Iscom is an efficient mucosal delivery system for Mycoplasma mycoides subsp. mycoides (MmmSC) antigens inducing high mucosal and systemic antibody responses

The purpose of this study was to explore the iscom as a mucosal delivery system for Mycoplasma mycoides subsp. mycoides small colony (MmmSC) antigens. BALB/c female mice were immunised intranasally (i.n.) twice, 8 weeks apart with three different doses (3, 10 and 20 μg) or subcutaneously (s.c.) with 3 μg of M. mycoides antigens incorporated into iscoms. Mycoplasma cells were administered s.c. twice, 8 weeks apart at a dose of 3 μg or i.n. at 10 μg as for iscoms. Both i.n. and s.c. modes of immunisation with iscoms induced prominent primary serum antibody responses in a dose-dependent manner, which were efficiently boosted. Compared to whole mycoplasma cells, iscoms enhanced the total Ig and IgG subclass (IgG1, IgG2a and IgG2b) responses in serum and in lungs greatly, and this enhancement was more prominent after i.n. than after s.c. immunisation. By the i.n. mode of immunisation iscoms containing mycoplasma antigens induced a 60-fold higher IgA response in lungs than the whole cell antigen. Iscoms also induced substantially higher total Ig and IgG subclass responses in the lungs. By Western blot a reduced number of bands (7) were detected in lung secretion after both i.n. and s.c. immunisations with iscoms compared to a high number of bands (more than 30) detected by serum antibodies. Interestingly i.n. immunisation with iscoms induced antibodies in lungs as well as in serum to mycoplasma cell antigens which differed from those induced by s.c. immunisation as revealed by the Western blot patterns.     

Complete Genome Sequences of Mycoplasma leachii Strain PG50T and the Pathogenic Mycoplasma mycoides subsp. mycoides Small Colony Biotype Strain Gladysdale

Mycoplasma mycoides subsp. mycoides small colony biotype (SC) is the high-consequence animal pathogen causing contagious bovine pleuropneumonia. We report the complete genome sequences of the pathogenic strain M. mycoides subsp. mycoides SC Gladysdale and a close phylogenetic relative, Mycoplasma leachii PG50(T), another bovine pathogen of the M. mycoides phylogenetic clade.     

Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp. mycoides small colony (MmmSC) vaccine cultures

The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C. The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month. After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1). In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).     

One Test Microbial Diagnostic Microarray for Identification of Mycoplasma mycoides subsp. mycoides and Other Mycoplasma Species

The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri strains. Specifically, we observed that some Italian Mycoplasma mycoides subsp. mycoides strains were positive for two out of the three Mycoplasma mycoides subsp. capri genes, differently from what has been observed for other European or African Mycoplasma mycoides subsp. mycoides strains. This study highlighted the use of microarray technology as a simple and effective method for a single-step identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. The opportunity to discriminate several mycoplasmas in a single analysis enhances diagnostic rapidity and may represent a useful tool to screen occasionally mycoplasmas affecting animal farming in territories where diagnostic laboratory support is limited. The heat-map of the hybridization results of the comparative genomic hybridizations DNA-designed chip clearly indicates that the microarray performs well for the identification of the tested Mycoplasma mycoides subsp. mycoides reference and field strains, discriminating them from other mycoplasmas.     

Uptake and utilization of deoxynucleoside 5''-monophosphates by Mycoplasma mycoides subsp. mycoides

Mycoplasma mycoides subsp. mycoides has been shown to possess an unusual capacity for the uptake and utilization of exogenous deoxyribonucleoside 5'-monophosphates intact without prior dephosphorylation. In this study, it was found that once inside the cell, deoxyribonucleoside 5'-monophosphates were rapidly phosphorylated to the triphosphate level and incorporated into DNA. Catabolism of deoxyribonucleoside 5'-monophosphates was also observed. Competition studies indicated that a single uptake system with a higher affinity for deoxyribonucleotides mediates the uptake of nucleoside 5'-monophosphates.     

