Mycoplasma hyopneumoniae is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP). Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, Mycoplasma hyopneumoniae, we performed comparative protein profiling of three M. hyopneumoniae strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422.
In 2DE comparisons, we were able to identify differences in expression levels for 67 proteins, including the overexpression of some cytoadherence-related proteins only in the pathogenic strains. 2DE immunoblot analyses allowed the identification of differential proteolytic cleavage patterns of the P97 adhesin in the three strains. For more comprehensive protein profiling, an LC-MS/MS strategy was used. Overall, 35% of the M. hyopneumoniae genome coding capacity was covered. Partially overlapping profiles of identified proteins were observed in the strains with 81 proteins identified only in one strain and 54 proteins identified in two strains. Abundance analysis of proteins detected in more than one strain demonstrates the relative overexpression of 64 proteins, including the P97 adhesin in the pathogenic strains.
Our results indicate the physiological differences between the non-pathogenic strain, with its non-infective proliferate lifestyle, and the pathogenic strains, with its constitutive expression of adhesins, which would render the bacterium competent for adhesion and infection prior to host contact.
Mycoplasma hyopneumoniae is associated with swine respiratory diseases. Although gene organization and regulation are well known in many prokaryotic organisms, knowledge on mycoplasma is limited. This study performed a comparative analysis of three strains of M. hyopneumoniae (7448, J and 232), with a focus on genome organization and gene comparison for open read frame (ORF) cluster (OC) identification. An in silico analysis of gene organization demonstrated 117 OCs and 34 single ORFs in M. hyopneumoniae 7448 and J, while 116 OCs and 36 single ORFs were identified in M. hyopneumoniae 232. Genomic comparison revealed high synteny and conservation of gene order between the OCs defined for 7448 and J strains as well as for 7448 and 232 strains. Twenty-one OCs were chosen and experimentally confirmed by reverse transcription–PCR from M. hyopneumoniae 7448 genome, validating our prediction. A subset of the ORFs within an OC could be independently transcribed due to the presence of internal promoters. Our results suggest that transcription occurs in ‘run-on’ from an upstream promoter in M. hyopneumoniae, thus forming large ORF clusters (from 2 to 29 ORFs in the same orientation) and indicating a complex transcriptional organization.
ORF cluster; intergenic regions; cotranscription; transcriptional units
Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis live in swine respiratory tracts. M. flocculare, a commensal bacterium, is genetically closely related to M. hyopneumoniae, the causative agent of enzootic porcine pneumonia. M. hyorhinis is also pathogenic, causing polyserositis and arthritis. In this work, we present the genome sequences of M. flocculare and M. hyopneumoniae strain 7422, and we compare these genomes with the genomes of other M. hyoponeumoniae strain and to the a M. hyorhinis genome. These analyses were performed to identify possible characteristics that may help to explain the different behaviors of these species in swine respiratory tracts.
The overall genome organization of three species was analyzed, revealing that the ORF clusters (OCs) differ considerably and that inversions and rearrangements are common. Although M. flocculare and M. hyopneumoniae display a high degree of similarity with respect to the gene content, only some genomic regions display considerable synteny. Genes encoding proteins that may be involved in host-cell adhesion in M. hyopneumoniae and M. flocculare display differences in genomic structure and organization. Some genes encoding adhesins of the P97 family are absent in M. flocculare and some contain sequence differences or lack of domains that are considered to be important for adhesion to host cells. The phylogenetic relationship of the three species was confirmed by a phylogenomic approach. The set of genes involved in metabolism, especially in the uptake of precursors for nucleic acids synthesis and nucleotide metabolism, display some differences in copy number and the presence/absence in the three species.
The comparative analyses of three mycoplasma species that inhabit the swine respiratory tract facilitated the identification of some characteristics that may be related to their different behaviors. M. hyopneumoniae and M. flocculare display many differences that may help to explain why one species is pathogenic and the other is considered to be commensal. However, it was not possible to identify specific virulence determinant factors that could explain the differences in the pathogenicity of the analyzed species. The M. hyorhinis genome contains differences in some components involved in metabolism and evasion of the host’s immune system that may contribute to its growth aggressiveness. Several horizontal gene transfer events were identified. The phylogenomic analysis places M. hyopneumoniae, M. flocculare and M. hyorhinis in the hyopneumoniae clade.
