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

Identification of genes coding for B cell antigens of Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) by using phage display

<p>Abstract</p> <p>Background</p> <p>Contagious bovine pleuropneumonia (CBPP) is a mycoplasmal disease caused by <it>Mycoplasma mycoides </it>subsp. <it>mycoides </it>SC (<it>Mmm</it>SC). Since the disease is a serious problem that can affect cattle production in parts of Africa, there is a need for an effective and economical vaccine. Identifying which of the causative agent's proteins trigger potentially protective immune responses is an important step towards developing a subunit vaccine. Accordingly, the purpose of this study was to determine whether phage display combined with bioinformatics could be used to narrow the search for genes that code for potentially immunogenic proteins of <it>Mmm</it>SC. Since the production of IgG2 and IgA are associated with a Th₁cellular immune response which is implicated in protection against CBPP, antigens which elicit these immunoglobulin subclasses may be useful in developing a subunit vaccine.</p> <p>Results</p> <p>A filamentous phage library displaying a repertoire of peptides expressed by fragments of the genome of <it>MmmSC </it>was constructed. It was subjected to selection using antibodies from naturally- and experimentally-infected cattle. Mycoplasmal genes were identified by matching the nucleotide sequences of DNA from immunoselected phage particles with the mycoplasmal genome. This allowed a catalogue of genes coding for the proteins that elicited an immune response to be compiled. Using this method together with computer algorithms designed to score parameters that influence surface accessibility and hence potential antigenicity, five genes (<it>abc, gapN, glpO, lppB </it>and <it>ptsG</it>) were chosen to be expressed in <it>Escherichia coli</it>. After appropriate site-directed mutagenesis, polypeptides representing portions of each of these proteins were tested for immunoreactivity. Of these five, polypeptides representing expression products of <it>abc </it>and <it>lppB </it>were recognised on immunoblots by sera obtained from cattle during a natural outbreak of the disease.</p> <p>Conclusion</p> <p>Since phage display physically couples phenotype with genotype, it was used to compile a list of sequences that code for <it>Mmm</it>SC proteins bearing epitopes which were recognised by antibodies in the serum of infected animals. Together with the appropriate bioinformatic analyses, this approach provided several potentially useful vaccine or diagnostic leads. The phage display step empirically identified sequences by their interaction with antibodies which accordingly reduced the number of ORFs that had to be expressed for testing. This is a particular advantage when working with <it>Mmm</it>SC since the mycoplasmal codon for tryptophan needs to be mutated to prevent it from being translated as a stop in <it>E. coli</it>.</p

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

Tipologi Dan Morfologi Arsitektur Suku Banjar Di Kal-Sel

Tujuan penelitian ini adalah ingin mengetahui tipologi dan morfologi arsitektur daerah Suku Banjar di Kalimantan Selatan sehingga ketidakjelasan tipe arsitektur Banjar yang ada saat ini dapat dipecahkan secara ilmiah.Populasi dalam penelitian ini adalah rumah tradisional yang berumur rata-rata lebih dari 50 tahun. Sampel yang digunakan adalah sampel bertujuan (purposive sample) dengan pengumpulan data menggunakan metode bola salju (snow ball sampling). Analisis data, dimulai dengan menelaah seluruh data, reduksi data, menyusun data-data dalam satuan-satuan, mengkategorisasikan, dan memeriksa keabsahan data. Tahap analisis dilanjutkan dengan tahap penafsiran data. Bagian analisis yang terpenting adalah mengkategorisasikan yang didasarkan pada metode analisis komparatif.Hasil penelitian menunjukkan Tipomorfologi arsitektur suku Banjar dapat dijelaskan berdasar beragam tema yang mempengaruhi perkembangan arsitektur Suku Banjar, yaitu; berdasar kesamaan yang menjadi ciri khas (geometrik), berdasar pengaruh kebudayaan suku, berdasar pengaruh kepercayaan dan agama, berdasar tata ruang, berdasar struktur dan konstruksi, berdasar lokasi, dan berdasar ornamen/ ragam hias.Keberadaan masing-masing tema yang mempengaruhi pembentukan tipo- morfologi Suku Banjar di atas saling berhubungan erat antar satu dengan yang lainnya sehingga tidak bisa dilepaskan dalam pembentukan pemahaman

Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be grouped into a single subspecies

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be combined into one taxon on the basis of several contributions on both DNA sequence and protein analyses reported in the literature. Moreover, for the differentiation and identification of mycoplasmas of the "mycoides cluster", we investigated the rpoB gene, encoding the beta-subunit of the RNA polymerase. A segment of 527 bp of the rpoB gene was amplified from 31 strains of ruminant mycoplasmas by PCR. The nucleotide sequences were determined and aligned, and accurate genetic relationships were calculated. Cluster analysis of rpoB DNA allowed species differentiation within the "mycoides cluster" and confirmed that M. mycoides subsp. capri and M. mycoides subsp. mycoides LC cannot be distinguished from each other. "Mycoplasma mycoides subsp. capri" is proposed as a common name for both subspecies

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