Available online at www.sciencedirect.com The Veterinary Journal The Veterinary Journal 174 (2007) 513–521 www.elsevier.com/locate/tvjl Review Molecular mechanisms of pathogenicity of mycoides subsp. mycoides SC

Paola Pilo 1, Joachim Frey *, Edy M. Vilei

Institute of Veterinary Bacteriology, University of Bern, Langgass-strasse 122, 3012 Bern, Switzerland

Accepted 13 October 2006

Abstract

Mycoplasma mycoides subsp. mycoides SC, the aetiological agent of contagious bovine pleuropneumonia (CBPP), is considered the most pathogenic of the Mycoplasma species. Its virulence is probably the result of a coordinated action of various components of an antigenically and functionally dynamic surface architecture. The different virulence attributes allow the pathogen to evade the host’s immune defence, adhere tightly to the host cell surface, persist and disseminate in the host causing mycoplasmaemia, efficiently import energetically valuable nutrients present in the environment, and release and simultaneously translocate toxic metabolic pathway products to the host cell where they cause cytotoxic effects that are known to induce inflammatory processes and disease. This strategy enables the mycoplasma to exploit the minimal genetic information in its small genome, not only to fulfil the basic functions for its replication but also to damage host cells in intimate proximity thereby acquiring the necessary bio-molecules, such as amino acids and nucleic acid pre- cursors, for its own biosynthesis and survival. Ó 2006 Elsevier Ltd. Open access under CC BY license.

Keywords: Mycoplasma mycoides subsp. mycoides SC; CBPP; Pathogenicity; Capsular polysaccharide; Lipoproteins; Adhesion factors; Variable surface proteins; Toxic metabolic pathway products; Catabolite repression

1. Introduction genetic resources and biosynthetic capacities, and adapt to an obligate parasitic lifestyle (Razin, 1997). Mycoplasma mycoides subsp. mycoides small colony The minimal cellular genome of also type (SC) is the aetiological agent of contagious bovine serves as a blueprint for the design of synthetic live organ- pleuropneumonia (CBPP), a severe infectious disease of isms (Check, 2002). In contrast to other pathogenic bacte- cattle (Food and Agriculture Organization, 2003). Myco- ria, where virulence is determined mainly by toxins, plasma species are the smallest self-replicating organisms cytolysins and invasins, no such typical primary virulence currently found on our planet. Their genomes range from genes have been found on the genomes of the ten Myco- 580 kb for Mycoplasma genitalium (Fraser et al., 1995)to plasmas species that have been sequenced completely (Fra- 1358 kb for Mycoplasma penetrans (Sasaki et al., 2002), ser et al., 1995; Himmelreich et al., 1996; Glass et al., 2000; while that of M. mycoides subsp. mycoides SC has a size Chambaud et al., 2001; Sasaki et al., 2002; Papazisi et al., of 1211 kb (Westberg et al., 2004). This 2003; Jaffe et al., 2004; Minion et al., 2004; Vasconcelos has led the Mycoplasma species to radically economise et al., 2005). Mycoplasmas seem rather to use intrinsic met- abolic and catabolic functions to cause disease in the affected host and to ensure the microbe’s survival (Abu-

* Groun et al., 1994; Baseman and Tully, 1997; Razin, Corresponding author. Tel.: +41 31 631 2414; fax: +41 31 631 2634. 1997; Vilei and Frey, 2001; Pilo et al., 2005). Analysis of E-mail address: [email protected] (J. Frey). 1 Present address: Department of Microbiology, Columbia University data from a severe mycoplasmal pathogen, such as Medical Center, New York, USA. M. mycoides subsp. mycoides SC, is expected not only to

1090-0233 Ó 2006 Elsevier Ltd. Open access under CC BY license. doi:10.1016/j.tvjl.2006.10.016 514 P. Pilo et al. / The Veterinary Journal 174 (2007) 513–521 unravel the mechanisms of pathogenicity which lead to and efficient vaccines. While the total virulence of M. myco- CBPP, but may also be useful in the better understanding ides subsp. mycoides SC can only be determined by expen- of other pathogenic Mycoplasma species. sive experimental infections of cattle, which are difficult to CBPP is the only animal disease belonging to the A list justify ethically, the various individual molecular mecha- of most severe infectious animal diseases (as defined by the nisms that contribute to virulence, such as adherence, anti- World Organisation for Animal Health – Office