Mycoplasma; Comparative genomics; Adhesins; Swine respiratory tract
Several Mycoplasma species have had their genome completely sequenced, including four strains of the swine pathogen Mycoplasma hyopneumoniae. Nevertheless, little is known about the nucleotide sequences that control transcriptional initiation in these microorganisms. Therefore, with the objective of investigating the promoter sequences of M. hyopneumoniae, 23 transcriptional start sites (TSSs) of distinct genes were mapped. A pattern that resembles the σ70 promoter −10 element was found upstream of the TSSs. However, no −35 element was distinguished. Instead, an AT-rich periodic signal was identified. About half of the experimentally defined promoters contained the motif 5′-TRTGn-3′, which was identical to the −16 element usually found in Gram-positive bacteria. The defined promoters were utilized to build position-specific scoring matrices in order to scan putative promoters upstream of all coding sequences (CDSs) in the M. hyopneumoniae genome. Two hundred and one signals were found associated with 169 CDSs. Most of these sequences were located within 100 nucleotides of the start codons. This study has shown that the number of promoter-like sequences in the M. hyopneumoniae genome is more frequent than expected by chance, indicating that most of the sequences detected are probably biologically functional.
Mycoplasma; promoter; transcription; sigma; matrix
Mycoplasma synoviae is a major pathogen of poultry, causing synovitis and respiratory infection. A cluster of 45- to 50-kDa membrane proteins is immunodominant in strain WVU-1853. Four distinct proteins were identified in this cluster by high-pressure liquid chromatography. Monoclonal antibodies and monospecific antisera against each established that they fell into two groups, MSPA and MSPB, each containing two members distinguishable by a difference in hydrophobicity. A 25- to 30-kDa membrane protein (MSPC) was shown to be antigenically related to the MSPB proteins. Considerable variation in the size and expression of MSPA and MSPB was observed among different strains of M. synoviae. Examination of expression in colonies of strain WVU-1853 established that both MSPA and MSPB (and MSPC) were phase variable. Immunostaining of MSPB (and MSPC) with monoclonal antibodies exhibited quantal variation, with three distinct levels observed between and within colonies. Hemadsorption by M. synoviae colonies was also found to be phase variable, with some colonies exhibiting sectorial expression of hemadsorption. Monospecific antisera against MSPA inhibited hemagglutination, but neither monoclonal antibodies nor monospecific antisera against MSPB could inhibit hemagglutination. However, loss of the capacity to hemadsorb by individual clones was associated with loss of expression of both MSPA and MSPB. These findings have elucidated the complexity of structure, function, and expression of the 45- to 50-kDa membrane protein cluster of M. synoviae, and they suggest that all members of the cluster may be involved in adhesion.
Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP), a mild, chronic pneumonia of swine. Despite presenting with low direct mortality, EP is responsible for major economic losses in the pig industry. To identify the virulence-associated determinants of M. hyopneumoniae, we determined the whole genome sequence of M. hyopneumoniae strain 168 and its attenuated high-passage strain 168-L and carried out comparative genomic analyses.
We performed the first comprehensive analysis of M. hyopneumoniae strain 168 and its attenuated strain and made a preliminary survey of coding sequences (CDSs) that may be related to virulence. The 168-L genome has a highly similar gene content and order to that of 168, but is 4,483 bp smaller because there are 60 insertions and 43 deletions in 168-L. Besides these indels, 227 single nucleotide variations (SNVs) were identified. We further investigated the variants that affected CDSs, and compared them to reported virulence determinants. Notably, almost all of the reported virulence determinants are included in these variants affected CDSs. In addition to variations previously described in mycoplasma adhesins (P97, P102, P146, P159, P216, and LppT), cell envelope proteins (P95), cell surface antigens (P36), secreted proteins and chaperone protein (DnaK), mutations in genes related to metabolism and growth may also contribute to the attenuated virulence in 168-L. Furthermore, many mutations were located in the previously described repeat motif, which may be of primary importance for virulence.
