The End of Antibiotics

A QUANTUM OF SCIENCE

As antibiotic after antibiotic becomes useless, where will we turn to fight the advancing tide of bacterial infection?

Most American adults are aware of the problem of antibiotic resistance: the rapid and aggressive evolution of pathogenic bacteria capable of tolerating even the most potent antibiotics that Western medicine has come up with. Less than a century ago penicillin would knock out any bacterial infection known; today we live in the age of MRSA, a strain of Staphylococcus aureus resistant to not just one but multiple antibiotics. Doctors are running out of ways to prevent even mild infections from becoming life-threatening because bacteria develop resistance can share genetic information with one another, spreading the resistance even further. While there are things we can do to slow the advance of antibiotic resistance, the pace of resistance is accelerating far beyond the ability of pharmaceutical companies to develop new drugs. Is there no hope?

One source of hope may be simpler - and far older - than we think. Mammals have a powerful adaptive immune system to protect them, but what about the lower animals, insects, and plants that lack a sophisticated immune system? How do they avoid succumbing to microbial infections? In the last thirty years scientists have begun to discover that living things have long since developed ways to fight bacteria. One of the most ancient and endemic of these are a class of proteins call antimicrobial peptides (AMP).

First a bit of nomenclature. The majority of your body is made of protein: a polymer of up to 21 different amino acids strung together in long chains. A shorter chain of amino acids (less than a hundred) is called a peptide. Both peptides and proteins have their sequence determined by genes: deoxyribonucleic acid (DNA), which is made up for four basic nucleic acids in repeating sequences. Antimicrobial peptides are encoded by special genes that first evolved, scientists estimate, over a hundred million years ago.

Scientists now know of over nine hundred antimicrobial peptides, and the number is growing at an exponential rate. The diversity of these peptides is astounding: even closely related species have a different set of AMP that little resemble those of even their closest evolutionary siblings. And almost every animal or insect examined has not merely one, but a palette of antimicrobial peptides in their genome, expressed as peptides found in epithelial tissues, blood cells, haemolymph and saliva. Not only the sequence of these peptides is diverse. Their structure also spans an amazing range of shapes, sizes and architectures. Some resemble a helix, others form two-dimensional sheets, and still others form complex rings and branches through multiple internal bonds. Further, many of these AMPs are processed by the cell after being translated from genes into peptides: glycosylation, amidation, halogenation and proteolytic cleavage are common modifications. Given the diversity of genes and structures, scientists have spent a long time trying to understand the unique effectiveness of AMP in combating microbial infections.

The first question that arose after the discovery of antimicrobial peptides was why resistant strains of bacteria had not evolved, especially since AMP genes were so evolutionarily ancient. The first key to this is the very diversity that has confounded scientists. Imagine if each species of plant and animal had evolved the genetic ability to produce between five and twenty broad-spectrum antibiotics, all of which were available for use as a kind of antibiotic "cocktail" whenever infection threatened! This diversity makes it very hard for bacteria to evolve resistance – just when it figures out how to evade one AMP, nineteen more are there to make sure it doesn't pass on that knowledge. Further, the target of antimicrobial peptides is almost impossible for bacteria to avoid: the bacterial membrane itself.

Bacterial membranes are made up of a double layer of molecules called phospholipids. Briefly, the "phospho" part of the membrane molecule is hydrophilic and prefers to be in water, thus facing both the inside and the outside of the bacterium. The "lipid" part of the membrane molecule is hydrophobic, preferring not to be exposed to water, and so gets sandwiched in the middle of the membrane. (Touch your four fingertips together in front of you; the fingers are like the lipid part of the membrane, the palms are like the phospho part. To the left of your hands is inside the cell, to the right is outside.) This makes an effective barrier because most molecules are either overall-hydrophilic or overall-hydrophobic; either way, the phospolipid bilayer will prevent both kinds of molecules from crossing into (or out of) the cell.

