Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system.

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents

Molecular epidemiology of Mycobacterium bovis isolates from South America.

One hundred seventy-eight isolates of Mycobacterium bovis were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the direct repeat element (DR) and the polymorphic GC-rich repeat sequence (PGRS) as probes. By combining the patterns generated by the two repeat DNA elements, 93 different patterns were observed. One hundred-one isolates were grouped in clusters, which include 25 different clusters. One pattern was the most frequently observed, clustering 18.5% of isolates. It was only found in the Center and northeast regions of Argentina and in one isolate from Paraguay. The isolates from Brazil analyzed here presented exclusive patterns (only found in a particular region). The number of exclusive patterns was high in all argentine regions: northeast 78%, center 81%, and Buenos Aires 81%.

An mpb-64 flanking sequence specific for Mycobacterium bovis.

A clone carrying a plasmid with the mpb-64 gene and 3' flanking sequences (plasmid pMBA122) was detected during the screening of a Mycobacterium bovis genomic library with sera from infected cattle. When the pMBA122 insert was used as a probe in Southern blots against PvuII-digested mycobacterial DNA, it distinguished the different M. tuberculosis complex species. This probe hybridized with a 7-kb band in M. tuberculosis, a 5-kb band in M. bovis and a 3-kb band in M. tuberculosis complex strains from wild seals. Smal genomic digestions enabled us to locate this polymorphic region downstream of the mpb-64 gene. In order to clone this particular region, we designed a pair of PCR primers. Unexpectedly, these primers amplified only M. bovis DNA; no amplification was seen in M. tuberculosis DNA. When the annealing temperature was lowered from 70 to 55 degrees C, an amplification product of the same size was obtained with M. tuberculosis. This product was cloned and sequenced, and showed partial homology to the M. bovis amplified fragment. Therefore, this region comprises M. bovis sequences with a lower homology with M. tuberculosis than other compared sequences. This suggests that a more precise differentiation method at the species level for the M. tuberculosis complex could be achieved using PCR directed to this region.

Comparison of different genetic markers for molecular epidemiology of bovine tuberculosis.

In the present study three different genetic markers were used in an RFLP study to differentiate Mycobacterium bovis (M. bovis) isolated in Argentina. The markers were: the insertion sequence IS6110, the direct repeat (DR) sequence flanking IS6110, and a polymorphic GC-rich repetitive sequence (PGRS), called pMBA2. Two restriction enzymes were used, PvuII for IS6110 and DR (DR/PvuII) and AluI for DR (DR/AluI) and pMBA2. DNA from 85 of M. bovis isolates was digested with PvuII and hybridized with IS6110 and Dr. IS6110 was not useful to differentiate M. bovis because most of the isolates contain a single monomorphic copy. The use of DR allowed a limited degree of differentiation. DNA from 44 of these isolates was also digested with AluI and hybridized with DR and pMBA2. In this condition these probes differentiated the isolates in many different RFLP types. By combining the patterns generated with DR/AluI and pMBA2 it was possible to increase the differentiation up to obtain 30 different RFLP types and 54% of the isolates were differentiated because they showed a unique pattern. Six isolates of M. bovis involved in two different outbreaks of bovine tuberculosis were correctly identified. Thus, DR and pMBA2 could be, at the moment, the probes of choice for comparisons of M. bovis isolates in different regions and for epidemiological surveillance of bovine tuberculosis.

Genetic characterization of mycobacteria from South American wild seals.

Tuberculosis has been recently diagnosed in four wild seals found stranded in the Atlantic coast of Argentina. By bacteriological studies and IS6110 hybridization, these isolates were characterized as belonging to the Mycobacterium tuberculosis complex. A genetic characterization using RFLP (Restriction fragment length polymorphism) and a species-specific probe of M. tuberculosis, called mtp40, showed hybridization with this probe on a single band. A similar band was also found in M. tuberculosis H37Rv. This showed a relationship between M. tuberculosis and the wild seal isolates. However these would also seem to belong to a different genetic group in the M. tuberculosis complex, since they do not grow on glycerol-egg containing medium (Lowenstein-Jensen) as typical M. tuberculosis strains usually do. Repeated sequences pMBA2, pTNB12, DR and IS6110 were used as probes to evaluate the epidemiological relationships between the 4 cases of tuberculosis. A low degree of polymorphism was observed, that suggested that these isolates were epidemiologically related.

Characterization of a novel Mycobacterium bovis secreted antigen containing PGLTS repeats.

Serum from naturally infected cattle was used to identify a novel Mycobacterium bovis antigen from an expression library. The first recombinant product identified was a fusion protein with lacZ (55 kDa). A clone containing the whole gene was also obtained. This clone expressed a 38-kDa protein. A rabbit serum against the recombinant antigen reacts in M. bovis supernatants with two proteins of 36 and 34 kDa. The new protein was called P36/P34. The gene cloned has a deduced amino acid sequence with a predicted molecular mass of 28 kDa, showing a characteristic signal sequence for exportation. The protein bears partial homology to a 28-kDa protein from M. leprae. An interesting feature of the P36/P34 sequence is that it contains several PGLTS repeats, which are not present in the M. leprae protein. Antigenic determinants seem also to be conserved between the two proteins because sera from leprosy patients recognized the recombinant M. bovis protein. The discrepancy among the molecular mass deduced from the sequence (28 kDa), that of the recombinant protein in Escherichia coli (38 kDa), and that of the native protein in M. bovis (36 and 34 kDa) could be attributed to posttranslational modifications or to the high proline content that may alter the migration properties of the protein. This antigen seems to be immunodominant during bovine tuberculosis, because 8 of 9 serum specimens from diseased cattle are reactive. The homology among the M. leprae 28-kDa protein, the protein described in this article, and a recently described M. tuberculosis protein suggests the existence of a new protein family in mycobacteria.