We studied the virulence attenuation mechanism of M. hyopneumoniae by comparative genomic analysis of virulent strain 168 and its attenuated high-passage strain 168-L. Our findings provide a preliminary survey of CDSs that may be related to virulence. While these include reported virulence-related genes, other novel virulence determinants were also detected. This new information will form the foundation of future investigations into the pathogenesis of M. hyopneumoniae and facilitate the design of new vaccines.
Mycoplasma hyopneumoniae; Genetic variation; Virulence attenuation; Sequence analysis; Repetitive sequences; Virulence factors
Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including Mycoplasma genitalium (n = 11), Mycoplasma pneumoniae (n = 5), Mycoplasma hominis (n = 5), Mycoplasma hyopneumoniae (n = 9), Myco plasma flocculare (n = 5), Mycoplasma hyosynoviae (n = 10), and Mycoplasma dispar (n = 5). AFLP templates were prepared by the digestion of mycoplasmal DNA with BglII and MfeI restriction endonucleases and subsequent ligation of corresponding site-specific adapters. The amplification of AFLP templates with a single set of nonselective primers resulted in reproducible fingerprints of approximately 60 to 80 fragments in the size range of 50 to 500 bp. The method was able to discriminate the analyzed strains at species and intraspecies levels as well. Each of the tested Mycoplasma species developed a banding pattern entirely different from those obtained from other species under analysis. Subtle intraspecies genomic differences were detected among strains of all of the Mycoplasma species analyzed. The extent of polymorphism varied markedly between the analyzed mycoplasmas, comprising pattern similarity levels from 61.7% detected among M. dispar strains to 95.9% detected among M. genitalium strains. The results of the present study provide evidence of the high discriminatory power of AFLP analysis, suggesting the possible applicability of this method to the molecular characterization of mycoplasmas.
The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an indirect enzyme-linked immunosorbent assay for the detection of antibodies in sera from convalescent pigs showed no correlation with clinical and pathological findings.
Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic and economically significant respiratory disease that affects swine production worldwide. M. hyopneumoniae adheres to and adversely affects the function of ciliated epithelial cells of the respiratory tract, and the cilium adhesin (Mhp183, P97) is intricately but not exclusively involved in this process. Although binding of pathogenic bacteria to glycosaminoglycans is a recognized step in pathogenesis, knowledge of glycosaminoglycan-binding proteins in M. hyopneumoniae is lacking. However, heparin and other sulfated polysaccharides are known to block the binding of M. hyopneumoniae to purified swine respiratory cilia. In this study, four regions within the cilium adhesin were examined for the ability to bind heparin. Cilium adhesin fragments comprising 653 amino acids of the N terminus and 301 amino acids of the C terminus (containing two repeat regions, R1 and R2) were cloned and expressed. These fragments bound heparin in a dose-dependent and saturable manner with physiologically significant binding affinities of 0.27 ± 0.02 μM and 1.89 ± 0.33 μM, respectively. Heparin binding of both fragments was strongly inhibited by the sulfated polysaccharides fucoidan and mucin but not by chondroitin sulfate B. When the C-terminal repeat regions R1 and R2 were cloned separately and expressed, heparin-binding activity was lost, suggesting that both regions are required for heparin binding. The ability of the cilium adhesin to bind heparin indicates that this molecule plays a multifunctional role in the adherence of M. hyopneumoniae to host respiratory surfaces and therefore has important implications with respect to the pathogenesis of this organism.
Sialidase activity varies widely among strains and tends to correlate with strain virulence in the avian pathogen Mycoplasma synoviae. To characterize the forms of selection acting on enzymes required for sialic acid scavenging and catabolism, the ratios of nonsynonymous (Ka) to synonymous (Ks) mutation frequency were calculated for codons in the sialidase gene of 16 strains of M. synoviae and for its nearly identical homolog in four strains of Mycoplasma gallisepticum. The Ka/Ks (ω) values for the linked genes required for nutritive N-acetylneuraminate catabolism (nanA, nagC, nanE, nagA, and nagB) from nine strains of M. synoviae were also determined. To provide context, ω was determined for all corresponding genes of 26 strains of Clostridium perfringens and Streptococcus pneumoniae. Bayesian models of sequence evolution showed that only the sialidase of M. synoviae was under significant (P < 0.001) diversifying selection, while the M. synoviae genes for N-acetylneuraminate catabolism and all genes examined from M. gallisepticum, C. perfringens, and S. pneumoniae were under neutral to stabilizing selection. Diversifying selection acting on the sialidase of M. synoviae, but not on the sialidase of M. gallisepticum or the sialidases or other enzymes essential for sialic acid scavenging in other Firmicutes, is evidence that variation in specific activity of the enzyme is perpetuated by a nonnutritive function in M. synoviae that is influenced by the genomic context of the organism.
