To paraphrase Abraham Lincoln, "We are engaged in a greatworldwide war, testing whether the human race can long endure."

No, not the threat of nuclear annihilation, but the great and growingshowdown of total germ warfare. So far, the germs are winning.

World War II launched the age of antibiotics, which promised to ridthe world of infectious diseases. To date, the score is one in favor _the eradication of smallpox _ vs. any number of new or resurgentlethal pathogens _ HIV, malarial parasites, gonorrhea, Ebola virus,legionnaire's disease, Lyme disease, and many more.

The hubris of taming the viral and bacterial killers is now up againstthe counter-insurgent campaign of the germs. The once-secretweapon the microbes are deploying is drug resistance.

Tuberculosis (TB) is the clearest, present microbial danger menacinghumankind today.

Its bacterial perpetrator, Mycobacterium tuberculosis, already hasdeveloped multidrug resistance to most of the antibiotics that oncecurbed this deadly infection.

* TB is today the largest single cause of death in the world from asingle infectious disease.

* It strikes 7.5 million new victims worldwide every year, and kills2.5 million people.

* In the U.S. alone, with 28,000 cases in a recent year, its upsurge islinked to the AIDS epidemic.

So far, the medical response to this menace has been a rearguardaction against M. tuberculosis resistance _ scrambling to developnew drugs, and treating patients with more of them in combinationtherapy. (See BioWorld Today, March 3, 1995, p. 1.)

Against these essentially tactical maneuvers, M. tuberculosis mountsa logistical arsenal of resistance factors. Like the ancient impregnablecity of Troy, the TB microbe is shielded on all sides by layered cellwalls and membranes that ward off invading antibiotics. Like archersbehind the walls of Troy, phalanxes of smart mutant proteins aimtheir weapons of resistance selectively at each and every antibacterialcompound, past, present and future, that could be sent against it.

Resistant pathogens repel boarding drugs by several mechanisms: bystrengthening their barrier walls against them, by pumping them out ifthey come in, or by blasting them with hostile enzymes.

Resistance Proteins Poised To Attack

These drug-defeating actions by once-vulnerable pathogens are nottriggered by the presence of antibiotics sent in against them. Rather,the TB bacterium spontaneously expresses preexisting resistanceproteins, which lie in wait for the drugs, poised to attack.

"Such a preexisting mutation would not rise unless the antibacterialcompound is present, and kills off all the drug-sensitive bacterialcells," said biochemist and cell biologist Eliezer Rapaport. "Thatleaves only the drug-resistant strains free to multiply."

Rapaport, a scientist at the Worcester Foundation for BiomedicalResearch, in Shrewsbury, Mass., is first author of a paper in thecurrent Proceedings of the National Academy of Sciences (PNAS),dated Jan. 23, 1996. It lays out Worcester's antisense-based strategyfor besting the logistics of microbial drug resistance, starting withMycobacteria.

The article, titled "Antimycobacterial activities of antisenseoligodeoxynucleotide phosphorothioates in drug-resistant strains,"has as one co-author molecular biologist Paul Zamecnik, seniorscientist at the Foundation, and a pioneer in antisense technology.(See BioWorld Today, Jan. 12, 1996, p. 3.)

The group's strategy for subverting M. tuberculosis's drug resistanceis akin to that of the Trojan horse.

Worcester synthesized short antisense strings of modified DNA,designed to bind to key sequences in the bacterial genome, andprevent them from expressing those preexisting resistance proteins.Their first problem was how to get those antisense molecules topermeate the impermeable walls, and enter the cell.

"Our general approach," he explained, "involves linking ourantisense oligonucleotides to small molecules that the bacteria takeup actively." Two such interference-running compounds are biotinand dextro-cycloserine, which the bacterium's dextro-alaninereceptor takes up very rapidly. "Dextro-alanine," Rapaport observed,"is actively needed by bacteria for cell-wall synthesis."

A separate ploy employs ethambutol, a second-line oral therapeutic inTB clinics, which punches holes in the bacterial cell wall, allowingentry.

As reported in PNAS, these methods inhibited both resistant anddrug-vulnerable bacteria.

For starters, the Worcester team lined up a pinch-hitting microbe,Mycobacterium smegmatis, to stand in for M. tuberculosis in testingits anti-resistance approach. M. smegmatis is a harmless soilbacterium, also secreted in certain human skin folds.

"The reason people use it for the type of studies we are doing,"Rapaport said, "is that M. smegmatis shares certain genetic sequenceswith M. tuberculosis, but grows much faster, and it's non-pathogenicin man."

Such drug-efficacy tests in M. smegmatis, he added, "become visiblein culture in two to three days, whereas M. tuberculosis takes threeweeks."

Rapaport's antisense nucleotides target highly conserved sequenceson the preexisting mutation genes, which are common toMycobacteria in particular, and to many other microorganisms ingeneral.

Unlike all antibacterials in use today, Worcester's antisense anti-TBmolecules are not themselves susceptible to encountering drugresistance, because, Rapaport pointed out, "they do not respond topreexisting mutations involved in other types of resistance." That'sthe good news.

The even better news: "There is nothing that prevents this approachfrom being utilized in gram-negative bacteria as well as inMycobacteria." He and his colleagues are now "trying to apply it toEscherichia coli as a model gram-negative system."

If this broader vista comes true, Rapaport predicted, "our antisensestrategy will be equally useful in a lot of today's main bacterialproblems, for instance, hemophilus influenza, intestinal streptococci,which cause urinary tract infection, Staphylococcus species that causesurgical wound infections and meningitis. All of these bacteria havedrug resistance due to the rise of preexisting mutations ortransmissible plasmids."

He concluded: "We are trying now to pinpoint efficacy, to get thebest drug candidate, based on structure and what covalent linkagewould produce the best entry of the antisense nucleotide into thebacterial cell." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.