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Chapter 18 : Molecular Genetic Strategies for Identifying Virulence Determinants of

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Abstract:

This chapter is concerned with how knowledge of other bacterial pathogens may provide insight into the pathogenesis of tuberculosis. In 1882, Robert Koch’s landmark paper demonstrated that tuberculosis was caused by . The necessity for defining virulence determinants stringently can readily be illustrated from the authors' studies of . They have analyzed the H37Rv inserts from clones isolated from different experiments that had the ability to preferentially localize in spleen and found several clones with overlapping DNA fragments. The introduction of linear DNA fragments into both and BCG results in their incorporation primarily in nonhomologous sites around the chromosome. It has long been appreciated that there are significant homologs of many virulence genes of gram-negative pathogens, even in different genera. Clearly, the demonstration that such homologs are involved in mycobacterial virulence will depend on fulfillment of Koch's molecular postulates, namely, mutation and transfer to avirulent strains.

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18
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Figures

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Figure 1

Macrorestriction analyses of genomes. The chromosomes of H37Ra and H37Rv and and BCG strains were isolated and digested with Following digestions, the DNA fragments were separated by pulsed-field gel electrophoresis. Note the 475-kb band present in the virulent H37Rv strain but absent in the avirulent H37Ra strain. Molecular weight markers are multimers of the bacteriophage λ genome.

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18
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Image of Figure 2.
Figure 2.

Virulence complementation assay. Cosmid genomic libraries of the virulent strain are constructed in an integrating cosmid vector and introduced into an avirulent mutant such as H37Ra or BCG. The resulting library of recombinant clones is injected into mice. Theoretically, clones that restore virulence will have a selective advantage and be enriched for in the mouse.

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18
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Image of Figure 3.
Figure 3.

Characterization oH37Rv DNA fragments that confer an in vivo growth advantage in spleen on H37Ra. Several overlapping clones that confer a selective growth advantage upon transformation into H37Ra have been identified.

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18
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Image of Figure 4.
Figure 4.

Strategy for constructing transposon libraries. A mycobacterial transposon is introduced on a suicide delivery vector, and clones that have obtained the transposon containing a selectable marker gene randomly inserted in the genome are selected for.

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18
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Image of Figure 5.
Figure 5.

In vivo expression technology for mycobacteria. The basic strategy has been described by Mekalanos and colleagues (Mahan et al., 1993) for spp. An appropriate auxotroph is identified and found not to grow in an animal host. A genomic library from the virulent organism is constructed in a vector in a unique restriction site present immediately upstream of the complementing gene. The reporter operon is used to identify genes that are turned on constitutively that are then removed from the library. The remaining library is passed through animals, and only those recombinant clones that are expressed in vivo will grow in the selective environment of the mouse.

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18
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References

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43. Snapper, S.,, R. E. Melton,, S. Mustafa,, T. Kieser,, and W. R. Jacobs Jr.. 1990. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4: 1911 1919.
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Tables

Generic image for table
Table 1.

Comparison of Koch's postulates and Koch's molecular postulates

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18
Generic image for table
Table 2

Properties of a virulent organism

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18
Generic image for table
Table 3.

Strategies successfully used to identify virulence genes

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18
Generic image for table
Table 4.

Why insertional mutagenesis?

Citation: Jacobs, Jr. W, Bloom B. 1994. Molecular Genetic Strategies for Identifying Virulence Determinants of , p 253-268. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch18

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