Mycoplasma hyopneumoniae strain 168, a pathogenic strain prevalent in China, was isolated in 1974. Although this strain has been widespread for a long time, the genome sequence had not been determined. Here, we announce the complete genome sequence of M. hyopneumoniae strain 168.
Mycoplasma synoviae strain MS-H, developed by chemical mutagenesis of the Australian field strain 86079/7NS, is a live temperature-sensitive (ts+) vaccine used for control of M. synoviae infection in poultry worldwide. Genetic basis of temperature sensitivity and attenuation of MS-H has not been revealed thus far. Comparison of the complete genome sequence of MS-H, its parent strain 86079/7NS and two non-temperature sensitive (ts–) reisolates of MS-H revealed a mutation in a highly conserved domain of GTP binding protein Obg of MS-H, with reversion in ts– MS-H reisolates. Nucleotide change from G to A at position 369 of the obg gene resulted in an alteration of glycine to arginine at position 123 in Obg fold. Further analysis of the complete obg gene sequence in several MS-H reisolates revealed that a Gly123Arg substitution was associated with alteration in temperature sensitivity phenotype of MS-H. A second mutation, C to T at position 629, in obg gene was found in some of the MS-H reisolates and appeared to suppress the effects of the Gly123Arg substitution. In silico analysis of point mutations revealed that Gly123Arg has highly destabilizing effect on the MS-H Obg structure that can potentially abolish its biological functions in vivo especially at non-permissive temperature. Findings of this study implicate Obg alteration (Gly123Arg) as one of the possible causes of MS-H attenuation/temperature sensitivity and warrant further investigations into exploring the role of Obg-like proteins, an evolutionarily conserved protein from human to bacteria, in the biology of mycoplasmas.
We investigated the effects of intact pathogenic Mycoplasma hyopneumoniae, nonpathogenic M. hyopneumoniae, and Mycoplasma flocculare on intracellular free Ca2+ concentrations ([Ca2+]i) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca2+]i of 103 ± 3 nM (n = 217 cells). The [Ca2+]i increased by 250 ± 19 nM (n = 47 cells) from the basal level within 100 s of the addition of pathogenic M. hyopneumoniae strain 91-3 (300 μg/ml), and this increase lasted ∼60 s. In contrast, nonpathogenic M. hyopneumoniae and M. flocculare at concentrations of 300 μg/ml failed to increase [Ca2+]i. In Ca2+-free medium, pathogenic M. hyopneumoniae still increased [Ca2+]i in tracheal cells. Pretreatment with thapsigargin (1 μM for 30 min), which depleted the Ca2+ store in the endoplasmic reticulum, abolished the effect of M. hyoneumoniae. Pretreatment with pertussis toxin (100 ng/ml for 3 h) or U-73122 (2 μM for 100 s), an inhibitor of phospholipase C, also abolished the effect of M. hyopneumoniae. The administration of mastoparan 7, an activator of pertussis toxin-sensitive proteins Gi and Go, increased [Ca2+]i in ciliated tracheal cells. These results suggest that pathogenic M. hyopneumoniae activates receptors that are coupled to Gi or Go, which in turn activates a phospholipase C pathway, thereby releasing Ca2+ from the endoplasmic reticulum. Thus, an increase in Ca2+ may serve as a signal for the pathogenesis of M. hyopneumoniae.
