Mycobacterial Genetics: The Sensibility of Antisense

Samuel C. Woolwine and William R. Bishai

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland

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Mycobacterium tuberculosis, the etiologic agent of human tuberculosis (TB), remains at the forefront of global human suffering despite its virtual eradication in the developed nations of the world. The World Health Organization (WHO) Report 2001 estimated 8.4 million new cases of TB in 1999, with the overwhelming majority found among the poorest nations in Africa, Southeast Asia, and South America (1). In addition, haphazard treatment in many of these same areas has led to alarming incidence rates of multidrug-resistant TB. There is a clear impetus today for accelerating TB research on all fronts. Research on the pathogenesis of M. tuberculosis has been difficult due to, among other things, the long generation time of the organism, its propensity for clumping, animal models of infection that do not adequately reflect the human disease, and a lack of genetic tools enjoyed by researchers in other bacterial fields. The latter obstacle is rapidly disappearing. There are now a variety of plasmids, phages, and mutagenesis strategies available for manipulating M. tuberculosis (2). In addition, the sequencing of both pathogenic and nonpathogenic species is paving the way for mycobacterial genomics (3, 4).

Much emphasis in the field of bacterial pathogenesis has been placed on identifying virulence genes. However, standard approaches falter when a virulence gene is also an essential gene. How can one use genetics, based on the isolation and characterization of mutants, to study the functions of essential genes? In the December 15, 2001 issue of AJRCCM, Edwards and colleagues enlisted anti-sense technology to reduce the expression of sodA, encoding an iron-cofactored superoxide dismutase (SodA) in M. tuberculosis H37Rv (5). This methodology represents one way to functionally "knock out" an otherwise essential gene by transcribing an RNA strand complementary to the mRNA of the target gene. The two strands hybridize with one another and are degraded. Although not as complete as a true genetic mutation, this strategy has been employed successfully to attenuate Staphylococcus aureus in a mouse infection model (6), to identify essential genes in S. aureus (7), and utilized in related mycobacteria to manipulate gene expression (8, 9).

The rationale for targeting SodA is based on its enzymology and on the fact that it is produced in abundance by M. tuberculosis. Superoxide dismutase converts superoxide anion into hydrogen peroxide and oxygen. Reactive oxygen intermediates such as superoxide are believed to be an important facet of innate host immunity. As far as levels of production, SodA is one of the 12 major proteins found in M. tuberculosis culture filtrates (10). According to a recent report, although SodA does not appear to be actively exported (11), it nevertheless accumulates in the culture filtrate to an impressive degree by virtue of its high level of expression and extracellular stability. Edwards and colleagues (5) discovered that by introducing plasmids expressing sodA anti-sense transcripts, levels of SodA could be greatly reduced in virulent M. tuberculosis. They also observed slower growth of this strain in liquid culture, which is consistent with the hypothesis that sodA is an essential gene.

When the sodA anti-sense M. tuberculosis strain was used to infect mice, the authors observed a five-log reduction in colony-forming units in the lungs and spleens as compared with mice infected with the same virulent strain containing a vector control at 4 wk of infection. In addition, histopathologic examination of the lungs showed that mice infected with the sodA anti-sense strain exhibited an early mononuclear cell infiltration at 1 wk after infection, which was not seen in control mice. This infiltrate subsided at 2 wk and then returned at 4 wk. There was also a difference in the number of apoptotic cells seen in the two sets of lungs; mice infected with the sodA anti-sense strain displayed significant numbers of apoptotic cells, whereas the control mice had few. Apoptosis is thought to be one way in which a host rids itself of intracellular pathogens. The authors discuss a possible role of superoxide dismutase in inhibiting innate host responses, suggesting that superoxide may be involved not so much in direct killing of M. tuberculosis as in inducing expression of cytokines (via nuclear factor kappa B activation) and apoptosis.

On a final note, Edwards and colleagues (5) introduce their article with the idea of attenuating pathogens in efforts to construct new vaccines and make comparisons of their sodA strain to the vaccine strain BCG. Although anti-sense is an attractive genetic tool, it is doubtful that the technology has a role in producing live-attenuated vaccines. Because you always get what you select for, the slow in vitro growth and the in vivo attenuation of the sodA anti-sense strain indicate strong selective pressure for loss of the plasmid harboring the anti-sense gene construct. Live vaccines attenuated by anti-sense approaches are unlikely to have the genetic stability necessary for use in humans. Nevertheless, as Edwards and colleagues (5) indicate, anti-sense is a sensible strategy for manipulating essential genes, particularly in a genetically difficult microbe such as M. tuberculosis. The technology promises to be a tool of increasing value in dissecting the intricate microbial virulence mechanisms of the tubercle bacillus.

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