant library in the natural sheep host using a negative selection method. Having successfully identified putative factors involved in the colonization of local infection and lymphogenic sites, the current study assessed mutants unable to spread systemically in sheep after experimental intramammary infection. Analysis of distant body sites for complete absence of mutants via SSM PCR revealed that additional set of genes, such as pdhB, oppC, oppB, gtsB, MAG1890, MAG5520 and MAG3650 are required for systemic spreading apart from those that were necessary for initial colonization. Additional in vitro studies with the mutants absent at these systemic sites confirmed the potential role of some of the respective gene products concerning their interaction with host cells. Mutants of pdhB, oppC and MAG4460 exhibited significantly slower growth in the presence of HeLa cells in MEM medium. This first attempt to identify genes exclusively required for systemic spreading provides a basis for further in-depth research to understand the exact mechanism of chronicity and persistence of M. agalactiae.

Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro.

Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae's induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection.

Sheep primary cells as in vitro models to investigate Mycoplasma agalactiae host cell interactions.

Appropriate infection models are imperative for the understanding of pathogens like mycoplasmas that are known for their strict host and tissue specificity, and lack of suitable cell and small animal models has hindered pathogenicity studies. This is particularly true for the economically important group of ruminant mycoplasmas whose virulence factors need to be elucidated for designing effective intervention strategies. Mycoplasma agalactiae serves as a useful role model especially because it is phylogenetically very close to M. bovis and causes similar symptoms by as yet unknown mechanisms. Here, we successfully prepared and characterized four different primary sheep cell lines, namely the epithelial and stromal cells from the mammary gland and uterus, respectively. Using immunohistochemistry, we identified vimentin and cytokeratin as specific markers to confirm the typical cell phenotypes of these primary cells. Furthermore, M. agalactiae's consistent adhesion and invasion into these primary cells proves the reliability of these cell models. Mimicking natural infections, mammary epithelial and stromal cells showed higher invasion and adhesion rates compared to the uterine cells as also seen via double immunofluorescence staining. Altogether, we have generated promising in vitro cell models to study host-pathogen interactions of M. agalactiae and related ruminant pathogens in a more authentic manner.

Simultaneous Identification of Potential Pathogenicity Factors of Mycoplasma agalactiae in the Natural Ovine Host by Negative Selection.

Mycoplasmas possess complex pathogenicity determinants that are largely unknown at the molecular level. Mycoplasma agalactiae serves as a useful model to study the molecular basis of mycoplasma pathogenicity. The generation and in vivo screening of a transposon mutant library of M. agalactiae were employed to unravel its host colonization factors. Tn4001mod mutants were sequenced using a novel sequencing method, and functionally heterogeneous pools containing 15 to 19 selected mutants were screened simultaneously through two successive cycles of sheep intramammary infections. A PCR-based negative selection method was employed to identify mutants that failed to colonize the udders and draining lymph nodes in the animals. A total of 14 different mutants found to be absent from ≥ 95% of samples were identified and subsequently verified via a second round of stringent confirmatory screening where 100% absence was considered attenuation. Using this criterion, seven mutants with insertions in genes MAG1050, MAG2540, MAG3390, uhpT, eutD, adhT, and MAG4460 were not recovered from any of the infected animals. Among the attenuated mutants, many contain disruptions in hypothetical genes, implying their previously unknown role in M. agalactiae pathogenicity. These data indicate the putative role of functionally different genes, including hypothetical ones, in the pathogenesis of M. agalactiae. Defining the precise functions of the identified genes is anticipated to increase our understanding of M. agalactiae infections and to develop successful intervention strategies against it.

Disruption of the pdhB pyruvate dehydrogenase [corrected] gene affects colony morphology, in vitro growth and cell invasiveness of Mycoplasma agalactiae.

The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae.

In vitro and in vivo cell invasion and systemic spreading of Mycoplasma agalactiae in the sheep infection model.

