Molecular mechanisms of virulence and antigenicity of Mycoplasma mycoides
subsp. mycoides SC: Conclusions for prevention and control of CBPP

Joachim Frey, Paola Pilo, Edy M. Vilei

Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland.

Introduction

Mycoplasma mycoides subspecies mycoides SC (Mmm SC), the etiological agent of contagious bovine pleuropneumonia (CBPP), is a highly virulent Mycoplasma species. In spite of the fact that this pathogen has been identified more than 100 years ago, its molecular mechanisms of pathogenicity and its virulence factors are still poorly known. However, in order to design novel rational diagnostic tools and safe and efficient vaccines to control this disease, it is essential to know the mechanisms of pathogenicity of this organism. So far no particular primary virulence factors such as toxins or invasins have been evidenced in Mmm SC, nor have primary virulence genes been among those Mycoplasma species whose full genomic sequences had been determined. This might be due to the extremely small genome of mycoplasmas leading them to a drastic economization in genetic resources which are reduced to essential functions of life (Razin et al., 1998). Our current studies indicate that mycoplasmas seem to have adopted efficient structural surface antigens and basic metabolic pathway functions as virulence effectors to cause a disease.

Major surface antigens and their potential impact in virulence

Among the prominent surface antigens detected on the cellular membrane of mycoplasmas, and in particular of Mmm SC, are a large number of lipoproteins, variable surface antigens, ABC transporter proteins, metabolic pathway enzymes, adhesins and polycarbohydrates such as galactan.

Lipoproteins, in particular of mycoplasmas, are expected to play a role in mechanisms of pathogenicity since they are known to induce pro-inflammatory cytokines and might adopt the function of lipopolysaccharides which are missing in mycoplasmas (Mühlradt and Frisch, 1994; Herbelin et al., 1994; Brenner et al., 1997; Marie et al., 1999; Calcutt et al., 1999). Furthermore, lipoproteins are in general strongly antigenic proteins that might be valuable targets for specific and sensitive serodiagnosis. Their analysis in mycoplasmas, therefore, deserves particular attention. Variable surface antigens (Vsp) have been analysed extensively in a large number of mycoplasmas including Mmm SC (Persson et al., 2002), Mycoplasma bovis, Mycoplasma agalactiae, Mycoplasma penetrans, Mycoplasma gallisepticum and Mycoplama pulmonis (Rosengarten and Wise, 1990; Citti and Rosengarten, 1997; Glew et al., 2000). Vsps seem to play an important role in the mycoplasmas to escape the host’s immune system. They have recently also been shown to modulate the susceptibility of mycoplasmas to complement killing hemadsorption and adherence (Simmons and Dybvig, 2003).

ABC carbohydrate transporters are common components in bacterial membranes since they serve the organism for import of various components used as energy source and nutrition and for the export of metabolic substances. Most metabolic pathway enzymes are cytoplasmlocated. Surface located metabolic pathway enzymes are commonly involved in metabolic steps that are closely related to uptake mechanisms or for elimination of substances badly tolerated in the cytoplasm. Blocking of surface located metabolic pathway enzymes by antibodies reduces or inhibits the ability of the organism to grow, a procedure that is currently used in the growth inhibition test.

Adhesins play a crucial role in the primary steps employed by mycoplasmas while interacting with their host eukaryotic cells using specific mammalian membrane receptors, a process that is assumed to be followed by either the invasion of host cells by mycoplasmas or by the production of signals by the mycoplasmas to subvert and damage the host cells.

Polycarbohydrates (glycans) are assumed to give the mycoplasma physico-chemical resistance against the host immune defence, but have also been shown to be involved in pathogenic mechanisms.