Interna- genic variation, impact of capsular polysaccharides to tional des Epizooties, OIE) caused by a bacterium as a allow persistence and dissemination in the host, or cyto- transmissible agent. CBPP has the potential for very seri- toxic activity, can be studied by means of diverse in vitro ous and rapid spread across national borders. It is cur- immunoassays, cellular studies, or experimental mouse rently responsible for major losses in livestock production infection assays. in Africa and, therefore, has serious socio-economic conse- The recently established sequence of the complete gen- quences and is of major importance in the international ome of M. mycoides subsp. mycoides SC did not reveal trade of animals and animal products. In most other parts any primary virulence factors such as toxins or invasins of the world, CBPP was eradicated using drastic policies of (Westberg et al., 2004). Therefore, the molecular mecha- stamping out cattle in infected areas, control of cattle nisms of pathogenicity of M. mycoides subsp. mycoides movement, and quarantine (Turner, 1954, 1959). The re- SC, as well as of most other Mycoplasma species, are ‘‘hid- emerging outbreaks of CBPP in some European countries den’’ and require a consideration of the particularities of at the end of the last century demonstrated the constant metabolic pathways, surface antigens, and their regulation. threat of the disease to industrialized countries and The present review will give a short overview of the molec- required the re-introduction of expensive eradication mea- ular mechanisms of pathogenicity that have been unrav- sures (Egwu et al., 1996). elled to date and discuss their impact on the virulence of The basic requirements to control CBPP are highly effi- M. mycoides subsp. mycoides SC. In spite of the host spec- cient diagnostic tests that detect M. mycoides subsp. myco- ificity of M. mycoides subsp. mycoides SC, many basic ides SC not only in symptomatic animals but also in molecular mechanisms of virulence are also expected to asymptomatic carriers, together with vaccine strategies that be found in other Mycoplasma species. DNA sequence are able to reduce the infectious pressure in infected areas analysis has revealed that the genomes of mycoplasmas by preventing the pathogen from achieving host infection, consist of a minimal set of genes encoding the essential colonization, and multiplication (Thiaucourt et al., 1998; functions of life, which are conserved in most species and Thiaucourt, 2002). In this context, it is worth noting that subspecies. the live vaccines currently used to control CBPP in different regions of Africa were derived from strain T1 of M. myco- 2. Immunopathogenicity ides subsp. mycoides SC, which was developed in Tanzania in the 1950s. The efficacy of T1 vaccine strains have been The principal pathological consequence of M. mycoides evaluated on several occasions and they have shown subsp. mycoides SC infections is a massive inflammatory advantages but also drawbacks (Hubschle et al., 2002). reaction mainly restricted to the lungs of the affected cattle, For instance, cattle that were infected experimentally with leading to death by respiratory distress caused by lung con- strain T1/44 (or that were in contact with T1/44-infected solidation in up to 30% of cases (Provost et al., 1987; Food animals) developed severe lung lesions characteristic for and Agriculture Organization, 2003). In most infected cat- CBPP (Mbulu et al., 2004). Molecular analyses confirmed tle, however, CBPP is manifested in a chronic form, which the identity of re-isolated T1/44, therefore the observed allows the host to recover from disease but to remain a pathologies were attributed to this particular vaccine potential carrier of the pathogen and so a threatening res- strain. Furthermore, inactivated whole cell vaccines ervoir of M. mycoides subsp. mycoides SC (Provost et al., appeared to exacerbate the effects of CBPP in adult cattle; 1987; Food and Agriculture Organization, 2003). as a result there have been few challenges with inactivated Dedieu and collaborators (2005a) recently studied the vaccines (Nicholas et al., 2004). difference between the acute and chronic progression of To cause disease or to be virulent, M. mycoides subsp. CBPP in cattle at the molecular level. The authors per- mycoides SC possesses particular mechanisms to adhere formed a kinetic analysis of the response of