Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen.
Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines.
The results are presented of a horizontal epidemiological survey of intestinal infections of children aged between six months and 15 years in three adjacent villages in northern Bangladesh. On the basis of 203 stool sample examinations, the prevalence of Ascaris lumbricoides, Trichuris trichiura, hookworm and amoebic infections was estimated as 68, 56, 53 and 19%, respectively. Age-specific prevalence data indicated that approximately 90% of the children were harbouring patent Ascaris infections by the time they were four years old and there was some evidence to suggest differences in the pattern of age-prevalence between male and female children. The intensity of Ascaris infection was found to rise to its maximum value within the first four years of life. No significant differences were detected in the mean worm burdens of children aged between four and 15 years. Each child in this age-group harboured on average 10 worms. The frequency distribution of numbers of A. lumbricoides per host was found to be overdispersed, with a value of the negative binomial parameter, k, of 0.44. The degree of aggregation was found to be approximately the same for each age-class of the population between one and 15 years (0.26 less than or equal to k less than or equal to 0.82). No evidence was found to suggest a density-dependent reduction in the weight of either male or female Ascaris within the range one to 43 worms per host.(ABSTRACT TRUNCATED AT 250 WORDS)
Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.Tuberculosis is a worldwide health problem that causes approximately 3 million deaths each year (13); however, little is known about the molecular basis of the pathogenesis of tuberculosis. The disease is caused by infection with Mycobacterium tuberculosis; tubercle bacilli are inhaled and then ingested by alveolar macrophages. As is the case with most pathogens, infection with M. tuberculosis does not always result in disease. The infection is often arrested by a developing cell-mediated immunity resulting in the formation of microscopic lesions, or tubercles, in the lung. If cell-mediated immunity does not limit the spread of M. tuberculosis, caseous necrosis, bronchial wall erosion, and pulmonary cavitation may occur (5). The factors that determine whether infection with M. tuberculosis results in disease are incompletely understood.The ability to transfer and express recombinant DNA among the mycobacteria, which has been recently demonstrated (11,20), enables the usage of molecular genetics to elucidate pathogenic mechanisms. However, the present lack of evidence of homologous recombination in the pathogenic mycobacteria prevents the application of allele exchange systems (12). We sought to develop an in vivo genetic complementation system that utilizes integrating shuttle cosmid libraries to identify potential virulence genes of M. tuberculosis. One of the first examples of in vivo selection for virulent bacteria was demonstrated by the classic work of Griffith in 1928 (7). Griffith observed that as a result of genetic exchange between bacteria, virulent, capsulated pneumococci were recovered from mice infected with a mixture of live attenuated, noncap-* Corresponding author. Mailing address: § Present address: PathoGenesis Corporation, Seattle, WA 98119. sulated pneumococci and heat-killed capsulated pneumococci (7).A strategy to identify virulence determinants by genetic complementation requires (i) two strains that are genetically similar, (ii) a phenotype associated with virulence, and (iii) gene transfer systems. An existing pair of M. tuberculosis strains, H37Rv (virulent) and H37Ra (avirulent), distinguishable by their ability to cause disease in animal models (17), was used. H37Ra and H37Rv were derived from the same clinical isolate in 1934 (17,21), and puls...
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