Lipoproteins of M. mycoides subsp. mycoides SC

Currently, a few lipoproteins of Mmm SC have been characterized (Table 1). Most of them are strong major antigens and are readily detected in serum of infected cattle on immunoblots. Lipoprotein A of Mmm SC is strongly conserved among mycoplasmas of the M. mycoides cluster, hence it cannot be used as a specific target for serodetection (Monnerat et al., 1999). Its role in Th1 and Th2 immunity is currently under investigation. Lipoprotein B is found only in Mmm SC strains belonging to the African/Australian cluster, but it is not found in strains isolated from the re-emerging European outbreaks in 1980 - 2000. It is, however, also present in other mycoplasmas of the M. mycoides cluster (Vilei et al., 2000). The role of two further lipoproteins LppC and LppD is under investigation. Lipoprotein Q (LppQ) seems to be a particular lipoprotein of Mmm SC as it is specific to this organism. It has a particularly strong antigenic N-terminal part which is located on the outer surface of the membrane, while its Cterminal part is involved in membrane anchoring (Abdo et al., 2000). The high specificity and strong antigenicity of LppQ have been exploited for the development of a robust indirect ELISA test for serological diagnosis and for epidemiological investigations of CBPP (Bruderer et al., 2002). Structural analysis of LppQ showed strong analogies to proteins with super-antigenic character (Abdo et al., 2000). A recent study has shown that cattle immunized with purified recombinant LppQ, using different adjuvant methods, were significantly more susceptible to challenge with Mmm SC than cattle that were not vaccinated with LppQ (Otto Huebschle, personal communication^[16]). Hence, LppQ is assumed to play an adverse reaction in vaccination similar to the peptidoglycan-associated lipoprotein PalA of Actinobacillus pleuropneumoniae, which was shown to inhibit completely beneficial effects of efficient subunit vaccines when animals were vaccinated simultaneously with PalA (van den Bosch and Frey, 2003). In this respect, it must be noted that the currently used life vaccines express LppQ, a matter to be considered in the development of new vaccine strains.

Variable surface proteins

A variable surface protein, designated Vmm, has recently been discovered in Mmm SC (Persson et al., 2002). Vmm is a protein of 16 kDa and is specific to Mmm SC. It is expressed by nearly all strains that were analyzed, where it showed a reversible ON-OFF phase variation at a frequency of 9 × 10^-4 to 5 × 10^-5 per cell generation. Genes resembling the vmm gene were also found in other species of mycoplasma, but the Vmm-like proteins in these species could not be detected with a specific monoclonal antibody directed to Vmm of Mmm SC. The function of Vmm is currently not known, but it is suggested to provide the pathogen flexibility on its outer surface and to help escape the host’s immune system.

Table 1. Currently known antigens of M. mycoides subsp. mycoides SC.

ABC transporter proteins

ABC transporter proteins are integral membrane proteins found in prokaryotes. They play a key role in the import and export of nutrition and energy sources and of metabolites and toxic substances across the membrane. The minimal genomes of Mycoplasma species contain an astonishingly large number of ABC transporter genes (Razin et al., 1998). In Mmm SC type strain PG1, more than 30 ABC transporter genes have been discovered (Westberg, 2003). At present, only the glycerol uptake system GtsABC has been investigated in detail (Vilei and Frey 2001). In Mmm SC, this ABC transporter is involved in active glycerol uptake and glycerol phosphorylation. Glycerol is metabolized in Mmm SC after phosphorylation to dihydroxyacetone phosphate (DHAP) by an oxidative process leading to the release of the highly toxic compound H₂O₂. Blocking the glycerol uptake proteins GtsABC by specific antibodies results in a significant reduction of H₂O₂ production. Hence, it is suggested that the glycerol uptake system GtsABC is indirectly involved in virulence of Mmm SC. In this respect, it is important to note that, due to a deletion, the new emerging European strains of Mmm SC lack the gtsB and gtsC genes. These strains produce significantly lower amounts of H₂O₂ and also seem to be less virulent than African strains that possess the full gtsABC operon and are highly virulent.