Relationship between Mycoplasma mycoides subsp. mycoides ('large-colony' strains) and M. mycoides subsp. capri, as indicated by numerical analysis of one-dimensional SDS-PAGE protein patterns

Twenty-five strains classified as Mycoplasma mycoides subsp. mycoides LC or subsp. capri have been compared by one-dimensional SDS-PAGE of their cellular proteins. A computerized numerical analysis revealed that the protein patterns of all but two aberrant strains formed one large phenon that separated clearly from representatives of the four other members of the 'M. mycoides cluster' at a similarity level (S) of 66% and which remained undivided at up to 78% S. At higher similarity levels, these strains fell heterogeneously into mixed sub-phenons containing strains of both subspecies. Serological comparisons by immunofluorescence largely confirmed the subspecies designations of the test strains, but also showed that some were serologically intermediate between subsp. mycoides and subsp. capri, being cross-reactive with both. These results confirm and enlarge upon those of our earlier studies indicating the protein-pattern inseparability of subsp. capri and subsp. mycoides LC strains and their distinctiveness from the classical M. mycoides subsp. mycoides SC strains and other members of the 'M. mycoides cluster'. As also recognized by other workers, subsp. mycoides LC and subsp. capri strains appear to comprise one large group, wherein those most readily identifiable as either type lie at either end of a serological spectrum that also contains serologically cross-reactive strains. Our observations therefore suggest the lines along which the three groups classified at present within the species M. mycoides (SC and LC strains of subsp. mycoides; subsp. capri) might eventually be reclassified, subject to direct genomic comparisons.     

Isolation of Mycoplasma mycoides subsp. mycoides (LC variant, Y-Goat) from naturally aborted bovine fetuses

Seven strains of Mycoplasma mycoides subsp. mycoides (LC, Y-Goat) were isolated from 4 of 85 (4.7%) cases of bovine abortion. The biochemical characters of all the isolates were homogenous (except for one sucrose negative isolate), but variation in sensitivity to neomycin, kanamycin and streptomycin were noted. The only gross lesions of internal organs in aborted fetuses were congestion of liver and lungs, and hemorrhagic patches on the heart surface. Significant microscopic lesions were encountered in lungs (edema of the interlobular septae, thickened alveolar wall, lymphocytic infiltration, septal hyperplasia and alveolar infiltration with neutrophils and macrophages), liver (mild lymphocytic infiltration in the hepatic triad, hypertrophy of Von-kupffer cells), kidney (degenerative necrosis and desquamation of tubular epithelium, and the marked lymphocytic infiltration in the interstitium), spleen (depleted lymphoid tissues, infiltration of lymphocytes, macrophage and plasma cells in the red pulp) and heart (lymphocytic myocarditis). The observations of this study focus on the role of M. mycoides subsp. mycoides (LC variant) in bovine abortions.     

A rapid biochemical test to aid identification of Mycoplasma mycoides subsp. mycoides small colony (SC) strains

The ability to utilize maltose, as determined by measurement of oxygen uptake, is used to differentiate Mycoplasma mycoides subsp. mycoides small colony (SC) and M. capricolum subsp. capripneumoniae (all strains negative) from other members of the M. mycoides cluster (M. mycoides subsp. capri, M. mycoides subsp. mycoides large colony (LC), M. capricolum subsp. capricolum; and bovine serogroup 7; 94% of strains positive). Rapid tests for maltose utilizing ability were developed, based on hydrolysis of a chromogenic alpha-glucosidase (maltase) substrate (p-nitrophenyl-alpha-D-glucopyranoside, colourless) to give a brightly coloured product (p-nitrophenol, yellow). On agar plates, colonies of maltose-utilizing strains became coloured within 40 min.     

