1 Biology Department, McGill University, 1205 Ave Dr Penfield, Montreal, Québec, Canada H3A 1B1
2 Laboratoire Ecologie, Systématique et Evolution, bât. 362 Université Paris-Sud, 91405 Orsay cédex, France
Correspondence
Graham Bell
graham.bell{at}mcgill.ca
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ABSTRACT |
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Background |
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Diversity of AMPs |
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The deadliness of RAMPs, and of non-RAMPs such as the polymyxins, is attributable to their interaction with very general biochemical characteristics of bacterial cell membranes. In particular, the negatively charged phospholipid head groups on the outer surface of bacterial membranes render them highly vulnerable to electrostatic and hydrophobic interactions with RAMPs, whereas eukaryotic cell membranes, with little or no net charge, are almost immune. Consequently it is argued it will be difficult for bacteria to evolve resistance to RAMPs. Zasloff (2002) points out that RAMPs have remained effective against bacterial infection for millions of years, confounding the general belief that bacteria, fungi and viruses can and will develop resistance to any conceivable substance. Thus Schroder (1999)
writes that .. it seems to be difficult for micro-organisms to acquire resistance, making these peptides very attractive for therapeutic use as antibiotics, while Hancock & Chapple (1999)
claim that It is also very difficult to raise mutants resistant to these cationic peptides, and there are very few naturally resistant bacteria.
It is very important that these claims turn out to be true. The evolution of resistance to any antibiotic makes it less useful in treating disease, of course. Quite incidentally, it also deprives any organism that produces it of part of its antibacterial armoury. This would not normally be a matter for concern; but in the case of antimicrobial peptides, we ourselves are the producers. The evolution of resistance to human antimicrobial peptides, therefore, may have much more serious consequences than the evolution of resistance to conventional antibiotics, because our ability to resist infection might be permanently compromised. Before these substances are released for general use, it is, in our view, important to be quite sure that we are justified in dismissing the possibility that bacteria will evolve resistance to them.
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Resistance to RAMPs |
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Resistance to RAMPs seems to vary greatly in specificity: in some cases it is highly specific and protects bacteria against only a narrow range of host peptides, whereas other cases involve mechanisms that confer broad resistance to many types of RAMP. The yeast mutants studied by Thevissen et al. (2000), for example, were cross-resistant to other defensins which are structurally similar to insect RAMPs but not to chemically unrelated antifungal agents. In a similar fashion, plasmid pSK1 of Staphylococcus aureus confers resistance to human platelet microbicidal protein 1 via the efflux protein encoded by qacA, but not to nisin or neutrophil
-defensin (Kupferwasser et al., 1999
). Genes that mediate resistance to epidermin in Staphylococcus epidermidis also mediate resistance to the similar peptide gallidermin from Staphylococcus gallinarum, but not to the less similar lantibiotic nisin or the insect RAMP melittin (Otto et al., 1998
). Nisin-resistant Listeria and Clostridium, on the other hand, are cross-resistant to chemically unrelated bacteriocins (Crandall & Montville, 1998
).
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Mechanisms of resistance |
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Modification of outer cell layers
The incorporation of components with reduced anionic charge obstructs the original aggregation of RAMPs on the cell membrane. In Gram-positive bacteria such as staphylococci, substitution of positively charged alanine residues into cell wall teichoic acids reduces deposition of RAMPs onto the cell surface. Transposon inactivation of the dlt operon creates strains with reduced alanine content that are highly susceptible to human defensin, several animal RAMPs, and even to bacterial RAMPs such as nisin (Peschel et al., 1999).
Failure to penetrate the outer membrane
The cell membrane can readily be modified so as to diminish the effectiveness of RAMPs. The most obvious route is that lower concentrations of anionic phospholipids will enhance resistance. Substitution of lysine into the membrane phospholipids of Staph. aureus reduces the net negative charge and causes reduced loading of RAMPs; inactivating the locus responsible, mprF, greatly increases susceptibility to killing by neutrophils (Peschel et al., 2001). Similar genes are found in many other bacteria. An increased content of aminoarabinose also decreases the charge on the membrane and confers a certain level of resistance to RAMPs (Shafer et al., 1984
). Resistance of Staph. aureus to human platelet microbicidal protein is caused by elevated levels of long-chain unsaturated lipids that cause greater membrane fluidity (Bayer et al., 2000
).
