Edy M. Vilei

A Metabolic Enzyme as a Primary Virulence Factor of Mycoplasma mycoides subsp. mycoides Small Colony

During evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast, Mycoplasma species, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenic Mycoplasma species lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenic Mycoplasma species cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant in Mycoplasma mycoides subsp. mycoides Small Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l-alpha-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with M. mycoides subsp. mycoides SC in the presence of physiological amounts of glycerol, H2O2 is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.

Genetic and Biochemical Characterization of Glycerol Uptake in Mycoplasma mycoides subsp. mycoides SC: Its Impact on H2O2 Production and Virulence

ABSTRACT Highly virulent strains of Mycoplasma mycoides subsp.mycoides SC belonging to the African cluster contain an operon with the genes gtsA, gtsB, andgtsC, encoding membrane ATP binding cassette transporter proteins GtsA, GtsB, and GtsC, which are involved in glycerol transport. Strain Afadé from the African cluster incorporated [U-14C]glycerol with a time-dependent increase. The less virulent strain L2 of the European cluster, which lacksgtsB and gtsC, failed to incorporate glycerol. Antibodies against GtsB noncompetitively inhibited glycerol uptake.l-α-Glycerophosphate was not transported by M. mycoides subsp. mycoides SC. It is postulated to be synthesized by phosphorylation of glycerol during transport and subsequently metabolized further to dihydroxyacetone phosphate accompanied by release of H2O2. Peroxide production in glycerol-containing growth medium was high for the African strain Afadé but very low for the European strain L2. Virtually no H2O2 was produced by both strains without glycerol. Hence, the efficient glycerol uptake system found in the virulent strain of the African cluster leads to a strong release of peroxide, a potential virulence factor which is lacking in the less virulent European strains. M. mycoides subsp.mycoides SC might have adopted, as a strategy for virulence, a highly efficient uptake system for glycerol which allows the production of an active metabolic intermediate that damages host cells.

Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri

The Mycoplasma mycoides cluster consists of six pathogenic mycoplasmas causing disease in ruminants, which share many genotypic and phenotypic traits. The M. mycoides cluster comprises five recognized taxa: Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), M. mycoides subsp. mycoides Large Colony (MmmLC), M. mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp). The group of strains known as Mycoplasma sp. bovine group 7 of Leach (MBG7) has remained unassigned, due to conflicting data obtained by different classification methods. In the present paper, all available data, including recent phylogenetic analyses, have been reviewed, resulting in a proposal for an emended taxonomy of this cluster: (i) the MBG7 strains, although related phylogenetically to M. capricolum, hold sufficient characteristic traits to be assigned as a separate species, i.e. Mycoplasma leachii sp. nov. (type strain, PG50(T) = N29(T) = NCTC 10133(T) = DSM 21131(T)); (ii) MmmLC and Mmc, which can only be distinguished by serological methods and are related more distantly to MmmSC, should be combined into a single subspecies, i.e. Mycoplasma mycoides subsp. capri, leaving M. mycoides subsp. mycoides (MmmSC) as the exclusive designation for the agent of contagious bovine pleuropneumonia. A taxonomic description of M. leachii sp. nov. and emended descriptions of M. mycoides subsp. mycoides and M. mycoides subsp. capri are presented. As a result of these emendments, the M. mycoides cluster will hereafter be composed of five taxa comprising three subclusters, which correspond to the M. mycoides subspecies, the M. capricolum subspecies and the novel species M. leachii.

Genomic and antigenic differences between the European and African/Australian clusters of Mycoplasma mycoides subsp. mycoides SC

Mycoplasma mycoides subsp. mycoides small-colony type (SC), the aetiological agent of contagious bovine pleuropneumonia (CBPP), can be grouped into two major, epidemiologically distinct, clusters. One cluster contains strains isolated from different European countries since 1980 and a second cluster contains African and Australian strains collected over the last 50 years. Genetic analysis of representative strains from the two clusters revealed a genomic segment of 8.84 kb, located close to a copy of IS1296, which is present in all strains of the African cluster but lacking in all strains of the European cluster. This segment contains a copy of IS1634, a gene for a potential lipoprotein, IppB, open reading frames encoding a putative surface-located membrane protein and a hypothetical proline-rich membrane protein, and two open reading frames showing similarity to putative ABC transporters. The product of the IppB gene, lipoprotein B (LppB), has an apparent molecular mass of 70 kDa and was shown to be surface located. It is detected with monospecific antibodies in all strains of the African cluster tested, but not in European-cluster strains. DNA sequence analysis of the splicing site at which European strains differ from African-cluster strains by the lack of the 8.84 kb segment showed that the European cluster has arisen by deletion from a strain of the African cluster. Hence, M. mycoides subsp. mycoides SC strains isolated in different European countries from the newly reemerging outbreaks of CBPP, which occurred after the eradication of the epizootic in Europe in the middle of the 20th century, represent a phylogenetically newer cluster that has been derived from a strain of the older cluster of M. mycoides subsp. mycoides SC which is still endemic on the African continent.

Characterization of P40, a cytadhesin of Mycoplasma agalactiae.

ABSTRACT An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGATrp codon to the universal TGGTrp codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.

