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One limiting factor in studies of tuberculosis and leprosy is the difficulty of genetic analysis and manipulation of mycobacteria. Two approaches were adopted for the construction of vectors, based on different Escherichia coli plasmids and using Mycobacterium smegmatis as the host. In both cases we found that the original E. coli plasmid is capable of being replicated in M. smegmatis, yielding chloramphenicol-resistant colonies. One such plasmid has been recovered from a M. smegmatis transformant and used to re-transform both M. smegmatis and E. coli to chloramphenicol resistance. This plasmid is indistinguishable from the original plasmid by restriction analysis, and can be used as a shuttle vector for the genetic manipulation of mycobacterial species.
Mol Microbiol 1989 Jan
PMID:Transformation of Mycobacterium smegmatis with Escherichia coli plasmids carrying a selectable resistance marker. 265 39

In addition to providing a powerful approach for identifying bacterial factors required for full infectivity and disease production, genetic analysis of Legionella pathogenesis should also lend critical insight into the biology of the macrophage and into the pathogenesis of other intracellular parasites. The interaction between L. pneumophila and the macrophage exhibits many features found in a wide variety of prokaryotic and eukaryotic intracellular human pathogens. For example, binding to complement receptors has been shown to occur for Mycobacterium tuberculosis, M. leprae, Leishmania donovani, Leishmania major and Histoplasma capsulatum. Coiling phagocytosis has been observed during entry of L. donovani. Phagosomes that contain Toxoplasma gondii or M. tuberculosis fail to fuse with lysosomes and, in the case of T. gondii, have been shown to remain close to neutral pH. Although the molecular bases for these phenomena are unknown, their functional similarities to the L. pneumophila-macrophage interaction provide optimism that generally applicable principles are involved. The genetic techniques reviewed here will provide the molecular tools with which such questions of a general biologic nature can be framed and eventually answered. Together with more traditional methods in biochemistry, microbiology and cell biology, molecular genetics offers a robust means toward identifying and understanding the bacterial factors involved in the pathogenesis of Legionnaires' disease. Molecular studies of L. pneumophila can also help address questions concerning the epidemiology, diagnosis and prevention of disease. For example, the distribution of virulence factors might help explain and predict the attack rates of different L. pneumophila strains or Legionella species. Moreover, bacterial genes/factors that are shown to be conserved in Legionella strains could be used to develop such diagnostic tools as DNA probes. Novel types of vaccines consisting of genetically constructed, avirulent L. pneumophila strains or subunit vaccines based on the molecular characterization of virulence factors might be developed and tested as protective immunogens. In this way, the capacity to analyze and to manipulate L. pneumophila genetically may facilitate the use of Legionnaires' disease as a model infection for studying protective cell-mediated immunity. Apart from its clinical significance as the etiologic agent of Legionnaires' disease, L. pneumophila may be a key to broader understandings in microbial pathogenesis and human cell biology and immunology. Although the extremely complex processes of bacterial infection and virulence are best understood when a variety of experimental approaches are employed, we believe that the evolving molecular genetic techniques reviewed here will be critical elements in many important breakthroughs in the future.
Mol Biol Med 1989 Oct
PMID:Genetics and molecular pathogenesis of Legionella pneumophila, an intracellular parasite of macrophages. 269 60

DNA probes were used to identify restriction-fragment-length polymorphisms (RFLPs) in DNA samples, demonstrating that the Mycobacterium avium complex could be clearly divided into M. avium and Mycobacterium intracellulare strains. Less than 2% DNA base substitution was found between M. avium strains, whereas the M. intracellulare strains had greater than 15% base substitution. The Johne's disease bacillus, Mycobacterium paratuberculosis (American type strain), was found to be distinguishable from the M. avium complex serotypes examined. Strain 18 was found to be identical to M. avium. The rat leprosy bacillus, Mycobacterium lepraemurium, was found to be very closely related, but not identical, to M. avium.
Mol Microbiol 1987 Nov
PMID:The use of DNA probes identifying restriction-fragment-length polymorphisms to examine the Mycobacterium avium complex. 289 88

A DNA probe which hybridizes to all pathogenic species of slow-growing mycobacteria has been used to identify restriction-fragment-length-polymorphisms (RFLPs) in Bam Hl digests of chromosomal DNA from members of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex. The RFLP patterns so produced were found to fall into distinct categories which were representative of each of the three species. Except for two doubtful isolates, strains of M. avium were found to fall into two related RFLP-types, one of which contained the vast majority of the strains tested. In contrast, M. intracellulare strains were found to be more heterogeneous. For these strains, we found one major RFLP-type and one subsidiary type which appears to be a sub-set of the first. We also found two further RFLP-types which contained serovars 7 and 18 respectively. We conclude from this that M. avium, M. intracellulare and M. scrofulaceum are three distinct species and that serovars belonging to the 'intermediate group' of Meissner and Anz belong to the species M. avium. Utilizing these criteria, we examined a number of isolates from the 'ambiguous' serovar 9 and found that of eight strains tested, six typed as M. avium and two typed as M. intracellulare.
Mol Cell Probes 1988 Dec
PMID:Speciation of organisms within the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex based on restriction fragment length polymorphisms. 290 75