The secretion of RAMPs is often induced by host recognition of Gram-negative bacteria mediated by the binding of receptors such as CD14 to lipid A of the bacterial outer cell membrane; in turn, bacteria have systems for detecting the presence of host tissues and modifying their cell membrane so as to evade recognition. In Salmonella, the signal transduction pathway PhoP/PhoQ mediates resistance to RAMPs and survival within host tissues. It controls the expression of a large group of genes, including about 15 that encode outer-membrane proteins. PhoP-activated genes are responsible for the survival of the cell in environments such as host macrophages, principally through modifying the composition of lipid A and thus changing the conformation of the membrane surface, making it less vulnerable to detection and destruction by the host. There is also a group of PhoP-repressed loci, which encode proteins involved in host tissue invasion. PhoP/PhoQ is essential for pathogenesis, and strains in which the system is inactivated are avirulent (Miller et al., 1990). The variety of loci controlled by the system and the complexity of their interactions provides a rich field for the evolution of different kinds of resistance to RAMPs. Many and perhaps all other Gram-negative bacteria have PhoP/PhoQ homologues and are virulent because of their ability to modify their cell membranes (Oyston et al., 2000
).
Export of peptides from the cell
Efflux systems may remove peptides from the cytoplasm. Those described so far fall into four main categories: ATP-binding proteins, major facilitator proteins, resistancenodulationdivision proteins and small multidrug resistance proteins. Many organisms can express more than one efflux system. All of these efflux systems can provide the basis of resistance to RAMPs. In staphylococci, the plasmid pSK1 confers resistance to several classes of antibiotics. It includes the qacA locus, which encodes a member of the major facilitator group of proteins that is capable of exporting a broad range of structurally dissimilar organic cations from the cell. The plasmid confers resistance to platelet microbicidal protein 1, a RAMP expressed in human neutrophils (Kupferwasser et al., 1999). Moreover, strains isolated from endovascular infections are more resistant to tPMP-1 than strains isolated from soft-tissue abscesses (Bayer et al., 1998
), suggesting that resistance can evolve in bacterial populations within the body. Lantibiotics such as epidermin are expelled from the cytoplasmic membrane of Staph. epidermidis by ATP-dependent translocases encoded by the three genes epiE, epiF and epiG (Otto et al., 1998
). These are similar to the ABC transporters with conserved ATP-binding cassettes responsible for the uptake or excretion of a broad variety of substrates in a wide range of organisms. Specific resistance to lantibiotics in staphylococci is also conferred by small membrane-associated proteins such as PepI, although their mechanism of action remains obscure (Pag et al., 1999
). Efflux pumps that confer resistance to RAMPs have been reported from a range of other organisms. Resistance to protegrin in Neisseria gonorrhoeae, for example, is associated with a plasmid-encoded efflux pump similar to those involved in resistance to other antibiotics (Shafer et al., 1998
).
Proteolysis
Once inside the cell, RAMPs are difficult to target specifically for destruction. Nevertheless, a few instances have been reported. The pgtE locus of Salmonella typhimurium, for example, is activated in host tissues by PhoP and encodes an outer-membrane protease that cleaves -helical peptides and thus protects the cell against RAMPs such as defensins (Guina et al., 2000
).
It seems possible that resistance to RAMPs, far from being unlikely and uncommon, is widespread in nature and readily induced in the laboratory. Indeed, it has become clear in the last few years that resistance to RAMPs is a normal and necessary component of pathogenesis in Salmonella (Groisman et al., 1992) and Staphylococcus (Peschel & Collins, 2001
). Resistance does not invariably evolve. The widespread use of nisin as a food preservative or of polymyxin B as a topical antibiotic has not led to any dramatic increase in levels of resistance. Rather, the evidence suggests caution in accepting claims that resistance will not evolve.