Generally regarded as extracellular pathogens, molecular mechanisms of mycoplasma persistence, chronicity and disease spread are largely unknown. Mycoplasma agalactiae, an economically important pathogen of small ruminants, causes chronic infections that are difficult to eradicate. Animals continue to shed the agent for several months and even years after the initial infection, in spite of long antibiotic treatment. However, little is known about the strategies that M. agalactiae employs to survive and spread within an immunocompetent host to cause chronic disease. Here, we demonstrate for the first time its ability to invade cultured human (HeLa) and ruminant (BEND and BLF) host cells. Presence of intracellular mycoplasmas is clearly substantiated using differential immunofluorescence technique and quantitative gentamicin invasion assays. Internalized M. agalactiae could survive and exit the cells in a viable state to repopulate the extracellular environment after complete removal of extracellular bacteria with gentamicin. Furthermore, an experimental sheep intramammary infection was carried out to evaluate its systemic spread to organs and host niches distant from the site of initial infection. Positive results obtained via PCR, culture and immunohistochemistry, especially the latter depicting the presence of M. agalactiae in the cytoplasm of mammary duct epithelium and macrophages, clearly provide the first formal proof of M. agalactiae's capability to translocate across the mammary epithelium and systemically disseminate to distant inner organs. Altogether, the findings of these in vitro and in vivo studies indicate that M. agalactiae is capable of entering host cells and this might be the strategy that it employs at a population level to ward off the host immune response and antibiotic action, and to disseminate to new and safer niches to later egress and once again proliferate upon the return of favorable conditions to cause persistent chronic infections.

Role of Vpma phase variation in Mycoplasma agalactiae pathogenesis.

Compared with other bacterial pathogens, the molecular mechanisms of mycoplasma pathogenicity are largely unknown. Several studies in the past have shown that pathogenic mycoplasmas are equipped with sophisticated genetic systems that allow them to undergo high-frequency surface antigenic variations. Although never clearly proven, these variable mycoplasma surface components are often implicated in host immune evasion and adaptation. Vpma surface lipoproteins of the ruminant pathogen Mycoplasma agalactiae are encoded on a genomic pathogenicity island-like locus and are considered as one of the well-characterized model systems of mycoplasma surface antigenic variation. The present study assesses the role of these phase-variable Vpmas in the molecular pathogenesis of M. agalactiae by testing the wild-type strain PG2 in comparison with the xer1-disrupted Vpma 'phase-locked' mutants in sheep infection models. The data clearly illustrate that although Xer1 recombinase is not a virulence factor of M. agalactiae and Vpma phase variation is not necessary for establishing an infection, it might critically influence the survival and persistence of the pathogen under natural field conditions, mainly due to a better capacity for dissemination and evoking systemic responses. This is the first study where mycoplasma 'phase-locked' mutants are tested in vivo to elucidate the role of phase variation during infection.

Xer1-mediated site-specific DNA inversions and excisions in Mycoplasma agalactiae.

Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5'-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery.

Critical role of RPI1 in the stress tolerance of yeast during ethanolic fermentation.

Stress tolerance of yeast Saccharomyces cerevisiae during ethanolic fermentation is poorly understood due to the lack of genetic screens and conventional plate assays for studying this phenotype. We screened a genomic expression library of yeast to identify gene(s) that, upon overexpression, would prolong the survival of yeast cells during fermentation, with the view to understand the stress response better and to use the identified gene(s) in strain improvement. The yeast RPI1 (Ras-cAMP pathway inhibitor 1) gene was identified in such a screen performed at 38 degrees C; introducing an additional copy of RPI1 with its native promoter helped the cells to retain their viability by over 50-fold better than the wild type (WT) parent strain, after 36 h of fermentation at 38 degrees C. Disruption of RPI1 resulted in a drastic reduction in viability during fermentation, but not during normal growth, further confirming the role of this gene in fermentation stress tolerance. This gene seems to improve viability by fortifying the yeast cell wall, because RPI1 overexpression strain is highly resistant to cell lytic enzyme zymolyase, compared with the WT strain. As the RPI1 overexpression strain substantially retains cell viability at the end of fermentation, the cells can be reused in the subsequent round of fermentation, which is likely to facilitate economical production of ethanol.