Contour clamped homogeneous field (CHEF) agarose gel electrophoresis (AGE), ramped to give linear separation of DNA molecules of 600-1600 kilobase pairs (kbp), was used to determine mobilities for full-sized genomic DNA of the serotype standard strains of the human genital mollicutes, Ureaplasma urealyticum relative to yeast chromosomal DNA markers. Indicated genome sizes (in kbp) were 760 for the four biotype 1 strains and 840-1140 for eleven biotype 2 strains. Other estimates were: 720 for Mycoplasma hominis, 1070 for Mycoplasma hyopneumoniae, 890 for Mycoplasma flocculare, 1180 and 1350 for Mycoplasma mycoides subsp. mycoides Y and GC1176-2, respectively, and 1650 and 1580 for Acholeplasma laidlawii B and PG 8, respectively. These data supplement previous evidence from CHEF AGE that the genomes of the Mycoplasmataceae are diverse in size with some larger than previously estimated from DNA renaturation kinetics.
In the present investigation we developed a method for the detection of Mycoplasma hyopneumoniae in bronchoalveolar lavage fluid (BALF) of pigs by PCR with a primer pair flanking a DNA fragment of 853 bp specific for M. hyopneumoniae. Several methods were tested to eliminate the amplification inhibitors present in BALFs. The best results were obtained by the extraction of the DNA from the BALFs. By the PCR performed with the extracted DNA, 102 CFU of M. hyopneumoniae could be detected in 1 ml of BALF from specific-pathogen-free swine experimentally inoculated with M. hyopneumoniae. DNA from 11 other mycoplasma species and 17 cell-walled bacterial species colonizing the respiratory tracts of pigs was not amplified. In a field study BALFs from 40 pigs from farms with a history of chronic pneumonia were tested for M. hyopneumoniae by cultivation and by PCR (i) with BALFs incubated in Friis medium and (ii) with DNA extracted from the BALFs. In addition, PCR was performed with postmortem lung washings from 19 of the 40 pigs, and immunofluorescence tests were carried out with sections of lungs from 18 of the 40 pigs. M. hyopneumoniae could not be detected in 18 of the 40 pigs by any of the five methods tested. The remaining 22 pigs showed a positive reaction by the PCR with DNA extracted from the BALFs and variable positive reactions by the other tests. A complete correspondence could be observed between the immunofluorescence test result and the result of PCR with DNA. The investigation shows that the PCR with DNA extracted from BALFs is a suitable technique for the sensitive and specific in vivo detection of M. hyopneumoniae.
A reservoir of pseudogene alleles encoding the primary adhesin VlhA occurs in the avian pathogen Mycoplasma synoviae. Recombination between this reservoir and its single expression site was predicted to result in lineages of M. synoviae that each express a different vlhA allele as a consequence of host immune responses to those antigens. Such interstrain diversity at the vlhA expression site, including major differences in the predicted secondary structures of their expressed adhesins, was confirmed in 14 specimens of M. synoviae. Corresponding functional differences in the extent to which they agglutinated erythrocytes, a quantitative proxy for VlhA-mediated cytadherence, were also evident. There was a >20-fold difference between the highest- and lowest-agglutinating strains and a rheostatic distribution of intermediate phenotypes among the others (Tukey-Kramer honestly significant difference [HSD], P < 0.001). Coincubation with the sialic acid analog 2-deoxy-2,3-didehydro-N-acetylneuraminate inhibited hemagglutination in a pattern correlated with endogenous sialidase activity (r = 0.91, P < 0.001), although not consistently to the same extent that erythrocyte pretreatment with sialidase purified from Clostridium perfringens did (P < 0.05). The striking correlation between the ranked hemagglutination and endogenous sialidase activities of these strains (Spearman's r = 0.874, P < 0.001) is evidence that host-induced vlhA allele switching indirectly drives sequence diversity in the passenger sialidase gene of M. synoviae.
