Antibiotics Arms Race Heats Up

© 2002 Wiley Periodicals, Inc.  AT DEATH'S DOOR: Negatively stained Pseu-domonas aeruginosa (A) untreated, (B) treated with amphipathic a helical lytic peptide dia-stereomer (containing both L- and D-amino acids), and (C) treated with with the all L-amino acid peptide. All were treated at 60% of their minimal inhibitory concentration (MIC). At or above the MIC, significant lysis occurs (not shown). (Y. Shai, "Mode of action of membrane active antimicrobial peptides,"

By Jack Lucentini | September 8, 2003

© 2002 Wiley Periodicals, Inc.
 AT DEATH'S DOOR: Negatively stained Pseu-domonas aeruginosa (A) untreated, (B) treated with amphipathic a helical lytic peptide dia-stereomer (containing both L- and D-amino acids), and (C) treated with with the all L-amino acid peptide. All were treated at 60% of their minimal inhibitory concentration (MIC). At or above the MIC, significant lysis occurs (not shown). (Y. Shai, "Mode of action of membrane active antimicrobial peptides," Biopolymers (Petp Sci), 66:236-48, 2002)

The next generation of antibiotics could greatly benefit medicine, many researchers say. It also could, some warn, be quite dangerous. Prompting this debate is a class of drugs based on antimicrobial peptides that all animals and plants produce to fight infections. Studies over the past 15 years have found that these peptides, called AMPs, are powerful germ-killers.

But their most remarkable property, proponents say, is that they rarely spur the evolution of resistant microorganisms.1 If true, this could herald a medical breakthrough. Antibiotic-resistant parasites are rendering existing drugs increasingly useless, toughening the biomedical arms race against pathogens. These peptides might turn the tide.

Research on AMPs is growing yearly.2 Such peptides might boost innate infection protections, lessening the need for traditional antibiotics. Under study for indications from acne to sepsis, medical development of AMPs progresses despite setbacks, including some disappointing clinical trial results. (See RAMPs on Trial) But scientists are raising alarms, too. AMPs aren't necessarily resistance-proof, they warn, and if resistance emerges to particular animal-derived AMPs, called RAMPs, it could be disastrous. Cross-resistant germs might evolve, partially invulnerable to human immunity, compromising defenses permanently.

That effective AMP resistance rarely occurs in nature, write Graham Bell and Pierre-Henri Gouyon, is "the strongest reason for believing that resistance will not evolve after all." They argue that, possibly, because microbes in nature encounter so many different AMPs, they have little chance to adapt to any one. Clinical use of the peptides might change the balance.

A GAUNTLET THROWN Bell, an evolutionary biologist at McGill University, Canada, and Gouyon of Université Paris-Sud, France, raise the concerns in Microbiology.3 AMPs have "great promise," they write, but "both experimental evidence and theoretical arguments" suggest resistance could arise.

AMP proponents are undeterred. Indeed, AMP pioneer Michael Zasloff issued a challenge to Bell: Apply a drug Zasloff developed, called Pexiganan, to any microbes, for any length of time. "I'll bet this peptide will not elicit resistance," Zasloff declares. Bell has accepted, and the two will work and publish their findings together.

Zasloff, dean of research and translational science at Georgetown University, Washington, DC, says there is no documented case of viable, AMP-resistant microbes emerging because of human tinkering. And for good reasons, he adds: First, the peptides have existed for eons, so germs have already mustered the best defenses they can. The human gut churns out grams of AMPs per day, which, biologically speaking, is huge. Saliva, white blood cells, and mucous membranes unload additional killer peptides. Since these molecules are nearly identical in all humans, they must be at least as old as Homo sapiens, argues Zasloff, and microbes have had at least that long to evolve resistance. "Why aren't we seeing people dying?" Zasloff asks. "There's something fundamentally wrong with [Bell's] logic."

Microbes may be perennially impotent against AMPs, Zasloff says, because the peptides strike their "Achilles' heel"--their cell membranes. AMPs appear to recognize these based on key differences between them, and the host organisms' cell membranes. The germs have no good answer, Zasloff says: Membrane structures are too finely tuned to the intricate workings of the little worlds they enclose to permit much change. "The membrane constitutes the environment of the cell," says Zasloff. "Changing the membrane can be a drastic change for a cell to deal with."

These considerations do not reassure everyone. Austin Hughes, of the University of South Carolina, Columbia, who has studied the evolution of mammalian AMPs, concedes that resistance may seldom arise. But the consequences could be serious, he adds, and thorough testing is advisable. "You can't be sure whether resistance will come out. It might not even come from the [pathogen] you're targeting; it could be one that no one has thought of. If it ends up in people having some current infection, that could wreak havoc," he says. "You might have to do in vitro studies with a wide array of bacteria and animal pathogens to test for resistance."

Bell says he doesn't rule out AMP safety; he just wants proof. Biologists once dismissed concerns about resistance to conventional antibiotics, he notes, before armies of drug-resistant microbes burst forth.