The exception, of course, are antimicrobial peptides. Regardless of the diverse sequences they possess, all AMP have the characteristic of being "amphipathic" – possessing both hydrophobic and hydrophilic sequences of amino acids in the same peptide. Carefully spaced, these amino acids first carpet the bacterial membrane, merging with the hydrophilic phospho exterior of the membrane and thinning it. The AMP then swing around so that their hydrophobic amino acids can merge with the lipid layer of the membrane. As this happens, pores begin to form in the membrane and more AMP are able to slip inside the bacterium, repeating the process from the inside. Pores expand and soon the membrane collapses, destroying the bacterium and freeing the AMP to find another target.

Because the target of the AMP is the bacterial membrane, this strategy has been effective for a remarkably long time – more than a hundred million years in some cases (Nicolas, 2003). The phospholipid membrane was one of the earliest innovations of the primordial unicellular world, and unlike genes and protein, it is not easily susceptible to evolutionary advance. While bacteria have learned how to add or subtract elements from their membranes – and do, often, to disguise themselves from the immune system – they cannot alter the basic phospholipid building block of their membranes.

There is even more to the story. In higher animals, their repertoire of AMP work in concert with their immune systems, giving T-cells and antibodies greater access to invade bacterial cells and defeat them – and more importantly, to recognize them and store that memory against future invasions. Additionally, commensal bacteria (the "helpful bacteria" that do things like help humans digest their food) seem to be considerably more tolerant of AMP and even play a role in stimulating the body to produce AMPs and keep the defenses active. This is part of why you are more likely to develop an infection after a course of treatment with broad-spectrum antibiotics that kill these commensal bacteria as well as the pathogenic ones.

So if antibiotics are unable to keep up with the rate of resistance and AMP are so much better, why aren’t we already using them? The answer is that pharmaceutical companies are exploring many potential AMP therapies but their nature makes them difficult to use as drugs. AMPs work in a mass-action fashion, mobbing a single bacterium with hundreds of copies and dozens of different specific AMPs; because of this, effective dosages of AMP are quite high and can result in potentially toxic side-effects. Still, topical uses of AMP are the most accessible application and several are in clinical trials or consideration by the FDA. Oral and systemic AMP drugs are still in development as scientists search for molecules with high activity that could be supplied in lower dosages, but the use of AMP in combination with antibiotics has resulted in some exciting drug cocktails of considerable potency.

AMP also have an array of very exciting applications even outside direct use as drugs, however. AMP molecules have been used as imaging probes, allowing doctors to better visualize the spread of a bacterial infection in the body. Biofilms of AMP have been shown to protect surgical instruments against airborne bacteria that could infect patients on the operating table. AMP genes have been introduced into crops such as tobacco and potatoes to noticeable improvement in their disease resistance. AMP constructs have also been shown to aid considerably in combating STDs, as well as the pulmonary infections associated with cystic fibrosis. A novel class of human therapeutics is even being explored that works by stimulating natural AMP production.

Antibiotics are powerful tools, but more and more doctors and researchers are turning to alternatives as resistance to antibiotics renders them ineffective. In the future, expect to see greater emphasis placed on peptide pharmaceuticals as an expanding answer to the threat of pathogenic bacterial infections – an answer that Nature has already tried and found effective for the last hundred million-odd years.

For more inforation:
Molecular strategies in biological evolution of antimicrobial peptides. P Nicolas, D Vanhoye, M Amiche – Peptides, 2003 [Link]

Antimicrobial Sequences Database (Tossi group, University of Trieste)

Antimicrobial peptides of multicellular organisms. Zasloff, Nature, 415 (24) 2002 [Link]

Antimicrobial Activity and Stability to Proteolysis of Small Linear Cationic Peptides with D-Amino Acid Substitutions. Hamamoto et al. Microbiology and Immunology
Vol. 46 (2002) , No. 11 pp.741-749 [Link]


© AQOS / Peter Smalley, 2009
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