cattle to an to the specific host tissue, to evade the host’s immune infection by M. mycoides subsp. mycoides SC and showed defence, to enable persistence and dissemination in the that in animals that recovered from disease, the CD4 infected animal, and to cause inflammation and disease Th1-like T-cell response to M. mycoides subsp. mycoides signs through cytotoxicity. The loss of any of these mech- SC was maintained over a long period (until slaughter of anisms can lead to attenuation or loss of virulence. Hence, the animals in the experiment). In contrast, in animals that a detailed knowledge of the molecular mechanisms of path- developed an acute disease, progression of the symptomatic ogenicity of M. mycoides subsp. mycoides SC is both a pre- CBPP was associated with a decreased ability of the periph- requisite to develop accurate diagnostic methods that allow eral blood mononuclear cells to produce interferon (Dedieu pathogen detection and differentiate it from closely related et al., 2005a). These data indicated that the susceptibility of bacterial species residing within the host, and to design safe cattle to infection by M. mycoides subsp. mycoides SC is P. Pilo et al. / The Veterinary Journal 174 (2007) 513–521 515 associated with switching the immune response to a mode 3. Secreted polycarbohydrates and capsular polysaccharide that allows dissemination of the pathogen and progression of immunopathologies. Molecules located on the outer surface or in the plasma In an in vitro study on the apoptotic effect of M. myco- membrane of Mycoplasma species, such as lipoproteins, ides subsp. mycoides SC on bovine peripheral blood mono- capsular polysaccharides and biofilms composed mainly nuclear cells, Dedieu et al. (2005b) demonstrated that of fibrillar polycarbohydrates surrounding cells, are gener- viable M. mycoides subsp. mycoides SC could induce strong ally assumed to protect the pathogen from the bactericidal morphological changes in the mononuclear cells, as evi- activity of complement and other host defence functions, denced by a reduction of cell size and increase of cell gran- and to trigger the inflammatory process in the infected host ularity, while heat-killed M. mycoides subsp. mycoides SC (Fig. 1). Biofilms lead to adhere among themselves had no effect. This cytopathic effect was shown to be the and to form micro-colonies on mucosal surfaces and inert cause of apoptosis of the mononuclear cells triggered by materials that are mostly resistant to bactericidal activity live M. mycoides subsp. mycoides SC or by a substance of the host and to antibiotics. However, M. mycoides released by mycoplasmas. However, the triggering effect subsp. mycoides SC does not form biofilms as shown in a measured by mycoplasma free culture supernatants was recently published study, where 25 strains from different rather low (Dedieu et al., 2005b), suggesting that the trig- origins and with variable degrees of virulence were tested gering agent has a short half-life or acts only over very for biofilm production (McAuliffe et al., 2006). short distances. The capsular polysaccharide galactan, composed of 6-O- Several other reports suggest that potentially secreted b-D-galactofuranosyl-D-galactose (Plackett and Buttery, substances of M. mycoides subsp. mycoides SC may be 1964), was shown to increase the virulence of the strongly the cause of the immunopathological effects of CBPP. attenuated M. mycoides subsp. mycoides SC vaccine strain However, the results of genomic DNA sequence analysis KH3J when added to the inoculum prior to experimental of M. mycoides subsp. mycoides SC (Westberg et al., infections of cattle (Hudson et al., 1967). Moreover, But- 2004), as well as genome sequences of other Mycoplasma tery et al. (1976) showed that intravenous injection of species, did not reveal any particular candidate molecules galactan from M. mycoides subsp. mycoides SC to calves that could be directly identified as molecular triggers for produced transient apnoea, increased pulmonary arterial apoptosis or cell necrosis. pressure and pulmonary oedema, indicating that capsular