Metabolic pathway enzymes

Metabolic pathway enzymes were generally well conserved during the evolution of prokaryotes. Therefore, many metabolic pathway genes of Mmm SC can be gained from the genomic sequence (Westberg, 2003). Currently, our laboratory is investigating the L - a - glycerophosphate oxidase GlpO of Mmm SC in detail. GlpO is the central enzyme involved in the metabolism, after phosphorylation, of both glycerol imported by the active GtsABC transporter and glycerol imported via the GlpF function to DHAP and H₂O₂. GlpO is membranelocated and allows to synthesize H₂O₂ directly on the surface of the organism and to release it to the environment and to the host cell, respectively, where it causes injury during infection (P. Pilo, E. M. Vilei, J. Frey, unpublished results). Preliminary studies using a cellular model based on calf epithelial cells revealed that the cytoxicity of Mmm SC is strongly reduced concomitantly to blocking H₂O₂ production, if the mycoplasmas are treated with monospecific polyclonal anti-GlpO antibodies (P. Pilo, E.M. Vilei, J. Frey, unpublished results). These studies show that GlpO could be an interesting target for the development of vaccines against CBPP.

Adhesins

Adhesins play a highly important role in the early steps of pathogenicity of most microorganisms. Since mycoplasmas do not secrete toxins that could act at long distances, adhesion is particularly important in the virulence of Mmm SC. Adhesion plays a central role in the intimate interactions with mammalian cells for long periods, assumed to trigger a cascade of signals which are transduced to the host cell and cause inflammation (Razin et al., 1998). Adhesins also seem to be the basic principles of host specificity of Mmm SC for bovines since the other virulence factors, such as toxic metabolic substances and intermediates, are not expected to have any host predilection. Although several adhesins have been identified in various Mycoplasma species (Razin et al., 1998; Belloy et al., 2003), adhesins of Mmm SC have not yet been detected in spite of their potential primordial role in immune protection.

Polycarbohydrates

Polycarbohydrates, or glycans, have been postulated to play a role in virulence of Mmm SC, supposedly by protecting the mycoplasmas from the devasting actions of the cell immune system and by giving them the possibility to closely associate with the host’s target cells (Gourlay and Shifrine, 1966). However, neither the chemical composition of the polycarbohydrates nor their direct role in virulence has been described for Mmm SC so far. Still certain monoclonal antibodies that are highly specific for the species of Mmm SC seem to react specifically with epitopes of polycarbohydrate material.

Conclusions for prevention and control

The knowledge about the molecular mechanisms of pathogenicity of Mmm SC has been significantly improved over the last four years, although only few virulence mechanisms are currently known. In particular, there is still no knowledge about the factors involved in the adhesion process, a key step of infection, which would most probably represent an ideal target in immuno-protection, and prevention of CBPP. On the other hand, evidence becomes available on specific targets of Mmm SC that are supposed to over-induce the host’s immune system and are consequently considered to cause adversary reactions in vaccines. Such reactions can enhance, rather than reduce, the symptoms caused by an infection by Mmm SC.

The corresponding antigens should therefore not be included in vaccines, or their genes must be deleted from vaccine strains. Other antigens, in contrast, are expected to induce antibodies that can block certain metabolic activities, thus avoiding the production of cytotoxic substances. The currently available molecular genetic methods are able both to generate targeted knockout mutants and to complement and genetically modify Mmm SC in the view of production of improved vaccine strains (Cordova et al., 2002). Furthermore, efficient expression systems allow the production of substantial amounts of recombinant proteins from Mmm SC in heterologous bacterial hosts for use in subunit vaccines or as novel diagnostics (Abdo et al., 2000; Bruderer et al., 2002). It is therefore important to design general strategies or novel concepts of vaccines against CBPP (Table 2), which have to fulfil a broad range of requirements: They must protect efficaciously the animals against infection with Mmm SC and prevent from CBPP; they must fulfil veterinary medical and biological safety requirements; they should be economical in production and application; and, finally, they must be socially and politically acceptable.

Table 2. Basic types of vaccines

*Only endogenous modifications no foreign DNA
**often submitted to similar regulations as GMOs

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