Recombinant Surface Proteomics as a Tool to Analyze Humoral Immune Responses in Bovines Infected by Mycoplasma mycoides Subsp. mycoides Small Colony Type

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC)¹ is the causative agent of contagious bovine pleuropneumonia (CBPP), a severe respiratory disease in cattle. It is a disease requiring official declaration to the World Organization for Animal Health (OIE) and that causes vast problems in Africa with severe socioeconomic consequences (1, 2). In 2006, 15 African countries reported 186 outbreaks of CBPP to the OIE. CBPP was eradicated from Europe in the beginning of the 20th century (3) but has reemerged in every decade since (4). Eradication was largely facilitated by slaughtering infected herds, which is still considered as the most efficient means of disease control and was successfully performed in Botswana in 1995 (5). However, this campaign was directly correlated to increased malnutrition in children (6) and is also considered to be too expensive for other African countries (2, 7). The use of chemotherapy in CBPP control is a debated subject, has long been discouraged, and is even illegal in some countries (1), mainly because of the risk of creating silent carriers of the disease (8). However, new antibiotics have shown positive effects (9), but extensive vaccinations are still considered the preferred option for prevention and control of CBPP in Africa (2, 10, 11). The vaccines currently in use are based on live attenuated M. mycoides SC strains and have several disadvantages such as short term immunity (12), poor protection as indicated in recent trials (4, 13), and even pathogenicity (13, 14).The two currently available tests for serological diagnosis of CBPP recommended by the OIE, the complement fixation test (15) and a competitive ELISA (16), are based on whole cell M. mycoides SC. For subcellular components of the organism, the genome sequence of M. mycoides SC strain PG1 (17) offers an emerging possibility to improve both diagnostic and therapeutic approaches with selected antigens. However, as for the 10 other Mycoplasma genomes sequenced, the genome sequences per se did not reveal any primary virulence factors common in other bacteria, such as adhesins or toxins (18). The few known molecular mechanisms of pathogenicity were recently reviewed (18) and include five lipoproteins studied in detail: LppA (19, 20), LppB (21), LppC (22) LppQ (23), and Vmm (24). Of these, LppQ has been used to develop an indirect ELISA (25), and Vmm, a variable surface protein, has recently been studied along with five novel putative variable surface proteins as recombinant proteins expressed in Escherichia coli (26). That study demonstrated the feasibility of producing recombinant surface proteins from M. mycoides SC in E. coli and screening for antibodies in sera from CBPP-affected bovines by Western and dot blotting.To explore further the immunogenicity of the M. mycoides SC surface proteome, a platform for multiplexed analysis of proteins using minute serum samples such as bead-based array systems (27) is desirable. One method is available from Luminex Corp. and uses spectrally distinguishable beads (28) to form an array in suspension. The array is analyzed in a flow cytometer-like instrument and can perform up to 100 simultaneous assays in a single reaction well. This platform has recently been used to determine binding specificities to antigens produced in a similar fashion (29) and to profile antibodies in serum toward six antigens of Mycobacterium tuberculosis (30).The aim of this study was to develop a rapid and highly multiplex method for affinity analysis of antibody levels in serum samples from CBPP-affected bovines against recombinant M. mycoides SC surface proteins. To facilitate this, a large set of surface proteins were cloned, expressed in E. coli, and purified. Furthermore, the bead-based assay conditions had to be optimized and verified for detection of immunoglobulin levels in bovine sera. This methodology would enable monitoring and protein-specific characterization of humoral immune responses during CBPP infections. As a secondary aim, the study was expanded to include specific IgG, IgA, and IgM responses in sera from a vaccine study with time series sampling from each animal over 8 months, covering prevaccination and 4 months postinfection.     

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Aims:  The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.
Methods and Results:  The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r ² = 0·93) between mycoplasma counts determined by FC (cells ml⁻¹) and by traditional plate count method (CFU ml⁻¹). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10⁴ cells ml⁻¹ and 10³ CFU ml⁻¹, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).
Conclusion:  Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.
Significance and Impact of the Study:  These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).     

Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display

The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.     

Kinetics of utilization of organic substrates by Mycoplasma mycoides subsp. mycoides in a salts solution: a flow-microcalorimetric study

The metabolism of various organic substrates by suspensions of Mycoplasma mycoides subsp. mycoides in a salts solution was followed by microcalorimetry. Enthalpy changes associated with metabolism were in good agreement with theoretical values. Substrate utilization showed Michaelis kinetics, allowing saturation constants (Km) and maximum specific rates of substrate utilization (Vmax) to be determined. In cells grown on a complex medium containing glucose, Km values were: glucose, fructose, N-acetylglucosamine, glycerol and pyruvate, less than 5 microM; lactate, 20 microM; glucosamine, 130 microns, and mannose, 1 mM. Values of Vmax for glycerol, pyruvate and lactate were similar and approximately twice those for glucose, mannose, glucosamine and N-acetylglucosamine; Vmax for fructose was one-quarter of that for glucose. In cells grown on complex medium in which glucose was replaced by mannose, glucosamine or N-acetylglucosamine, Vmax and Km for the respective growth sugars and for glucose were not significantly affected. However, in cells grown in the presence of fructose, Vmax for fructose increased to the value observed for glucose. It is suggested that M. mycoides is adapted to, and is constitutive for, the utilization of a single sugar (glucose), and a single amino sugar (N-acetylglucosamine), but that in the presence of fructose a fructose-utilizing pathway is induced.     

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 × 10⁵ M⁻¹ which indicates a strong binding close to that of antibody.     

Identification using phage display of peptides promoting targeting and internalization into HPV-transformed cell lines

'High-risk' human papilloma viruses (HPVs) cause cervical tumours. In order to treat these tumours therapeutic approaches must be developed that efficiently target the tumour cells. Using phage display, we selected tumour-targeting peptides from a library of constrained nonamer peptides presented multivalently on pVIII of M13. Three different consensus peptide sequences were isolated by biopanning on HPV16-transformed SiHa cells. The corresponding phage-peptides targeted and were internalized in HPV16 transformed SiHa and CaSki cells as well as in HPV18-transformed HeLa cells, but failed to bind a panel of normal or transformed cell lines. Two of the three selected peptides targeted cells only when presented on phage particles in a constrained conformation. However, all three peptides retained their targeting capacity when presented on the reporter protein enhanced green fluorescent protein (EGFP) in a monovalent form. These peptides may be useful for the design of drug or gene delivery vectors for the treatment of cervical cancer.     

Relationships between strains of Mycoplasma mycoides subspp. mycoides and capri studied by two-dimensional gel electrophoresis of cell proteins.

Strains of Mycoplasma mycoides subsp. mycoides have been divided into small colony (SC) and large colony (LC) types (Cottew & Yeats, 1978). The protein patterns of representative strains of these two types and of M. mycoides subsp. capri were compared by a high resolution, two-dimensional gel electrophoretic method. The results suggest that the LC strains are more closely related to M. mycoides subsp. capri than to the SC strains of M. mycoides subsp. mycoides.     

Identification of a collagen-binding protein from Necator americanus by using a cDNA-expression phage display library.