Fig. 1. Schematic representation of the various virulence pathways of M. mycoides subsp. mycoides SC. The mycoplasma is represented in yellow, the host cells in light blue. The upper pathway, which is currently the best characterized, is based on glycerol import and metabolism. GtsABC represents the membrane-located ATP-binding cassette (ABC) transporter system, which incorporates and phosphorylates imported glycerol to glycerol-3-phosphate (G3P). The bypass pathway that allows diffusion of glycerol via the glycerol facilitator factor (GlpF) and the subsequent phosphorylation by the glycerol kinase (GlpK) has not been evidenced directly, but the corresponding genes, glpF and glpK, have been found by cloning and DNA sequence analysis, and their functionality was supported by strains lacking functional gtsABC genes, which can import and phosphorylate glycerol. The membrane-located L-a- glycerophosphate oxidase (GlpO) represents the central enzyme in this virulence pathway and is able to translocate H2O2 or reactive oxygen species (ROS) into the host cell. The adhesins that seem necessary for the close contact are still hypothetical. The variable surface antigen (Vmm) is supposed to play a role in evading the host’s immune defence. Capsular polysaccharide has been proposed to be produced by most Mycoplasma species and is expected to induce cytokine production. Lipoprotein LppQ is highly antigenic and also seems to be involved in the induction of inflammatory processes by superantigen-like properties. Catabolism of glucosides by the phosphotransferase system (PTS) and 6-phospho-b-glucosidase (Bgl) is known to repress virulence of many pathogens. 516 P. Pilo et al. / The Veterinary Journal 174 (2007) 513–521 polysaccharide of this species has a direct cytopathic effect SC strains, is also found in other, less pathogenic Myco- and seems to lead to the contraction of blood vessels, which plasma species (Pilo et al., 2003). Hence, the role of these may initiate thrombosis. The impact of capsular polysac- lipoproteins in virulence is still unclear. LppQ is a lipopro- charide on virulence was also shown by growth inhibition tein specific to M. mycoides subsp. mycoides SC. It has a tests, which primarily measure serum resistance, and by a particularly strong antigenic N-terminal domain located mouse infection model, which mainly measures the capac- on the outer surface of the membrane, while its C-terminal ity of the Mycoplasma strain to cause bacteraemia. domain is involved in membrane anchoring (Abdo et al., March et al. (1999) showed that a strain of M. mycoides 2000). subsp. mycoides SC that produces low amounts of capsular The high specificity and strong antigenicity of LppQ polysaccharide was much more sensitive to growth inhibit- have been exploited for the development of a robust indi- ing antisera than strains that produced larger amounts of rect ELISA test for serological diagnosis and for epidemi- polysaccharide. Furthermore, the same strains that pro- ological investigations of CBPP (Bruderer et al., 2002). duced large amounts of capsular polysaccharide also gener- Structural analysis of LppQ showed strong similarities to ated a significantly longer duration of bacteraemia in a proteins with super-antigenic character (Abdo et al., mouse infection assay than the strain with little capsular 2000). A recent study has shown that cattle immunized polysaccharide (March and Brodlie, 2000). Hence, capsular with purified recombinant LppQ, using different adjuvant polysaccharide seems to play a role in the capacity of per- methods, were significantly more susceptible to challenge sistence and dissemination of M. mycoides subsp. mycoides with M. mycoides subsp. mycoides SC than cattle that were SC in the infected host (Fig. 1). This process is independent not vaccinated with LppQ (Nicholas et al., 2004). Hence, of the strain’s capacity to release H2O2 upon glycerol LppQ is assumed to play an adverse reaction in vaccina- uptake and metabolism, a mechanism that affects the cyto- tion, similar to the peptidoglycan-associated lipoprotein toxicity of M. mycoides subsp. mycoides SC (Vilei and PalA of Actinobacillus pleuropneumoniae, which was shown Frey, 2001; Pilo et al., 2005). For this reason, the virulence to completely inhibit the beneficial effects of efficient sub- (as measured using the mouse infection assay) and the abil- unit vaccines when animals were vaccinated simultaneously ity of the different strains of M. mycoides subsp. mycoides with PalA (van den Bosch and Frey, 2003). LppQ may SC to metabolize glycerol were assumed to be unconnected, therefore contribute to the immunopathologies induced as reported previously (March and Brodlie, 2000). by M. mycoides subsp. mycoides SC (Fig. 1).