The Origin of the ‘Mycoplasma mycoides Cluster’ Coincides with Domestication of Ruminants

The ‘Mycoplasma mycoides cluster’ comprises the ruminant pathogens Mycoplasma mycoides subsp. mycoides the causative agent of contagious bovine pleuropneumonia (CBPP), Mycoplasma capricolum subsp. capripneumoniae the agent of contagious caprine pleuropneumonia (CCPP), Mycoplasma capricolum subsp. capricolum, Mycoplasma leachii and Mycoplasma mycoides subsp. capri. CBPP and CCPP are major livestock diseases and impact the agricultural sector especially in developing countries through reduced food-supply and international trade restrictions. In addition, these diseases are a threat to disease-free countries. We used a multilocus sequence typing (MLST) approach to gain insights into the demographic history of and phylogenetic relationships among the members of the ‘M. mycoides cluster’. We collected partial sequences from seven housekeeping genes representing a total of 3,816 base pairs from 118 strains within this cluster, and five strains isolated from wild Caprinae. Strikingly, the origin of the ‘M. mycoides cluster’ dates to about 10,000 years ago, suggesting that the establishment and spread of the cluster coincided with livestock domestication. In addition, we show that hybridization and recombination may be important factors in the evolutionary history of the cluster.

Antibiotic‐induced expression of a cryptic cpb2 gene in equine β2‐toxigenic Clostridium perfringens

The cpb2 gene of β2‐toxigenic Clostridium perfringens isolated from horses, cattle, sheep, human and pigs was sequenced. The cpb2 gene of equine and other non‐porcine isolates differed from porcine isolates by the absence of an adenine in a poly A tract immediately downstream of the start codon in all non‐porcine C. perfringens strains. This deletion involved formation of a cryptic gene harbouring a premature stop codon after only nine amino acid codons, while the full β2‐toxin protein consists of 265 amino acids. Immunoblots carried out with antibodies directed against a recombinant β2‐toxin showed the absence of expression of the β2‐toxin in equine and the other non‐porcine strains under standard culture conditions. However, treatment of C. perfringens with the aminoglycosides gentamicin or streptomycin was able to induce expression of the cpb2 gene in a representative equine strain of this group, presumably by frameshifting. The presence of the β2‐toxin was revealed by immunohistology in tissue samples of small and large intestine from horses with severe typhlocolitis that had been treated before with gentamicin. This result may explain the finding that antibiotic treatment of horses affected by β2‐toxigenic C. perfringens leads to a more accentuated and fatal progression of equine typhlocolitis. Clinical observations show a reduced appearance of strong typhlocolitis in horses with intestinal complications admitted to hospital care since the standard use of gentamicin has been abandoned. This is the first report on expression of a bacterial toxin gene by antibiotic‐induced ribosomal frameshifting.

Molecular Epidemiology of Mycoplasma conjunctivae in Caprinae: Transmission across Species in Natural Outbreaks

ABSTRACT Mycoplasma conjunctivae is the etiological agent of infectious keratoconjunctivitis, a highly contagious ocular infection that affects both domestic and wild Caprinae species in the European Alps. In order to study the transmission and spread of M. conjunctivae across domestic and wild Caprinae populations, we developed a molecular method for subtyping and identifying strains of M. conjunctivae. This method is based on DNA sequence determination of a variable domain within the gene lppS, a gene that encodes an antigenic lipoprotein of M. conjunctivae. This domain of lppS shows variations among different strains but remains constant upon generations of individual strains on growth medium and thus allows identification of individual strains and estimation of their phylogenetic intercorrelations. The variable domain of lppS is amplified by PCR using primers that match conserved sequences of lppS flanking it. Sequence analysis of the amplified fragment enables fine subtyping of M. conjunctivae strains. The method is applicable both to isolated strains and to clinical samples directly without requiring the cultivation of the strain. Using this method, we show that M. conjunctivae was transmitted between domestic and wild animals that were grazing in proximate pastures. Certain animals also presented infections with two different strains simultaneously.

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

ABSTRACT 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.

Prevalence and distribution of the insertion element ISMag1 in Mycoplasma agalactiae

In characterising Mycoplasma agalactiae strains from various European countries and from Africa, a new insertion sequence (IS), ISMag1, which is related to IS of the family of IS30 insertion elements, has been identified by DNA sequence analysis and Southern blot hybridisation. ISMag1 has a size of 1515bp, and contains inverted repeats of 3bp and a gene encoding the putative transposase on a single open reading frame. ISMag1 is present only in the rarely isolated serotypes E, F, G and H of M. agalactiae, where it is found in 1 to approximately 30 copies. The different patterns obtained by hybridisation of a labelled probe of ISMag1 to genomic DNA cut with various restriction enzymes correlate to some extent to the different serotypes and to variations of the nucleotide sequences of the uvrC genes of the different strains. Based on uvrC sequences, the strains of M. agalactiae carrying ISMag1 form a cluster, separate from the other strains. IS patterns obtained with ISMag1 allow a fine subtyping of the serotypes E, F, G and H of M. agalactiae for epidemiological studies. The potential role of ISMag1 and of its copy numbers on virulence and persistence of the respective strains requests further studies.