The primary structure of 5S ribosomal RNA has been determined in five species belonging to the genus Mycobacterium and in Micrococcus luteus. The sequences of 5S RNAs from Actinomycetes and relatives point to the existence in this taxon of a bulge on the helix that joins the termini of the molecule. An attempt was made to reconstruct bacterial evolution from a sequence dissimilarity matrix based on 142 eubacterial 5S RNA sequences and corrected for multiple mutation. The algorithm is based on weighted pairwise clustering, and incorporates a correction for divergent mutation rates, as derived by comparison of sequence dissimilarities with an external reference group of eukaryotic 5S RNAs. The resulting tree is compared with the eubacterial phylogeny built on 16S rRNA catalog comparison. The bacteria for which the 5S RNA sequence is known form a number of clusters also discernible in the 16S rRNA phylogeny. However, the branching pattern leading to these clusters shows some notable discrepancies with the aforementioned phylogeny.
J Mol Evol 1987
PMID:Structure of 5S rRNA in actinomycetes and relatives and evolution of eubacteria. 311 42

Traditional methods used in identifying mycobacteria such as acid-fast bacillus stains and culture are often time-consuming, insensitive and non-specific. The isolation of DNA probes, coupled to a non-radioactive, e.g. biotin-based detection system, have the potential to foster the development of clinical assays for Mycobacterium tuberculosis and mycobacteria other than tuberculosis (MOTT) that are rapid, sensitive and specific. To this end, we have isolated two different probes: one which is specific for the Mtb complex and one which recognizes all other potentially pathogenic mycobacteria. The use of these probes in combination should allow the detection and differentiation of M. tuberculosis from MOTT. To isolate the first probe, we prepared a library of M. tuberculosis DNA fragments in a lambda EMBL phage vector. Recombinant phage were screened by plaque-lift hybridization procedures using nick-translated mycobacterial genomic DNA to identify sequences specific to the Mtb complex. Inserts from candidate recombinant phage were purified, nick-translated and hybridized against a wide variety of filter-bound mycobacterial and non-mycobacterial DNAs. Two clones were identified which hybridized to the closely related M. tuberculosis, M. bovis and M. microti but not to other species of mycobacteria. The second probe was isolated by preparing a library of M. malmoense DNA fragments in lambda EMBL and screening by plaque-lift hybridization. One clone was identified which, in addition to recognizing members of the Mtb complex, also hybridized to M. intracellulare, M. malmoense, M. scrofulaceum, M. simiae, M. xenopi, M. avium, M. szulgai, M. kansasii and M. haemophilum. None of the three clones hybridized to DNA from non-mycobacterial species.
Mol Cell Probes 1988 Jun
PMID:DNA probes for mycobacteria. I. Isolation of DNA probes for the identification of Mycobacterium tuberculosis complex and for mycobacteria other than tuberculosis (MOTT). 313 97

DNA-, RNA- and protein synthesis have been studied in Mycobacterium smegmatis cells infected with phage I3. The macromolecular synthesis continued until the end of latent period. Early RNA and protein synthesis were necessary prior to the commencement of DNA replication. The infecting phage DNA sedimented as larger than unit length of genome, after initiation of DNA synthesis. Although the host DNA was not degraded, 90 percent of the RNA synthesized after phage infection hybridized to phage DNA.
Mol Biol Rep 1981 Nov 30
PMID:Macromolecular synthesis in mycobacteriophage I3 infected cells. 617 40

This study was undertaken to identify biochemical alterations in serum, lymphoid organs, and peritoneal macrophages (PM) which reflect the histopathology of experimental Mycobacterium lepraemurium (MLM) infection in mice. A significant increase of acid phosphatase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and lysozyme was found in serum, spleen, and liver homogenates of mice infected intraperitoneally (ip) with MLM. PM from infected mice showed a substantially greater rate of secretion of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and acid phosphatase than PM from normal mice. There was, however, no significant difference in the ability of PM from BALB/c and C57BL/6 mice to secrete such enzymes in vitro. There was also a significant increase in all these enzymes in PM in the early stage of infection but they dropped to values lower than normal in the advanced stage of infection despite the fact that such cells increased in size and protein content as the infection progressed. Infected mice were also found to have progressively elevated levels of serum lactic dehydrogenase, glutamic oxaloacetic, and glutamic pyruvic transaminases which indicated damages of hepatocytes and other tissues. Values of other blood components were also reported. Both BALB/c and C57BL/6 strain of mice, which are susceptible to the ip route of MLM infection, showed an indistinguishable pattern of biochemical alterations as reflected by their similar histopathological changes in various organs. BALB/c mice, which are still susceptible to subcutaneous (sc) route of infection showed similar characteristic changes in various serum components as before. In contrast, C57BL/6 mice, which are resistant to MLM infection sc, showed insignificant alterations in most of these biochemical parameters.
Exp Mol Pathol 1984 Apr
PMID:Experimental murine leprosy: a biochemical study emphasizing lysosomal enzyme changes in vivo and enzyme secretion by macrophages in vitro. 636 59

Gene replacement by homologous recombination is a powerful tool for fundamental studies of gene function, as well as allowing specific attenuation of pathogens, but has proved difficult to achieve for Mycobacterium tuberculosis. We have used a plasmid-based test system to demonstrate the occurrence of homologous recombination in the tuberculosis vaccine strain Mycobacterium bovis BCG, and we have successfully replaced a target gene in BCG by homologous recombination, using a shuttle plasmid. Specific inactivation of selected genes will facilitate study of virulence factors and drug resistance as well as allowing rational attenuation of M. tuberculosis for the production of new vaccines.
Mol Microbiol 1995 May
PMID:Gene replacement by homologous recombination in Mycobacterium bovis BCG. 747 69

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an 'up-regulated' promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The availability of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.
Mol Microbiol 1995 Jun
PMID:Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae. 747 85


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