4. Surface proteins 5. Adhesion factors

Lipoproteins are expected to play a role as triggers in Adhesins play an important role in the early steps of mechanisms of pathogenicity, since they are known to have pathogenesis of most . Since mycoplasmas a central role in interactions between mycoplasmas and do not seem to secrete toxins that could act over long dis- eukaryotic cells, particularly with respect to adhesion. Fur- tances, adhesion is particularly important in mycoplasmal thermore, lipoproteins have been reported to stimulate the virulence. Adhesion plays a central role in the intimate release of pro-inflammatory cytokines (Mu¨hlradt and interactions of pathogenic mycoplasmas with mammalian Frisch, 1994; Herbelin et al., 1994; Brenner et al., 1997; cells for long periods and is assumed to trigger a cascade Marie et al., 1999; Calcutt et al., 1999). Since lipoproteins of signals, which are transduced to the host cell and induce are usually strongly antigenic proteins, they are also con- inflammation (Razin et al., 1998). Adhesins also seem to be sidered to be valuable targets for specific and sensitive sero- responsible for host specificity and tissue tropism. diagnosis. Currently, a few lipoproteins from M. mycoides Although several adhesins have been identified in various subsp. mycoides SC have been characterized in detail. Most Mycoplasma species (Sachse et al., 1996; Noormohammadi of them are major antigens and are readily detected in the et al., 1997; Razin et al., 1998; Kitzerow et al., 1999; serum of infected cattle on immunoblots. Sachse et al., 2000; Minion et al., 2000; Fleury et al., LppA is strongly conserved among mycoplasmas of the 2002; Belloy et al., 2003), specific adhesins have not yet ‘‘M. mycoides cluster’’ and it is expressed by highly virulent been detected in M. mycoides subsp. mycoides SC, but Mycoplasma species as well as by non-pathogenic or low- can be postulated to occur from the above considerations. pathogenic species (Monnerat et al., 1999a; Monnerat et al., 1999b). The role of LppA in Th1 and Th2 immunity 6. Variable surface proteins is currently under investigation. LppB is found only in strains belonging to the African/Australian cluster, but is Phase variation is a common mechanism among Myco- not found in strains isolated from the re-emerging Euro- plasma species and is thought to be involved in microbial pean outbreaks in 1980–1999. LppB is, however, also pres- survival, leading to the emergence of varied intra-clonal ent in other mycoplasmas of the ‘‘M. mycoides cluster’’, in populations that adapt quickly to new environments (Dyb- particular, in the bovine pathogen Mycoplasma sp. bovine vig and Voelker, 1996; Henderson et al., 1999). Some group 7 (Vilei et al., 2000). Another lipoprotein, LppC, Mycoplasma species possess an abundance of phase-vari- which is expressed by all M. mycoides subsp. mycoides able proteins, especially surface-exposed lipoproteins (Citti P. Pilo et al. / The Veterinary Journal 174 (2007) 513–521 517 and Wise, 1995; Citti et al., 2000; Ro¨ske et al., 2001; Den- panied by the release of a molecule of H2O2, was ison et al., 2005). identified as the membrane protein that plays a central role A variable surface protein, designated Vmm, has been in cytotoxicity of M. mycoides subsp. mycoides SC strains discovered in M. mycoides subsp. mycoides SC (Persson towards embryonic calf nasal epithelial (ECaNEp) cells et al., 2002). Vmm is a 16-kDa protein, specific to this (Pilo et al., 2005). In the presence of physiological concen- Mycoplasma species. It is expressed by nearly all strains trations of glycerol, relatively large amounts of H2O2 were analyzed, where it has shown a reversible ON–OFF phase released into the culture medium. When ECaNEp cells variation at a high frequency per cell generation. This var- were exposed to African M. mycoides subsp. mycoides SC iation is regulated at the transcriptional level by dinucleo- field strains in the presence of physiological concentrations tide insertions or deletions in a repetitive region of the of glycerol, H2O2 was rapidly detected in their cytosol and, promoter (Persson et al., 2002). Genes resembling the subsequently, cell death occurred. Only a weak cytotoxic vmm gene were also found in other species of mycoplasma, effect was observed when ECaNEp cells were infected with but analogous Vmm-like proteins in these species could not European strains. Culture supernatants from M. mycoides be detected with a specific monoclonal antibody directed to subsp. mycoides SC grown