Behavioral Adaptations

Specialized behavior allows bypassing the defensive system of the attacked organism. Thus, for example, thorns or spines do not deter all enemies because some are insensitive enough or have special techniques to ignore this type of defense.

Among herbivores some specialists prepare plant leaves before they start feeding by cutting the leaf veins or trenches in the leaf. Then they feed distant from the cuts. This ensures both a better food quality and safe feeding for the herbivore because the flow of resin, which can trap the insects or may expose them to toxic secondary metabolites in the fluid, is prevented. This specialized behavior is widespread among different insects, for example, caterpillars and beetles, indicating that it may have been acquired during evolution independently. Alternatively, some herbivores carefully concentrate their feeding between the leaf veins in order to avoid damage to plant organs that contain toxic secretions.

Similarly, herbivores feeding on plants, which contain poisonous coumarin derivates, have developed a behavioral adaptation protecting them from the toxicity of these compounds. Coumarin derivatives are phototoxic molecules which are activated by UV-light and subsequently damage DNA molecules. Some insects have developed a so-called leaf-rolling behavior which shields them from direct sunlight during feeding. This technique not only protects the insect from heat and loss of water, but may also reduce the phototoxicity of the plant's secondary metabolites.

Excretion

An efficient way to bypass defensive secondary metabolites is rapid excretion of the toxic compounds. For example, the neurotoxin nicotine from Nicotiana plants is rapidly excreted by Manduca sexta larvae. Thus, nicotine that indeed would also be toxic for these specialist larvae never reaches a critical concentration in the gut.

Similarly, reactive dialdehydes (2) derived from iri-doids such as oleuropein (1) have been found to be neutralized by large amounts of glycine (3) in the gut of privet moths. The high glycine content in the gut traps reactive dialdehydes, for example 2, by reacting to imines such as 4. Thus, the free amino acid prevents the reaction of the dialdehydes with amino groups of digestive proteins from the insect's gut (Figure 1). As a result the larvae ensure the functioning of their digestive enzymes in the presence of crosslinking and protein denaturing dialdehydes.

Also microorganisms make use of excretion by expressing efflux transporters that confer resistance against antibiotics. These microbial efflux mechanisms are of high medicinal interest as they play a major role in the bacterial resistance to established antibiotics. This forces the urgent need of new therapeutics. Even though it is conceivable, it has not been investigated so far whether and how microorganisms make use of these export systems to prevent intoxication by the secondary metabolites of competing micro- and macroorganisms in their natural environment.

Suppression of Defense Reactions

An elegant countermeasure against defense mechanisms is the suppression of defense by interfering with the signaling processes of the victim and thus inhibiting the subsequent onset of defense in the first place.

An instructive example for this strategy has been observed for plant herbivore interactions. Obviously the salivary secretion of lepidopteran larvae appears to be designed to suppress plant defense induction. For example, Helicoverpa zea larvae feeding on Nicotiana tabacum make use of the enzyme glucose oxidase in their salivary glands to suppress the amount of nicotine produced by the tobacco plants. Glucose oxidase generates gluconic acid and H2O2, the latter is assumed to be responsible for the suppression of nicotine production presumably by interfering with the signaling of the defense phytohor-mone jasmonic acid. Consequently, the introduction of glucose oxidase into the plant leaf results in an increased performance of the larvae.

Apart from herbivores, plants have to face attack by microorganisms. A complex innate immunity system enables them to recognize microbial pathogens and to initiate adequate responses (see Plant Defense). The innate immune system of plants is directed against a broad range of microbes as it recognizes unspecific pathogen-associated molecular patterns, so-called PAMPs. For example, plants recognize the ubiquitous flagellin from microbes via a receptor-mediated process. As a counter-defense, microorganisms evolved so-called effectors that suppress the plant's innate immune system. Some effectors are proteins interfering with the plant's immune perception system and its signaling cascades. The plant pathogen Pseudomonas syringae., for example, introduces more than 30 effector proteins with diverse enzymatic activity into the plant cell. One of these effector proteins is called AvrPto and interferes with the signaling of

COOH

Figure 1 Reaction of dialdehydes from iridoid origin with glycine (3) provides protection for the insect's proteins.

COOH

Figure 1 Reaction of dialdehydes from iridoid origin with glycine (3) provides protection for the insect's proteins.

PAMP receptors of plants and thus suppresses defense reactions. Bacteria which are unable to deliver their effector proteins to the plant cell are less virulent than pathogens with an intact secretion system.

