Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-α-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H2O2 into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly 12−Gly13−Gly 14−Ile15−Ile16−Gly 17. Recombinant GlpO lacking these six amino acids (GlpOΔFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpOΔFAD, similarly to anti-GlpO antibodies, neutralised H2O2 production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP

β-D-Glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H(2)O(2 )production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-β-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val(204), from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala(204). RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val(204), but not strains with the Bgl isoform Ala(204), do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., β-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val(204 )show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-β-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala(204). Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H(2)O(2 )production. Rather, the viability during addition of β-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val(204 )than for those with the isoform Ala(204). CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val(204 )with low hydrolysing activity are more prone to survive in environments that contain high levels of β-D-glucosides, thus contributing in some extent to mycoplasmaemia

Mycoplasma bovis shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides SC: Evolutionary and developmental aspects

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine hos

Cytotoxicity of Mycoplasma mycoides subsp. mycoides Small Colony Type to Bovine Epithelial Cells▿

The cytotoxicities of various strains of Mycoplasma mycoides subsp. mycoides small colony type (SC), the agent of contagious bovine pleuropneumonia (CBPP), were measured in vitro using embryonic calf nasal epithelial (ECaNEp) cells. Strains isolated from acute cases of CBPP induced high cytotoxicity in the presence of glycerol, concomitant with the release of large amounts of toxic H2O2 that were found to be translocated into the cytoplasms of the host cells by close contact of the Mycoplasma strains with the host cells. Currently used vaccine strains also showed high cytotoxicity and high H2O2 release, indicating that they are attenuated in another virulence attribute. Strains isolated from recent European outbreaks of CBPP with mild clinical signs, which are characterized by a defect in the glycerol uptake system, released small amounts of H2O2 and showed low cytotoxicity to ECaNEp cells. M. mycoides subsp. mycoides SC strain PG1 released large amounts of H2O2 but was only slightly cytotoxic. PG1 was found to have a reduced capacity to bind to ECaNEp cells and was unable to translocate H2O2 into the bovine cells, in contrast to virulent strains that release large amounts of H2O2. Thus, an efficient translocation of H2O2 into host cells is a prerequisite for the cytotoxic effect and requires an intact adhesion mechanism to ensure a close contact between mycoplasmas and host cells

First Report of Mycoplasma conjunctivae from Wild Caprinae with Infectious Keratoconjunctivitis in the Pyrenees (NE Spain)

Frequent outbreaks of infectious keratoconjunctivitis have been reported in wild Caprinae in Europe. While etiologic studies in the Alps indicate that the main etiologic agent is Mycoplasma conjunctivae, there are few reports from other mountain areas, such as the Pyrenees, where M. conjunctivae has never been reported. In 2006 and 2007, five adult Pyrenean chamois (Rupicapra pyrenaica; two males and three females) and one adult male European mouflon (Ovis orientalis musimon) were studied; they exhibited clinical symptoms of infectious keratoconjunctivitis such as blindness, corneal opacity, and ulceration. In three of the five chamois tested, and in the mouflon, Mycoplasma conjunctivae was identified from conjunctival swabs by means of a TaqMan(R) polymerase chain reaction based on the lipoprotein gene lppS. Cluster analysis indicated that the three southern chamois isolates form a cluster that is distinct from the mouflon isolate. This is the first report of M. conjunctivae in Pyrenean chamois, and it supports the hypothesis that M. conjunctivae also could be the main cause of infectious keratoconjunctivitis in areas other than the Alps, such as the Pyrenees

Genomic differences between type strain PG1 and field strains of Mycoplasma mycoides subsp. mycoides small-colony type

The recently accomplished complete genomic sequence analysis of the type strain PG1 of Mycoplasma mycoides subsp. mycoides small-colony type revealed four large repeated segments of 24, 13, 12, and 8 kb that are flanked by insertion sequence (IS) elements. Genetic analysis of type strain PG1 and African, European, and Australian field and vaccine strains revealed that the 24-kb genetic locus is repeated only in PG1 and not in other M. mycoides subsp. mycoides SC strains. In contrast, the 13-kb genetic locus was found duplicated in some strains originating from Africa and Australia but not in strains that were isolated from the European outbreaks. The 12- and 8-kb genetic loci were found in two and three copies, respectively, in all 28 strains analyzed. The flanking IS elements are assumed to lead to these tandem duplications, thus contributing to genomic plasticity. This aspect must be considered when designing novel diagnostic approaches and recombinant vaccines

Selective activation of TACI by syndecan-2

B-lymphocyte homeostasis and function are regulated by complementary actions of the TNFR family members TACI, BCMA, and BAFF-R, which are expressed by mature B cells. How these receptors are differentially activated is not entirely understood, because the primary ligand BAFF binds to all three. We searched for alternative ligands for TACI using recombinant TACI-Fc fusion protein as a probe and identified syndecan-2 as a new binding partner. TACI binding appears to require heparan sulfate posttranslational modifications of syndecan-2, because free heparin or pretreatment with heparitinase blocked the interaction. Syndecan-2 bound TACI but bound neither BAFF-R nor BCMA. Transfected cells expressing syndecan-2 activated signaling through TACI, as indicated by an NFAT-specific reporter. Syndecan-1 and syndecan-4 were also able to induce TACI signaling in a similar manner. This is the first identification of ligands that selectively activate TACI without simultaneously triggering BCMA or BAFF-R. This finding may help explain the alternative outcomes of signaling from this family of receptors in B cells