in the presence of glycerol, and Vmm of M. mycoides subsp. mycoides SC. The function of thus containing up to 150 lMH2O2, or buffers or axenic Vmm is currently unknown, but repeating elements in var- culture media supplemented with 150 lMH2O2 had no iable membrane proteins of mycoplasmas have been sug- cytotoxic effect on ECaNEp cells. In these cases, H2O2 or gested to increase the pathogen’s ability to adhere to host the accompanying reactive oxygen species (ROS) could cells and to evade the host immune response (Peterson not be detected in the cytosol of the eukaryotic cells (Pilo et al., 1995; Sachse et al., 2000)(Fig. 1). et al., 2005). Hence, close contact between mycoplasmas and host cells was necessary in order to successfully trans- 7. Toxic metabolic pathway products locate the toxic compounds from the glycerol metabolism into the host cells. In this context, it is worth noting that Oxygen uptake and H2O2 production were identified as most Mycoplasma species, including M. mycoides subsp. particular characteristics in fermentative Mycoplasma spe- mycoides SC, remain tightly attached to the surface of epi- cies and were expected to influence the virulence of patho- thelial cells but do not penetrate them (for review see Razin genic mycoplasmas (Cole et al., 1968; Brennan and et al., 1998). A possible mechanism of adhesion, particu- Feinstein, 1969; Wadher et al., 1990; Miles et al., 1991). larly in calf epithelial cells, could be the partial fusion of It was later discovered that M. mycoides subsp. mycoides the surface exposed epitopes of M. mycoides subsp. myco- SC strains isolated from the re-emerging European out- ides SC with the host cell wall (Pilo et al., 2005). breaks of CBPP in 1980–1999 produced much less H2O2 A proposed model for triggering cellular damage to when grown in the presence of glycerol than strains isolated eukaryotic cells is that glycerol present in the interstitial in the African and Australian continents and it was sug- fluid is incorporated actively via the highly active ABC gested that a glycerophosphate oxidase could represent a glycerol transporter GtsABC and is subsequently phos- significant virulence factor of M. mycoides subsp. mycoides phorylated into G3P. This, in turn, is oxidized by GlpO SC (Wadher et al., 1990; Houshaymi et al., 1997; Rice into DHAP, which enters in the glycolysis cycle of the et al., 2001). mycoplasma, and H2O2 is released. Facilitated by the inti- Recently, the role of glycerol metabolism in the viru- mate contact of the mycoplasma with the host cell mem- lence of M. mycoides subsp. mycoides SC was also consid- brane, H2O2 or ROS enters the host cell. Inside the host ered in our laboratory, based on the observation that cells, these toxic compounds act as powerful mediators of African strains contain an efficient active glycerol import cell injury and inducers of inflammatory processes system, GtsABC, specified by an ABC transporter protein, (Fig. 1). They are expected to damage the host either by while the European strains, which are considered less viru- directly impairing tissue cells or by inducing host gene lent (Abdo et al., 1998) and cause a disease that appears to expression, e.g. of pro-inflammatory genes via activation be largely chronic showing few clinical signs and low mor- of the nuclear factor kappa B (NF-jB) (Baeuerle and Hen- tality (Nicholas et al., 1996), are devoid of this transporter kel, 1994), or via the Fenton reaction (Crichton et al., protein (Vilei and Frey, 2001). Glycerol is metabolized 2002). after uptake and phosphorylation to dihydroxyacetone Interestingly, mycoplasmas have previously been shown phosphate (DHAP) by an oxidative process leading to to induce a respiratory burst in phagocyte cells (Koppel the release of the highly toxic compound H2O2. Blocking et al., 1984), suggesting that host-generated ROS might the glycerol uptake proteins GtsABC by specific antibodies further contribute to tissue damage. Furthermore, it was resulted in a significant reduction of H2O2 production shown that M. mycoides subsp. mycoides SC strains are (Vilei and Frey, 2001). Furthermore, it was shown that also able to induce the tumor necrosis factor alpha European strains that lack the GtsABC transporter pro- (TNF-a) in bovine alveolar macrophages (Jungi et al., duce significantly lower amounts of H2O2. 