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The evolution of antibiotic resistance |
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The simplest case would be a single chromosomal allele encoding resistance to a single substance. The population comprises resistant cells (symbolized R) that bear the allele, and susceptible (S) cells that do not. The environment, in an equally simple manner, consists of toxic (T) patches, where the substance is present, and permissive (P) patches, where it is not. At regular intervals cells are redistributed randomly among patches, with cells from the toxic patches contributing some fixed fraction K of the total population. The frequency of the resistant allele at equilibrium is then
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Evolution of resistance to RAMPs |
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Given that the RAMP treatment is effective, so that susceptible bacteria are inhibited in treated hosts, the evolution of resistance depends in the first place on the balance of two kinds of cost. The first is the cost of resistance in pristine habitats or on normal (non-treated) hosts, as before, which can be defined as CRP=(GSP-GRP)/GSP, where G is a growth rate. [For explicit definitions of parameters, see legend to Fig. 1.] The second is the cost of susceptibility in infected hosts, CSI=(GRI-GSI)/GRI. At one extreme, suppose that GSI=0, so that the native RAMP defences are fully effective in killing susceptible bacteria. This may provoke the evolution of resistance, but the situation is not worsened, or changed much, by the introduction of RAMP chemotherapy. At the other extreme, suppose that GSI=GRI, so that native RAMPs are completely ineffective. In this case, RAMP chemotherapy will cause the evolution of resistance in much the same way as conventional antibiotics. It is in the region between these two extremes that administering RAMPs to infected patients may provoke the evolution of resistance that would not otherwise occur.
For example, suppose that we set CSI=0·25, so that the growth rate of susceptible types is 25 % less than that of resistant types in infected hosts. There are roughly equal numbers of bacteria living on hosts and in the general environment, with moderate rates of movement between the two. Other parameters are chosen so as to be biologically reasonable (see legend to Fig. 1); for example, susceptible bacteria are barely able to grow in infected hosts (GSI=1·125), and the growth of resistant bacteria is less in treated hosts (GRT=1·2) than in untreated hosts (GRI=1·5), although it exceeds that of susceptible bacteria in both (GST=0, GSI=1·2). With no RAMP treatment, resistance is maintained at very low frequency by recurrent mutation if CRP>0·33 about, and spreads to high frequency if CRP<0·38 about. Between the two is a region where the outcome is heavily influenced by the stochastic nature of infection, and where the evolution of resistance may be long-delayed, if it occurs at all. The threshold value of CRP that permits the evolution of resistance is thus about 0·35. We can then investigate the effect of allocating each infected individual, immediately after the onset of infection, to a course of RAMP therapy with a probability of 0·5. In this case, the critical value of CRP is about 0·65. Thus the effect of the therapy is to enlarge the class of resistant mutants able to invade the population and be maintained at high frequency. This effect is quite a large one. Without therapy, only mutants able to grow at about two-thirds the rate of the susceptible types can invade. When half of infected hosts receive AMP therapy, on the other hand, resistant types that grow at about a third of the rate of the susceptible types can invade. Thus administering RAMPs to infected hosts can greatly relax the conditions for the evolution of resistance.
Whether RAMP therapy causes the spread of resistance in circumstances where it would otherwise not evolve depends on the combination of costs (Fig. 2). Without RAMP therapy, resistance spreads readily when CRP is low and CSI is high. Beyond a certain limit, it fails to spread at all. When RAMP therapy is applied, the evolution of resistance becomes almost insensitive to CSI and instead depends only on CRP; resistance will spread provided that CRP is sufficiently small, for almost any value of CSI. This is because a sufficiently small value of CRP causes the frequency of resistance at mutation-selection equilibrium to be sufficiently high to create a few infected individuals; when these are treated they will incubate populations of resistant bacteria that are then transmitted to uninfected hosts. The effect is to create a region in which CRP and CSI are both relatively low, in which resistance spreads only when RAMP therapy is administered. The size and shape of this region depends in detail on the parameter values chosen for the model, but its existence under reasonable combinations of values argues that the possibility that resistance may evolve should be taken seriously. The main ways in which the conclusion will be modified by changing parameter values are as follows.
