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We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts. 相似文献
3.
It has recently been advanced that Mycobacterium tuberculosis sigma factor K (SigK) positively regulates expression of the antigenic proteins MPB70 and MPB83. As expression of these proteins differs between M. tuberculosis (low) and Mycobacterium bovis (high), this study set out to determine whether M. bovis lacks a functional SigK repressor (anti-SigK). By comparing genes near sigK in M. tuberculosis H37Rv and M. bovis AF2122/97, we observed that Rv0444c, annotated as unknown function, had variable sequence in M. bovis. Analysis of in vitro mpt70/mpt83 expression and Rv0444c sequencing across M. tuberculosis complex (MTC) members revealed that high-level expression was associated with a mutated Rv0444c. Complementation of M. bovis bacillus Calmette-Guerin Russia, a high producer of MPB70/MPB83, with wild-type Rv0444c resulted in a significant decrease in mpb70/mpb83 expression. Conversely, a M. tuberculosis H37Rv mutant which expressed sigK but not Rv0444c manifested the M. bovis phenotype of high-level MPB70/MPB83 expression. Further support that Rv0444c encodes the anti-SigK was obtained by yeast two-hybrid studies, where the N-terminal region of Rv0444c-encoded protein interacted with SigK. Together these findings indicate that Rv0444c encodes the regulator of SigK (RskA) and mutations in this gene explain high-level MPT70/MPT83 expression by certain MTC members. 相似文献
4.
Classification and identification of Mycobacterium africanum by pyrolysis mass spectrometry 总被引:1,自引:0,他引:1
G Wieten J Haverkamp D G Groothuis L G Berwald H L David 《Journal of general microbiology》1983,129(12):3679-3688
Pyrolysis mass spectrometry was used to classify and identify strains of Mycobacterium africanum and of M. tuberculosis, M. bovis and M. bovis BCG. The multicharacter mass pyrograms were evaluated by computerized data handling procedures that were suited for classification and identification. The results revealed considerable heterogeneity among the African strains, which was shown to be linked to the geographic distribution of the strains. On the basis of a routine mass spectrometric identification key the African strains were identified without exception as belonging to, what is referred to as the 'Tuberculosis complex' (i.e. the clinically relevant group formed by strains of M. Tuberculosis, M. bovis and M. bovis BCG). Classification of the strains by means of discriminant analysis indicated an intermediate clustering for the majority of the African strains and overlap for some African strains with in particular M. bovis. It was concluded that from the mass spectrometric data a species status for the group of African strains was not justifiable. 相似文献
5.
Cytolytic T-cell responses from 63 normal blood donors were monitored in a Mycobacterium bovis BCG infection system in vitro. We wanted to know whether cultured dendritic cells were capable of potentiating the cytolytic T-cell responses to M. bovis BCG. Infected cultured dendritic cells were up to ten times more effective antigen-presenting cells than macrophages in proliferative assays, while cytolytic T-cell induction did not differ significantly between dendritic cells and macrophages. Separated CD4+ and CD8+ T-cell subsets contributed equally to lysis of infected targets. Experiments comparing wild-type M. bovis BCG strain with two new recombinant M. bovis BCG strains secreting listeriolysin revealed statistically significant higher maximal lysis values for recombinant M. bovis BCG. We conclude from our in vitro infection system with mycobacteria that dendritic cells are superior to macrophages in proliferative assays but equal to macrophages in their ability to induce cytolytic T-cell responses. Moreover, our data suggest that recombinant M. bovis BCG vaccine strains secreting listeriolysin improve cytolytic T-cell responses. 相似文献
6.
Complete nucleotide sequence of immunogenic protein MPB70 from Mycobacterium bovis BCG 总被引:15,自引:0,他引:15
Kunihiro Terasaka Ryuji Yamaguchi Kazuhiro Matsuo Akihiro Yamazaki Sadamu Nagai Takeshi Yamada 《FEMS microbiology letters》1989,58(2-3):273-276
The extracellular protein MPB70 is a heat-stable immunogenic protein which was found in the culture filtrate of Mycobacterium bovis BCG Japanese. We determined the complete nt and aa sequences of MPB70 and correlated with the previously reported data. The N-terminal sequence revealed that the signal peptide (SP) consisted of 30 aa and that the mature protein had 163 aa with a molecular weight of 16,305. The SP displayed a characteristic feature of an Ala-rich property which would be efficient in a SP function. 相似文献
7.
Alison J Woolford R.Glyn Hewinson Martin Woodward Jeremy W Dale 《FEMS microbiology letters》1997,148(1):43-48
The protein antigen MPB70 is a major component of culture supernatants of Mycobacterium bovis and is an active ingredient of bovine PPD used for skin-testing cattle for tuberculosis. We have shown that Mycobacterium kansasii possesses a similar gene that cross-reacts in a PCR test for M. bovis. Single strand conformational polymorphism analysis, and the DNA sequence of the PCR product, shows differences between M. kansasii strains, supporting the suggestion that M. kansasii is not a homogeneous species. 相似文献
8.