A phage display library was made starting from a cDNA library from the hematophagous human parasite Necator americanus. The cDNA library was transferred by polymerase chain reaction (PCR) cloning into phage display vectors (phagemids), using specially designed primers such that proteins would be expressed as fusions with the C-terminal part of the phage coat protein pVI. The vectors used are multicloning site variants of the original pDONG vectors described by Jespers et al. (1995). Electroporation of the ligation mixtures into electrocompetent Escherichia coli TGI cells yielded 3 x 10(8) pG6A, 1.9 x 10(8) pG6B, and 1 x 10(8) pG6C transfectants for N. americanus. The final libraries consisted of a mix of equal numbers of insert-containing phages from the A, B, and C libraries. Selection of phages for binding to human collagen was performed. Four rounds of panning on human collagens I and III resulted in a significant enrichment of collagen-binding phages from the N. americanus libraries. PCR analysis revealed various insert lengths; however, sequence determination indicated that all phages contained the same protein, albeit with different poly-A tail lengths. The encoded protein itself is a 135-amino acid protein (15 kDa), with no apparent homology to any other known protein. Next the protein was recloned into E. coli using the pET-15b-vector. Upon isopropyl-1-thio-beta-D-galactopyranoside induction, the recombinant protein, rNecH1, could be recovered by urea treatment from inclusion bodies. The rNecH1 protein binds to different collagens: human I > rat I > human III = calf skin I in a specific, dose-dependent, and saturable manner.     

Distinctive repertoire of contingency genes conferring mutation- based phase variation and combinatorial expression of surface lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides phylogenetic cluster

The generation of surface variation among many divergent species of Mollicutes (mycoplasmas) occurs through stochastic expression patterns of diverse lipoprotein genes. The size and wide distribution of such variable gene sets in minimal (approximately 0.6- to 1.4-Mb) mycoplasmal genomes suggest their key role in the adaptation and survival of these wall-less monoderms. Diversity through variable genes is less clearly established among phylogenetically similar mycoplasmas, such as the Mycoplasma mycoides cluster of ruminant pathogens, which vary widely in host range and pathobiology. Using (i) genome sequences from two members of this clade, Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides small colony biotype (SC), (ii) antibodies to specific peptide determinants of predicted M. capricolum subsp. capricolum gene products, and (iii) analysis of the membrane-associated proteome of M. capricolum subsp. capricolum, a novel set of six genes (vmcA to vmcF) expressing distinct Vmc (variable M. capricolum subsp. capricolum) lipoproteins is demonstrated. These occur at two separate loci in the M. capricolum subsp. capricolum genome, which shares striking overall similarity and gene synteny with the M. mycoides subsp. mycoides SC genome. Collectively, Vmc expression is noncoordinate and combinatorial, subject to a single-unit insertion/deletion in a 5' flanking dinucleotide repeat that governs expression of each vmc gene. All vmc genes share modular regions affecting expression and membrane translocation. In contrast, vmcA to vmcD genes at one locus express surface proteins with highly structured size-variable repeating domains, whereas vmcE to vmcF genes express products with short repeats devoid of predicted structure. These genes confer a distinctive, dynamic surface architecture that may represent adaptive differences within this important group of pathogens as well as exploitable diagnostic targets.     

用差异显示PCR法筛选与血管外膜细胞表型转化相关的基因

为筛选血管外膜成纤维细胞（adventitial fibroblast,AF）与肌成纤维细胞（myofibroblast,MF）间表型转化有关的基因,实验建立了大鼠胸主动脉AF和MF两种细胞模型,用差异显示聚合酶链反应（DD－PCR）技术获得表达差异片段,对差异片段进行克隆和测序分析,并用定量PCR和Northern blot对差别显示结果进行验证。用反义核酸转染技术观察骨桥蛋白（osteopontin,OPN）对AF迁移的影响。结果表明,两种表型细胞存在明显的基因表达差异,其中一个在MF下调的差异片段与GenBank中NADH脱氢酶亚单位5（NADH dehydrogenase subunit 5,Nd5）基因高度同源。另一个在MF上调的差异片段与OPN基因同源。上述差异表达结果被定量PCR及Northern blot证实。此外还有4个表达序列标志（expressed sequence-tag,EST）在GenBank中未查到同源序列。反义OPN寡脱氧核甘酸可抑制AF的迁移活动。结果提示,AF转化为MF可能与ND5基因下调、OPN上调及其它未知基因的表达改变有关。应用反义技术适度抑制OPN表达在防治血管重塑中具有重要作用。