© 2002 Wiley Periodicals, Inc.
 ROLL OUT THE RED: In the carpet model of membrane permeation, (A) Antimicrobial peptides bind the cell membrane with their hydrophobic surfaces (blue) facing the membrane and their hydrophilic surfaces (red) facing the solvent. When a threshold concentration is reached, (B) the membrane is permeated and can disintegrate (C). (Y. Shai, "Mode of action of membrane active antimicrobial peptides," Biopolymers (Petp Sci), 66:236-48, 2002)

A COSTLY VICTORY Bell and Gouyon propose a mathematical model showing that situations in which a microbe cannot develop resistance are rare. Such scenarios, they write, require an extraordinarily high "cost of resistance," the level of sacrifice an organism must make to adapt to a given challenge. Often, a mutation that protects it against one threat leaves it more vulnerable to another. That microbes face a prohibitive cost of resistance is precisely Zasloff's claim. While resilient microbes exist, he argues, they do not proliferate very effectively.

Experiments support this view, Zasloff says. Thomas Montville, a professor at Rutgers University, has studied resistance to nisin, an AMP made by bacteria. Nisin, already used as a preservative, functions much the way animal AMPs do, he says. Montville found that the cost of nisin is so high among Listeria monocytogenes, that resistant strains have no overall advantage over others.4 "Three to five years ago, I thought this was a big enough threat to do the research," Montville says. "Paradoxically, the nisin-resistant mutants we have are more sensitive to everything else. It cripples them to other preservatives."

Hints of possible bacterial resistance have appeared in some other AMP studies, says David Friedland, vice president of clinical and medical affairs at Micrologix Biotech, a Vancouver company developing two AMP drugs. But the effect was always too small to be conclusive, he says.

Better verified examples of AMP resistance occur in natural settings. A part of the innate immune system, AMPs are diverse; more than 800 have been described so far. When microbes invade, AMPs attack their membranes, possibly by recognizing distinctive characteristics of charge, hydrophobicity, and composition. Bacterial membrane surfaces are normally negatively charged, whereas those of most plant and animal cells are neutral. The peptides usually puncture or break up the membrane, causing leakage. They also may enter the cytoplasm and cause further damage.5

Not all AMPs are the subject of major resistance concerns, Bell and Gouyon say. The problematic ones are a subcategory called RAMPs, for ribosomally synthesized AMPs. Because multicellular organisms, including humans, make RAMPs, microbes that resist them could threaten humans. Less worrisome are non-RAMPs, the more distantly related peptides produced by prokaryotes. Some are already used as antibiotics.

Furthermore, if an AMP from a nonhuman elicits microbial resistance, the extent of danger to humans depends largely on the extent to which the germ is cross-resistant to other AMPs--human ones. That, in turn, depends on how closely the peptides in question are related. "The crucial question is cross-resistance," Bell says, because without it frog peptides, for example, would pose little danger to human health.

Microbes naturally deploy various mechanisms to resist AMPs. Some have changed their membranes to evade detection. Others expel invading peptides, or unleash proteases that destroy them.6

Just as resistance mechanisms abound, so do different AMPs. The sheer variety of both (nearly every species has its own special peptides) may attest, some researchers say, to a long, rapid evolutionary arms race. This alone might justify concerns that microbes can evolve resistance, Hughes says. But it's unknown whether rapid, in evolutionary terms, is rapid enough to cause problems for humans.

For now, AMP developers face decidedly shorter-term headaches: getting their drugs to market. Though several non-RAMPs are in commercial use, no RAMPs are. A combination of clinical trial disappointments and regulatory hitches has held up at least three drug candidates. In two of these cases, however, trial investigators judged the peptides' performance either as good as leading antibiotics, or, in the case of Zasloff's Pexiganan, almost as good.

Costs also slow progress. "These peptides are fairly expensive to make," Friedland says. Moreover, because systemic use of the drugs raises toxicity concerns, many studies are limited to topical applications. But the promise that AMPs are nearly resistance-proof remains "under-argued," contends Michael Yeaman, at Harbor-University of California Los Angeles Medical Center. Also promising, says Yeaman, is evidence that the peptides not only kill germs on their own, but enhance the potency of traditional antibiotics. The AMPs may accomplish this by facilitating entry of antibiotics into the cell.

To Bell and Gouyon, the peptides' very promise adds urgency to the resistance question. "It is very likely that RAMPs, including human RAMPs, can satisfy regulatory criteria and will be introduced into clinical practice in the near future," they write. "We should be prepared, therefore, for the less desirable side effects that will follow from the evolution of resistance."3

Jack Lucentini ( is a freelance writer in New York City.

1. R.E.W. Hancock et al., "The role of antimicrobial peptides in animal defenses," Proc Natl Acad Sci, 97:8856-61, 2000.

2. A.R. Koczulla et al., "Antimicrobial peptides: current status and therapeutic potential," Drugs, 63:389-406, 2003.

3. G. Bell et al., "Arming the enemy: the evolution of resistance to self-proteins," Microbiol-SGM, 149 (Pt 6):1367-75, June 2003.

4. E. De Martinis et al., "Influence of pH, salt, and temperature on nisin resistance in Listeria monocytogenes," J Food Protect, 60:420-3, 1997.

5. Y. Shai, "Mode of action of membrane active antimicrobial peptides," Biopolymers, 66:236-48, 2002.

6. M.R. Yeaman et al., "Mechanisms of antimicrobial peptide action and resistance," Pharmacol Rev, 55:27-55, 2003.

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