Also the oomycete Phytophthora sojae, a soybean pathogen, uses glucanase inhibitor proteins (GIPs) as effectors that specifically inhibit the action of plant endo-fl-1,3-glucanases. These endo-fl-1,3-glucanases play an important role in plant defense by degrading the pathogen's cell wall and thus generating fl-1,3/1,6-glucan elicitors, which induce plant defense reactions. GIPs are protease-like proteins; however, they do not exhibit enzymatic activity but probably inhibit the plant's glucanases by high-affinity protein-protein interactions.

However, in the coevolution of plants and their pathogenic attackers plants have evolved resistance gene products (R-proteins) in order to react to the pathogen's effector proteins. R-protein-mediated plant responses lead to an even enhanced defense response and a high resistance of plants against specific pathogens. The tomato protein Pto, for example, recognizes the bacterial effector protein AvrPto and thus induces a localized programmed cell death in the infected plant cells. This reaction inhibits further spread of the pathogen. The ongoing evolution of effector proteins in pathogens and of corresponding R-proteins in plants nicely reflects the dynamic arms race between the attacker and its prey leading to a coevolution of defense strategies.

Also in the interaction between plants and pathogenic viruses that enter the plant via insect feeding damage or through small wounds, defense and counterdefense strategies have evolved. To defend against viral infection, plants use RNA silencing. Small interfering RNAs (siRNA) are produced that bind to the viral RNA, thus labeling it for enzymatic degradation. At first RNA silencing is a localized process but there is an as yet unknown signal that transfers the silencing information to other cells. As countermeasures some viruses express proteins that interfere with this plant signaling pathway.

In humans viruses have the ability to undermine the natural defense system of their victim. One of the best-studied examples is the human immune deficiency virus (HIV); but also other viruses, such as the Ebola virus or the herpes virus, are efficiently disrupting the effective defense arsenal of the mammal's immune system. The HI-virus indeed reduces the number of lymphocytes, for example T-helper cells and macrophages, as it infects exactly these immune cells which are otherwise responsible for fighting viral infections. Eventually, the demise of the human immune system culminates in the acquired immune deficiency syndrome (AIDS). Also other viruses, which cause a persistent infection, for example, viruses of the herpes family, are able to hide from the immune system. Infected cells normally present viral peptides on their outer surface; these are detected by immune cells, which in turn kill the infected cell in order to prevent spreading of the virus. Herpes viruses can inhibit this system of antigen presentation; consequently, the infected cells will not be recognized by the immune system. Stress or other infection can reactivate the virus and lead to the outbreak of the well-known herpes symptoms.

A different way of suppressing defense responses is the synthesis of small molecules that are identical or analogs to the victims's own signaling molecules and thus disturb hormone-regulated signaling cascades. For example, blood sucking animals such as ticks and leeches produce compounds to suppress the mammals inflammatory and wound response, thus undermining the natural defense system of animals. While leeches use proteins such as hirudin that inhibit coagulation of the blood, many ticks synthesize eicosanoids such as prostaglandin E2 (5) in their salivary glands (Figure 2). These hormone-like molecules interfere with the mammals own prostaglandin signaling pathways, thus suppressing wound response and promoting a continuous blood flow.

In the interaction between plants and their enemies, the analogs of phytohormones play an important role (Figure 3). For example, coronatine (6) produced by Pseudomonas syringae mimics the hormone jasmonic acid and thus not only promotes jasmonic acid-like defense responses but interferes as well with the signaling pathway of other phytohormones such as abscisic acid or salicylic acid. Typically, an early immune response of plants is the closure of stomata, in order to prevent pathogens from entering the leaf intercellular space. By producing coronatine (6), the pathogen forces the opening of these microscopic holes, because the abscisic acid-controlled closure of the stomata is suppressed. In addition, coronatine (6) interferes with the plant's salicylate signaling pathway that would induce a systemic acquired resistance reaction against the pathogen. Similarly also gibberellins such as gibberellin A1 (7) from the fungal pathogen Gibberella fujikuroi and cytoki-nins such as zeatin (8) from pathogens disturb plant signaling processes.

Prostaglandin E2 5

Figure 2 Prostaglandin E2 (5) released by ticks to ensure continuous blood flow after biting its victim.

Figure 3 Analogs or phytohormones produced by microorganisms influencing plant signaling pathways.