The present investigation dealt with the identification of Mycobacterium tuberculosis and M. bovis by RD9 region and 500 bp fragment PCR assays. Eight M. tuberculosis and 5 M. bovis characterized and identified from 40 human sputum and 41 bovine lung specimens and 20 M. tuberculosis and 9 M. bovis strains maintained at Mycobacteria Laboratory, Indian Veterinary Research Institute were included in this study. In this way, 28 M. tuberculosis and 14 M. bovis strains and, for comparison and control purpose, M. tuberculosis H37Rv, M. bovis BCG, M. canetti, M. smegmatis, M. phlei, M. chelonae, M. kansasii, M. xenopi and M. avium were subjected to RD9 and 500 bp amplification by PCR. All M. tuberculosis strains, M. tuberculosis H37 Rv and M. canetti yielded a product of 333 bp which showed presence of RD9 region in these strains, whereas all M. bovis yielded a product of 206 bp with RD9 PCR assay. There was no ampli-fication product found in M. bovis BCG, M. xenopi, M. smegmatis, M. phlei, M. chelonae, M. kansasii, and M. avium. PCR based on 500 bp fragment showed a product of 500 bp in all M. bovis strains and M. bovis BCG. There was no amplification product of 500 bp found in M. canetti, M. smegmatis, M. phlei, M. chelonae, M. avium, M. kansasii, M. xenopi and was absent in all M. tuberculosis strains. The PCR assay results correlated 100% with the culture and biochemical results of the isolates. Our study suggested that PCR based on RD9 and 500 bp may effectively identify two closely related species of M. tuberculosis and M. bovis. 相似文献
9.
Philippe Lefèvrea Martine Braibanta Jean Contenta Philippe Gilota 《FEMS microbiology letters》1999,178(2):211-217
The structure and distribution of a Mycobacterium bovis BCG insertion element of the IS21 family were investigated. Several IS21-like elements found in mycobacterial genomes were separated in four types, following their nucleic acid similarities. The M. bovis BCG IS21 element is highly similar to IS1533 (class I), 70% similar to IS1534 (class II), 52% similar to IS1532 (class III) of Mycobacterium tuberculosis, and 54% similar to both an Mycobacterium avium serovar 2 and an M. avium silvaticum IS (class IV). The M. bovis BCG IS21 element of the class I appears to be present in a single copy in the genome of M. bovis BCG, M. bovis, M. tuberculosis and Mycobacterium africanum and to be absent from all other tested species of the Corynebacteria-Mycobacteria-Nocardia group. 相似文献
10.
Genome sequence of Mycobacterium bovis BCG Moreau, the Brazilian vaccine strain against tuberculosis
Gomes LH Otto TD Vasconcellos EA Ferrão PM Maia RM Moreira AS Ferreira MA Castello-Branco LR Degrave WM Mendonça-Lima L 《Journal of bacteriology》2011,193(19):5600-5601
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis, and the strains used worldwide represent a family of daughter strains with distinct genotypic characteristics. Here we report the complete genome sequence of M. bovis BCG Moreau, the strain in continuous use in Brazil for vaccine production since the 1920s. 相似文献
11.
B V Nikonenko M B Mezhlumova A M Moroz 《Biulleten' eksperimental'no? biologii i meditsiny》1990,110(11):526-528
Vaccination with M. bovis (BCG) essentially prolonged survival time (ST) of several strain mice, with the exception, of CBA/N, infected with M. tuberculosis H37Rv. ST of CBA/N, differing from CBA by xid mutation, was not prolonged by vaccination. Mouse strains with alternative alleles of BCG gene (s and r) and fzy gene as a genetic marker for Bcg5 were used for segregation analysis. It was shown that ST, the level of DTH reaction of mice infected with M. tuberculosis H37Rv, and protective effect of BCG vaccination did not depend on Bcg gene. However, Bcg gene, apparently, regulate the DTH response to PPD in mice only vaccinated with M. bovis (BCG). 相似文献
12.
Seixas FK Borsuk S Fagundes MQ Hartwig DD Da Silva EF Cerqueira GM Dellagostin OA 《Biological research》2010,43(1):13-18
Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18 kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector. 相似文献
13.