1996). TNF-a also acts as an inflammatory mediator and, The L-a-glycerophosphate oxidase (GlpO), which cata- in association with NF-jB, can act as primary signals lyzes the oxidation of glycerol-3-phosphate (G3P) accom- inducing apoptosis in host cells. The demonstration of 518 P. Pilo et al. / The Veterinary Journal 174 (2007) 513–521 the involvement of these two molecules in an apoptotic ence of the oedema factor and the lethal toxin (Brossier pathway triggered by M. mycoides subsp. mycoides SC in and Mock, 2001). It is therefore not surprising that M. host cells must be investigated. mycoides subsp. mycoides SC strains, such as KH3J, that produce low amounts of capsular polysaccharide and have 8. Catabolite repression of bacterial virulence lost most of their potential to cause bacteraemia in mice but still produce large amounts of H2O2 by their capacity A non-synonymous single nucleotide polymorphism to import and metabolize glycerol, have a strongly attenu- (SNP) in the bgl gene encoding the 6-phospho-b-glucosidase ated virulence (Lloyd et al., 1971; Dyson and Smith, 1976; (Bgl) has recently been detected. It differentiates African March et al., 1999; March and Brodlie, 2000; Rice et al., field strains from European field strains from the 1980– 2001). 1999 outbreaks, as well as currently used vaccine strains The various approaches and methods to study virulence of M. mycoides subsp. mycoides SC (Vilei and Frey, 2004). of M. mycoides subsp. mycoides SC target individual mech- Bgl is an intracellular enzyme associated with the phospho- anisms. To assess the overall virulence of M. mycoides enolpyruvate-dependent sugar:phosphotransferase system subsp. mycoides SC, all of these different mechanisms of (PEP–PTS), a multicomponent system involved in the pathogenicity would have to be considered. Currently, a simultaneous translocation and phosphorylation of sugars major handicap in the study of the basic principles of the by bacteria from both Gram-positive and Gram-negative different virulence mechanisms of M. mycoides subsp. genera. Sugars in general and sugar catabolites are known mycoides SC is the lack of a genetic basis for most of them. to mediate repression of bacterial virulence gene expression; In order for research on the molecular mechanisms of path- in particular, catabolism of b-glucosides is involved in the ogenicity of M. mycoides subsp. mycoides SC to progress, regulation of the virulence of bacterial pathogens, such as efficient tools for targeted mutagenesis and vectors able Listeria monocytogenes (Brehm et al., 1999) and Erwinia to trans-complement mutations, as have been constructed amylovora (Kerppola et al., 1987). Hence, it will be worth- for other related Mycoplasma species (Janis et al., 2005), while in the future to analyze the role of the bgl gene in will be essential. Such methodologies will not only be useful attenuated strains of M. mycoides subsp. mycoides SC, to drive basic research on molecular pathogenicity, but will and to investigate whether the resulting enzyme shows a also be valuable for the targeted construction of novel, effi- regulated activity that would be able to catabolically modu- cient, and safe vaccines against CBPP in areas where the late the mycoplasmal virulence characteristics. disease is endemic. In fact, vaccines currently administered to cattle are in the form of live bacteria and pose a poten- 9. Concluding remarks tial threat to livestock especially in areas so far free of CBPP. Their biological safety aspects require particular The pathogenicity of M. mycoides subsp. mycoides SC attention by implementing safe and non-reversible attenua- seems to be multifunctional, as would be expected from tion sites in new vaccine strains. knowledge of the molecular mechanisms of pathogenicity from other bacterial species. However, in contrast to the Acknowledgement latter, whose virulence is determined by special virulence factors such as toxins and invasins, the virulence factors The authors acknowledge the financial support of The of Mycoplasma species seem to be determined by intrinsic Wellcome Trust (London, UK), Grant No. 075804 ‘‘A metabolic or catabolic pathway functions or by proper genomics approach to understanding the immunopathol- constituents of the mycoplasmal outer surface. In M. myco- ogy of contagious bovine pleuropneumonia (CBPP): ides subsp. mycoides SC, a few virulence determinants have improvement of current live vaccines and the development been unravelled in recent years including capsular polysac- of next generation vaccines’’. charide, which seems to be involved in serum-resistance to give the organism the capacity to persist and disseminate in References the host. 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