Coronatine 6

Figure 3 Analogs or phytohormones produced by microorganisms influencing plant signaling pathways.

Molecular Adaptation

Apart from behavioral adaptation and suppression of defense mechanisms, molecular and enzymatic adaptation is the most common strategy which helps attackers to face defense compounds of their victims.

In order to attenuate the action of a toxin, its enzymatic breakdown or modification is used by many organisms. Especially cytochrome P450 enzymes are involved in such detoxification processes. Cytochrome P450 enzymes are able to oxidize various positions of molecules even at relatively unreactive moieties. After epoxidation or hydroxylation, the compounds may undergo further breakdown or are conjugated to polar molecules such as sugars and are subsequently excreted.

It has been shown that the expression of detoxifying P450 enzymes is inducible and consequently a prepared organism with high levels of P450 enzymes is better protected against toxic compounds. The lepidopteran Helicoverpa zea makes use of the plant's signaling system and recognizes the levels of plant defense-related hormones such as jasmonic acid and salicylate. When levels of these phytohormones are high, the caterpillar reacts with the upregulation of detoxifying cytochrome P450 enzymes in order to be prepared against upcoming plant defense compounds.

In the same line it was found that many lepidopteran larvae rapidly convert 12-oxo-phytodienoic acid (OPDA, 9), the precursor of jasmonic acid (Figure 4; see Plant Defense), to ixo-OPDA (10) by an enzymatically controlled process in their foregut. Although the significance of this reaction is not fully understood, it indeed suggests that insects recognize plant hormones. The conversion may serve their deactivation, because the structurally flat ixo-OPDA does not induce plant defense reactions such as the emission of volatiles. Furthermore, the isomerization could serve the insect as signal processing to initiate counterdefense.

The development of detoxifying enzymes by successful herbivores imposes an evolutionary pressure on the plant to develop new secondary defense metabolites. This coevolution of toxins with the upcoming of resistant feeders can be followed in the family of Apiaceae which produce phototoxic coumarins. Simple metabolites such as coumarin (11) or umbelliferone (12) are less toxic than the tricyclic furanocoumarins, for example, psoralen (13). The adaptation of insects to simple coumarins is suspected to have led to the development of linear furanocoumarins such as psoralen (13). Furanocoumarins destroy DNA when activated by UV-light. Generalist insects are able to consume plants producing hydroxy-coumarins, while they die on diets containing furanocoumarins. However, some insects evolved detoxification mechanisms for these linear furanocoumarins, which in turn forced some Apiaceae to produce in addition angular furanocoumarins such as angelicin (14). Angular furanocoumarins escape the detoxifying enzymes of most herbivores. Only highly specialized insects, such as the short-tailed swallowtail (Papilio brevicauda), are able to survive feeding on plants that contain angular furanocoumarins (Figure 5).

Figure 4 Isomerization of 12-oxophytodienoic acid (9) in the gut of lepidopteran larvae.

A consequent step of specialists that are resistant to toxins from other organisms is to make use the poison they take up with their food for their own defense (see Animal Defense Strategies). For example, Tyria jacobaeae larvae take up pyr-rolizidine alkaloids from highly toxic Senecio species on which they almost exclusively feed. The alkaloids are stored as inactive N-oxides which do not harm the insect itself.

Also in marine systems specialized herbivores are sequestering toxins from their food source. For instance, the opisthobranch mollusk Oxynoe olivacea is specialized on the macroalgae Caulerpa sp., which is toxic for most other marine herbivores because of its defense compound caulerpenyne (15) (see Defense Strategies of Marine and Aquatic Organisms). Upon wounding, the acetylated sesquiterpene caulerpenyne (15) is enzymatically converted to a highly reactive dialdehyde that destroys proteins by reaction with free side chain amino groups. However, O. olivacea is able to feed on Caulerpa sp. and somehow stores the caulerpenyne (15) and structurally closely related oxytoxin 1 (16) for its own defense. Compared to caulerpenyne (15) oxytoxin 1 (16) bears one acetyl residue less than 15 and thus contains already one aldehyde moiety (Figure 6). The detailed mechanism how O. olivacea manages to prevent caulerpenyne (15) from complete deacetylation has not been uncovered so far.

Figure 5 Evolution of plant secondary metabolites under the pressure of the herbivore's successful counterdefense measures.

Psoralen Angelicin

13 14

Figure 5 Evolution of plant secondary metabolites under the pressure of the herbivore's successful counterdefense measures.