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(b) The environmental model
If the number of bacteria that inhabit hosts is small relative to the number growing in the general environment, then the advantage of resistance is reduced because resistant bacteria are likely to be dispersed away from the hosts where they evolved. This does not affect the range of genotypes that spread, or the rapidity with which resistance evolves, but it reduces the final frequency of resistance. For example, if ten times as many bacteria grow in the general environment as on the host population, the final frequency of resistance falls from about 0·5 to about 0·1. The general environment might become polluted by the habitual use of RAMPs, however, creating patches in which resistant types have an advantage. This might occur, for example, through the use of RAMPs in agriculture or food processing. High levels of resistance to the non-RAMP bacitracin, for example, have been found in isolates from poultry, pigs and other domestic stock (Aarestrup et al., 1998) and from farm soil (Jensen et al., 2001
), from where it has spread to less likely environments such as bottled mineral water (Massa et al., 1995
). The mechanism of resistance involves an ABC transporter system that expels the peptide from the membrane (Podlesek et al., 2000
; Neumuller et al., 2001
). Suppose that polluted sites are only one-tenth as frequent as pristine sites, and that susceptible bacteria are 25 % less fit in these sites. With the same combination of parameters as before, the threshold cost of resistance in pristine sites rises from 0·65 to 0·75. Thus environmental pollution by particular RAMPs may extend the range of resistant genotypes that are able to spread.
(c) The transmission model
In many circumstances, it is likely that the bacteria dispersing from a host individual will encounter another host, rather than passing to the general environment, regardless of the relative numbers of bacteria in each. This promotes the evolution of resistance in the same way that habitat choice promotes local adaptation in simple models of heterogeneous environments. RAMP therapy magnifies this effect because of the proliferation of resistant bacteria in treated individuals. Moreover, because sick people (bearing different kinds of bacteria able to exchange genes through plasmids) tend to be aggregated in hospitals, the degree of transmission in such places is far higher than is assumed by supposing a random distribution.
(d) The infection model
The model describes a stochastic model of infection governed by a rate parameter. This parameter translates the number of resistant bacteria colonizing a host into a probability of infection, and consequently its effect on the evolution of resistance is essentially the same as that of the mutation rate.
(e) The treatment model
When infected individuals are more likely to be treated, the overall relative fitness and thus the final frequency of resistance increases.
Although the detailed behaviour of the model depends on the parameter set used, the evolution of resistance to RAMPs as a consequence of their use in therapy occurs over a broad range of parameter values. Moreover, natural populations seem likely to lie within this range. It is clear that resistance is not currently segregating at high frequency in most populations of pathogenic bacteria. Thus CSI must be relatively low, whereas CRP might be either low or high. The cost of resistance to conventional antibiotics is often surprisingly low, and can be further reduced by the spread of compensatory mutations after the establishment of resistance (Andersson & Levin, 1999). We do not know much about the cost of RAMP resistance, but there is no reason to suppose that it is out of line. Nisin-resistant strains of Listeria and Clostridium grow more slowly than wild-type on a range of standard media (Mazzotta et al., 2000
), for example, but the effect is not a large one. This would place populations in the region of the CSI-CRP phase space where RAMP therapy is most likely to trigger the evolution of resistance. Certainly, the argument that this is inherently unlikely to occur is without foundation.
If resistance is likely to evolve as a consequence of the widespread use of RAMPs, why are bacterial strains resistant to the great range of peptides produced by living organisms so rare? This is the strongest reason for believing that resistance will not evolve after all (Zasloff, 2002). The great diversity of RAMPs, which is one of the most striking features of this class of substances, could be interpreted in two ways, however. In the first place, RAMPs may have evolved independently in each species, or small group of closely related species. They would then provide a vast reservoir of antibiotics with almost unlimited potential to control bacterial populations. This seems very unlikely, because the systems for the induction and expression of RAMPs are strikingly similar in Drosophila and mammals (Hoffmann et al., 1999
). The second possibility is that bacteria evolve specific resistance rather easily, so that rare peptides are likely to be more effective. This would generate negative frequency-dependent selection driven by hostpathogen coevolution that would lead to rapid evolution at RAMP-encoding loci and thus great diversity among species. The population frequency of resistant types depends in part on the diversity of host defences. Wild populations of grasses are usually infested at low levels by a variety of pathogens that rarely cause serious disease, whereas the large-scale planting of cereal monocultures often elicits epidemics; natural communities of fungi produce a diversity of antimicrobial agents, but resistance remains low because a lineage of bacteria is rarely exposed for long enough, or in large enough numbers, for selection to be effective. If this interpretation be correct, then the therapeutic use of insect or fungal RAMPs might not be of great concern, any resistance that evolves as a consequence being highly specific, whereas the use of mammalian or human RAMPs would be correspondingly risky.
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Consequences of the evolution of resistance to human RAMPs |
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Coda |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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