Pulsed field gel electrophoresis of representatives of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains 总被引:1,自引:0,他引:1
Anne Varnerot Françoise Clément Marina Gheorghiu Véronique Vincent-Lévy-Frébault 《FEMS microbiology letters》1992,98(1-3):155-160
Abstract Using field inversion gel electrophoresis (FIGE), different Mycobacterium tuberculosis strains, such as phage prototypes, exhibit different DNA restriction patterns which are easy to compare. Virulent and avirulent variants of M. tuberculosis H37, as well as daughter strains of M. bovis BCG, display characteristic DNA profiles. BCG strains isolated from suppurative adenitis following vaccination of French patients showed patterns identical to the BCG Pasteur strain used for vaccination. These results demonstrate that FIGE of DNA restriction fragments generated by Dra I represents a suitable technique for the analysis of mycobacteria at a genomic level. The Dra I profiles allow the differentiation and precise identification of the BCG Pasteur, Glaxo, Russian and Japanese strains. 相似文献
14.
Survival of Mycobacterium bovis after ingestion by protozoa would provide an environmental reservoir for infection of cattle. We have shown that M. bovis survived ingestion by Acanthamoeba castellanii. In contrast, two strains of M. bovis BCG did not survive well within Acanthamoeba. 相似文献
15.
Immunogenic protein MPB57 from Mycobacterium bovis BCG: molecular cloning, nucleotide sequence and expression 总被引:15,自引:0,他引:15
Using a single-probe method, we have cloned the gene for an immunogenic MPB57 protein of Mycobacterium bovis BCG. The nucleotide sequence includes an ORF of 300 base pairs encoding a protein of 99 amino acids with an Mr of 10,818. This cloned gene was expressed in an Escherichia coli expression vector to give a mature protein which reacted with a polyclonal antibody raised against MPB57. 相似文献
16.
G.L. Cooper J.M. Grange J.A. McGregor J.J. McFadden † 《Letters in applied microbiology》1989,8(4):127-130
DNA was extracted and purified from 11 strains of Mycobacterium bovis isolated from cattle in Ireland. After digestion with restriction endonuclease PvuII and electrophoresis on an agarose gel, the separated DNA fragments were transferred to a nylon membrane and sequentially hybridized with three DNA probes derived from BCG.
None of the three probes detected restriction fragment length polymorphism (RFLP) within the 11 M. bovis strains, indicating a very close genetic relationship. One probe, pBCG12, detected RFLPs between the M. bovis strains and a reference PvuII digest of DNA from M. tuberculosis R37Rv, confirming that M. bovis and M. tuberculosis are closely related though genetically distinct. 相似文献
None of the three probes detected restriction fragment length polymorphism (RFLP) within the 11 M. bovis strains, indicating a very close genetic relationship. One probe, pBCG12, detected RFLPs between the M. bovis strains and a reference PvuII digest of DNA from M. tuberculosis R37Rv, confirming that M. bovis and M. tuberculosis are closely related though genetically distinct. 相似文献
17.
Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. 总被引:22,自引:0,他引:22
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G G Mahairas P J Sabo M J Hickey D C Singh C K Stover 《Journal of bacteriology》1996,178(5):1274-1282
The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection. 相似文献
18.
Redchuk TA Korotkevich NV Kaberniuk AA Oleĭnik ES Labyntsev AIu Romaniuk SI Kolibo DV Busol VA Komisarenko SV 《T?Sitologii?a i genetika》2010,44(5):23-30
Antibody responses to purified protein derivate PPD of tuberculin and to antigens MPB63 and MPB83 of Mycobacterium bovis were determined in bovine herd (94 adult animals). Statistical approach based on approximation by multiple Gaussians with Levenberg-Marquardt algorithm for analysis of antibody level distribution against antigens examined was provided. Our results confirm that indirect ELISA with recombinant MPB83 and MPB63 as well as conventional PPD could be used for test-systems development for detection of cow tuberculosis infection at the herd level. 相似文献
19.
DNA restriction endonuclease analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG 总被引:10,自引:0,他引:10
DNA preparations from two reference (H37Ra and H37Rv) and two wild strains of Mycobacterium tuberculosis and one re-isolated strain of Mycobacterium bovis BCG were analysed using 17 restriction endonucleases. The enzyme BstEII revealed the greatest differences between strains. Electrophoretic DNA patterns from the wild M. tuberculosis strains differed from each other and from the reference strains at relatively few positions. At the highest resolution attained, patterns from the two reference strains remained indistinguishable from each other. The pattern of the M. bovis BCG strain was substantially different from, but had many bands in common with, the M. tuberculosis patterns. 相似文献
20.
Culture filtrate proteins (CFPs) of M. bovis were produced by culturing the organism for a period of 3 weeks. These CFPs elicited a good delayed hypersensitivity (DTH) reaction in cattle and guinea pig. However, CFPs showed least cross-reactivity when tested in guinea pigs sensitized with M. bovis BCG and M. phlei. CFPs were found to be well recognized by T-cells of single intradermal test (SIDT) positive cattle and were also sero-reactive. Lymphocyte transformation assay revealed the culture filtrate antigens as major T-cell antigens. CFPs induced a good interferon gamma release as compared to conventional antigen, PPD, thereby highlighting their diagnostic capabilities. 相似文献