By David N. Leff

If the pathogen that causes TB were Mycobacterium tuberculosis Inc., its stock would have taken a severe tumble Tuesday in the competitive market of microbes vs. antibiotics.

For nearly half a century an anti-bacterial called isoniazid (isonicotinic acid hydrazide) has been the drug of choice for bankrupting M. tuberculosis, and saving people from the TB infection's lingering death. But in defense of its turf against all comers, the bacterium has evolved corporative resistance to this frontline anti-TB drug, and holds its own against every new competitor compound that the pharmaceutical industry throws at it.

If M. tuberculosis shares were quoted on the Big Board, its equity position would have fallen in microbial-investor reaction to a paper in the current Proceedings of the National Academy of Sciences (PNAS), dated Oct. 26, 1999. This high-tech threat to the TB bug bears the title, "Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization." Its senior author is microbiologist Gary Schoolnik at Stanford University.

Schoolnik made the point up front that, "The growing problem of drug resistance - combined with a global incidence of 7 million new cases per year worldwide - underscores the need for new anti-TB therapies." To which he added, "The publication last year of the complete sequence of the M. tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the drug discovery process."

That TB genome comprises 4,411,529 base pairs and contains 3,924 genes, known and unknown, as Nature for June 11, 1998, announced under the crisp title, "Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence." France's Pasteur Institute in collaboration with Britain's Sanger Center accomplished that feat.

To put that genome on a DNA chip, Stanford's Schoolnik picked up the challenge, in collaboration with co-author Patrick Brown of Stanford, a renowned microchip inventor. The microarray he constructed consisted of DNA fragments mounted on polymer-coated glass microscope slides. These provided templates for comparing patterns of gene activation before and after the bacterium was exposed to isoniazid.

TB Pathogen's Achilles' Heel Is A Fatty Wraparound

The name of their game was to fill in the blanks in understanding precisely how the antibiotic works in the bacterium, and how the latter fights back with resistance to the drug. Besides a tough cell wall, the TB microorganism has an up-front barrier, consisting of a waxy outer lipid envelope largely composed of mycolic acid, a fatty acid. Isoniazid breaches this defense by blocking a microbial fatty-acid enzyme that keeps the mycolic acid barrier in good repair.

"Although isoniazid is the drug most often given to TB patients," Schoolnik told BioWorld Today, "it is - paradoxically - also the first and main antibiotic against which the bacterium has mounted effective resistance. To counter this counterattack, isoniazid must be given in combination with two other drugs, which are also threatened with resistance inhibition.

"To identify the genes in the pathogen that act as inhibitors," he continued, "we followed the principle that the expression of each gene in the genome must be interrogated simultaneously in the presence and absence of inhibitor. To do so, we spotted the slides with DNA fragments representing 3,834 (97 percent) of the 3,924 open reading frames of the total genes in the genome.

"Our microarray findings," Schoolnik pointed out, "corroborated already-known biochemical data for isoniazid's antibacterial processes, and highlighted genes directly involved in its inhibition of the mycolic acid enzymes. These genes popped up as early as 20 minutes after treatment, within a fraction of the 24 to 36 hours required for M. tuberculosis cells to divide and multiply."

He underlined the significance of this phenomenon by observing, "Although the organism replicates more slowly than almost any other medically important bacteria, it nonetheless responds vigilantly within minutes to changes in its environment. These can be detected by microarray hybridization. And thus this method is particularly powerful for organisms like M. tuberculosis.

"For those in the mycobacterial field," Schoolnik went on, "who struggle with the slow replication rate of the organism, it certainly opens up a pathway into new kinds of experiments that can be done."

Revealed: New Antibiotic Drug Resistance Genes

The Stanford team's TB chip also turned up several other M. tuberculosis genes encoding enzymes that have not been biochemically identified but are similar in sequence to other proteins of known function. Some of these apparently recycle the fatty acids that pile up as a consequence of isoniazid's attack on the mycolic-acid cell envelope. And EfpA, an efflux protein that pumps the antibiotic out of the drug-resistant microbial cell, the PNAS paper proposes, "may be an appropriate novel drug target, especially because its gene is present only within the pathogenic members of the mycobacterial genus." Most notable of these lately is M. avium-intracellulare complex, a ferociously drug-resistant pathogen that opportunistically infects victims of AIDS.

Schoolnik and his co-authors then went on to test the response of an isoniazid-resistant M. tuberculosis strain. The antibiotic is a prodrug (precursor compound) that, once it's inside the cell, requires activation by a specific bacterial catalase called KatG. Drug resistance involves mutations that defuse KatG.

Therefore, the team turned to a look-alike antibiotic, ethionamide by name. "Unlike isoniazid," Schoolnik explained, "ethionamide does not need KatG in order to inhibit mycolate biosynthesis. Our microarray assay revealed that ethionamide treatment induces the same bacterial genes that isoniazid does."

Schoolnik and his co-authors are now "screening many other inhibitors of vital metabolic and biosynthetic pathways, including other TB drugs, for which the mode of action is not known. And we are using this information to construct high-throughput screens to identify entries in combinatorial libraries that might constitute new leads to antibiotics. It's a broad-based effort" he concluded, "which involves other academic institutions, including the NIH, Colorado State University and Glaxo Wellcome Pharmaceutical Co."