Newcastle disease is characterized by respiratory manifestations in association with nervous and/or digestive symptoms. Its prevention is done by vaccination with live attenuated (lentogenic strains) and/or killed vaccines. The lentogenic strains can lead to strong post-vaccination reaction, principally due to the presence of other pathogenic agents. Among them, Mycoplasma synoviae is worldwide important, mainly in Brazil. The dissemination of this agent in poultry flocks has been achieved due to difficulties in diagnosis and disease reproduction, virulence variations among different M.synoviae strains, and attribution of typical M.synoviae disease manifestation to other disease agents. This experimental study in SPF chicks (Gallus gallus), previously infected by M.synoviae and thereafter vaccinated against Newcastle disease, was done with the objective of evaluating M.synoviae pathogenicity through assessment of post-vaccinal respiratory reactions and serologic responses to Newcastle disease virus vaccine in the absence of environmental factors. A total of 86 three days old chicks were used, being 57 infected by eye and nostril drop, with chicken activated M. synoviae strain WVU 1853. Seven days later, 21 mycoplasma infected birds plus 29 not mycoplasma infected ones were vaccinated against Newcastle disease. As results, the not infected and vaccinated birds yielded, significantly, higher and longer lasting serologic responses to Newcastle disease vaccine virus than those infected and vaccinated. Similarly, the infected and vaccinated birds yielded lower serologic reactions to M.synoviae than those only mycoplasma infected. No post-vaccinal respiratory reaction was observed in the vaccinated birds.
vaccine; serology; Mycoplasma; Newcastle disease
Mycoplasma gallisepticum is a bacterial pathogen of poultry that is estimated to cause annual losses exceeding $780 million. The National Poultry Improvement Plan guidelines recommend regular surveillance and intervention strategies to contain M. gallisepticum infections and ensure mycoplasma-free avian stocks, but several factors make detection of M. gallisepticum and diagnosis of M. gallisepticum infection a major challenge. Current techniques are laborious, require special expertise, and are typically plagued by false results. In this study, we describe a novel detection strategy which uses silver nanorod array–surface-enhanced Raman spectroscopy (NA-SERS) for direct detection of avian mycoplasmas. As a proof of concept for use in avian diagnostics, we used NA-SERS to detect and differentiate multiple strains of avian mycoplasma species, including Acholeplasma laidlawii, Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma synoviae, and M. gallisepticum, including vaccine strains 6/85, F, and ts-11. Chemometric multivariate analysis of spectral data was used to classify these species rapidly and accurately, with >93% sensitivity and specificity. Furthermore, NA-SERS had a lower limit of detection that was 100-fold greater than that of standard PCR and comparable to that of real-time quantitative PCR. Detection of M. gallisepticum in choanal cleft swabs from experimentally infected birds yielded good sensitivity and specificity, suggesting that NA-SERS is applicable for clinical detection.
The ability of Mycoplasma hyopneumoniae to persist despite fluoroquinolone treatments was investigated with pigs. Groups of specific-pathogen-free pigs were experimentally infected with M. hyopneumoniae strain 116 and treated with marbofloxacin at the therapeutic dose (TD) or half of the therapeutic dose (TD/2) for 3 days. Results showed that, despite tissue penetration of marbofloxacin, particularly in the trachea and the tracheal secretions, the treatments did not have any influence on M. hyopneumoniae recovery from tracheal swabs. Mycoplasmas were also isolated from inner organs and tissues such as liver, spleen, kidneys, and bronchial lymph nodes. Recontamination of pigs via environment could not explain mycoplasma persistence after medication, as decontamination of pigs and allocation to a new disinfected environment did not have any significant effect on the phenomenon. A significant decrease in the susceptibility level to marbofloxacin of 12 mycoplasma clones reisolated after the treatments (TD/2 and TD) was observed. Two point mutations were found in the ParC quinolone resistance-determining region (QRDR) of DNA topoisomerase IV (Ser80→Phe and Asp84→Asn), and one point mutation was observed just behind the QRDR of ParC (Ala116→Glu). This is the first time that mutations in a gene coding for topoisomerase IV have been described for M. hyopneumoniae after in vivo marbofloxacin treatments in experimentally infected pigs. However, development of resistance is not sufficient to explain M. hyopneumoniae persistence in vivo since (i) marbofloxacin concentrations were above the marbofloxacin MIC of the wild-type strain and (ii) mycoplasmas reisolated after a single injection of marbofloxacin did not display an increased marbofloxacin MIC.
Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine. p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39°C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.
Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes the respiratory ciliated epithelial cells. Cilium adherence is mediated by P97, a surface protein containing a repeating element (R1) that is responsible for binding. Here, we show that the cilium adhesin is proteolytically processed on the surface. Proteomic analysis of strain J proteins identified cleavage products of 22, 28, 66, and 94 kDa. N-terminal sequencing showed that the 66- and 94-kDa proteins possessed identical N termini and that the 66-kDa variant was generated by cleavage of the 28-kDa product from the C terminus. The 22-kDa product represented the N-terminal 195 amino acids of the cilium adhesin preprotein, confirming that the hydrophobic leader signal sequence is not cleaved during translocation across the membrane. Comparative studies of M. hyopneumoniae strain 232 showed that the major cleavage products of the cilium adhesin are similar, although P22 and P28 appear to be processed further in strain 232. Immunoblotting studies using antisera raised against peptide sequences within P22 and P66/P94 indicate that processing is complex, with cleavage occurring at different frequencies within multiple sites, and is strain specific. Immunogold electron microscopy showed that fragments containing the cilium-binding site remained associated with the cell surface whereas cleavage products not containing the R1 element were located elsewhere. Not all secreted proteins undergo multiple cleavage, however, as evidenced by the analysis of the P102 gene product. The ability of M. hyopneumoniae to selectively cleave its secreted proteins provides this pathogen with a remarkable capacity to alter its surface architecture.
Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.
real-time PCR; Mycoplasma gallisepticum; Mycoplasma synoviae
Bacteria belonging to the class Mollicutes were among the first ones to be selected for complete genome sequencing because of the minimal size of their genomes and their pathogenicity for humans and a broad range of animals and plants. At this time six genome sequences have been publicly released (Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum-parvum, Mycoplasma pulmonis, Mycoplasma penetrans and Mycoplasma gallisepticum) and as the number of available mollicute genomes increases, comparative genomics analysis within this model group of organisms becomes more and more instructive. However, such an analysis is difficult to carry out without a suitable platform gathering not only the original annotations but also relevant information available in public databases or obtained by applying common bioinformatics methods. With the aim of solving these difficulties, we have developed a web-accessible database named MolliGen (http://cbi.labri.fr/outils/molligen/). After selecting a set of genomes the user can launch various types of search based on annotation, position on the chromosomes or sequence similarity. In addition, relationships of putative orthology have been precomputed to allow differential genome queries. The results are presented in table format with multiple links to public databases and to bioinformatic analyses such as multiple alignments or BLAST search. Specific tools were also developed for the graphical visualization of the results, including a multi- genome browser for displaying dynamic pictures with clickable objects and for viewing relationships of precomputed similarity. MolliGen is designed to integrate all the complete genomes of mollicutes as they become available.
Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen.
The P46 and P65 proteins of Mycoplasma hyopneumoniae are two membranous proteins carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the genes encoding entire P46 (1,260 bp) and P65 (1,803 bp) and N-terminally truncated P65c (1,200 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species that commonly colonize the porcine respiratory tract. Both amplified genes were then cloned into the pGEX-4T-1 vector to be expressed in Escherichia coli cells as recombinant fusion proteins with glutathione S-transferase (GST). Prior to generation of expression constructs, TGA nonsense codons, exceptionally used for tryptophan residues by M. hyopneumoniae, had been converted to TGG codons by PCR-directed mutagenesis. Following induction by IPTG (isopropyl-β-d-thiogalactopyranoside), both GST-P46 and GST-P65c recombinant fusion proteins were recovered by disrupting transformed cells by sonication, purified by affinity chromatography, and then cut with thrombin to release the P46 and P65c moieties. The enriched E. coli-expressed P46 and P65c proteins were used to immunize female BALB/c mice for the generation of anti-P46 and anti-P65c monoclonal antibodies (MAbs). The polypeptide specificities of MAbs obtained was confirmed by Western blotting with cell lysates prepared from the homologous strain. Cross-reactivity study of the anti-P46 and anti-P65c MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture, suggested that the MAbs obtained against both membranous proteins were directed against highly conserved species-specific epitopes. No reactivity to other mycoplasma species tested was demonstrated. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs that had been inoculated intratracheally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. Both anti-P46 and anti-P65c MAbs permitted effective detection by indirect immunofluorescence and indirect immunoperoxidase assay of M. hyopneumoniae in, respectively, frozen and formalin-fixed, paraffin-embedded lung sections from pigs that were killed after the 6- to 7-week observation period.