An elegant way to avoid a defense compound is to change the structure of its molecular target. This method of counterdefense is widely used among plants and animals. The rough skinned newt (Taricha granulosa), for example, is defended by the deadly poisonous sodium channel blocker tetrodotoxin (LD50 10 mgkg-1, see Plant Defense). Therefore, the rough skinned newts do not need to fear any predators but the garter snake Thamnophis sirtalis. This snake adapted its sodium channels by mutations so that tetrodotoxin binds less tightly to its target. Interestingly, there is evidence that this adaptation occurred independently in several snake populations: each developed a different set of mutations in a conserved region of the sodium channel that is crucial for the toxi-city of tetrodotoxin. As a consequence, the reaction of the newt against the snake's adaptation is to accumulate large amounts of tetrodotoxin that exceed the toxic amount needed to kill an unadapted organism by far.

In plant herbivore interactions, protease inhibitors are an impressive example to follow evolutionary adaptation by mutation. Protease inhibitors are not directly toxic themselves but are intended to inhibit digestive enzymes and lower the nutrient uptake by the herbivore. However, they are ineffective against some larvae, for example, Spodoptera frugiperda that adapted by changing the amino acid sequence of their proteases.

In particular, microorganisms are masters in inventing new molecular mechanisms to face challenges from their environment. Because of their short generation cycle their adaptation can even be followed within several years. In

Figure 7 ^-Lactamase inhibitor clavulanate (17) from Streptomyces clavuligerus.

Clavulanate 17

Figure 7 ^-Lactamase inhibitor clavulanate (17) from Streptomyces clavuligerus.

Figure 6 Caulerpenyne (15) and the structurally closely related oxytoxin 1 (16).
Vancomycin (18)

CH3 CH3

CONH2

CH3 H

CH3 H

Unmodified cell wall peptide fragment (19)

Modified cell wall peptide fragment (20)

Figure 8 Alteration of the binding efficiency of vancomycin to bacterial cell-wall fragments.

Modified cell wall peptide fragment (20)

Figure 8 Alteration of the binding efficiency of vancomycin to bacterial cell-wall fragments.

addition, the ability of microorganisms to exchange plas-mids supports the spreading of resistance mechanisms even among different species. Thus, also humans indirectly participate in the arms race of defensive mechanisms of microorganisms because it forces us to continuously search for new weapons against pathogens. Just 4 years after massive usage of penicillin, first resistant bacteria were observed that developed a ^-lactamase enzyme which destroyed penicillin. The ^-lactamase evolved from an alteration of the peptidoglycan synthase, an enzyme involved in the bacterial cell-wall synthesis. A next step in the defense arms race was the isolation of the ^-lactamase inhibitor clavulanate (17) from Streptomyces clavuligerus. This molecule does not exhibit any antibiotic activity by itself but blocks the ^-lactamase and thus ensures the function of penicillin antibiotics. Interestingly, S. clavuligerus produces a set of penicillins and penicillin-related nonribosomal peptides with antibiotic activity so that it is possible that the microorganism makes use of the combination of penicillins and the ^-lactamase inhibitor clavulanate (17) in competition with other microorganisms for living space; similarly as the combination of clavulanate (17) with penicillins made its way into pharmaceutical use (Figure 7).

Another example for the fast adaptation of microorga-nims against antibiotics is the development of resistance against the glycopeptide antibiotic vancomycin (18). This powerful antibiotic interferes with the cell-wall biosynthesis just like penicillins. Vancomycin (18) blocks the biosynthesis of the bacterial cell wall by binding to the pentapeptide side chain motif D-Ala-D-Ala (19) of the peptidoglucan cell-wall structure and thus prevents its polymerization. For a long time, it has been one of the antibiotics of last resort that stayed active against multi-resistant strains such as Streptoccocus aureus.

However, in 1987 the first vancomycin-resistant Enterococcus strains appeared. The inhibitor of the cellwall biosynthesis lost its efficiency against resistant strains because those microorganisms slightly modified the structure of the molecular target, that is, the peptidoglycan by replacing one D-alanine against D-lactate. This replacement ofjust one amide bond against an ester bond D-Ala-D-lactate (20) in the peptide side chains weakens the binding to the cell-wall fragment at factor 1000 and thus renders vancomycin (18) inefficient as inhibitor of the cell-wall biosynthesis (Figure 8).

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