chitin synthesis and degradation as targets for pesticide action

Archives of Insect Biochemistry and Physiology 22:245-261 (1 993)

Chitin Synthesis and Degradation as Targets for Pesticide Action Ephraim Cohen Department of Entornolo#, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel

Various pesticides are being used to destabilize, perturb, or inhibit crucial biochemical and physiological targets related to metabolism, growth, development, nervous communication, or behavior in pestiferous organisms. Chitin is an eukaryotic extracellular aminosugar biopolymer, massively produced by most fungal systems and by invertebrates, notably arthropods. Being an integral supportive component in fungal cell wall, insect cuticle, and nematode egg shell, chitin has been considered as a selective target for pesticide action. Throughout the elaborate processes of chitin formation and deposition, only the polymerization events associated with the cell membrane compartment are so far available for chemical interference. Currently, the actinomycetes-derived nucleoside peptide fungicides such as the polyoxins and the insecticidal benzoylaryl ureas have reached commercial pesticide status. The polyoxins and other structurally-related antibiotics like nikkomycins are strong competitive inhibitors of the polymerizing enzyme chitin synthase. The exact biochemical lesion inflicted by the benzoylaryl ureas i s still elusive, but a post-polymerization event, such as translocation of chitin chains across the cell membrane, i s suggested.

Hydrolytic degradation of the chitin polymer is essential for hyphal growth, branching, and septum formation in fungal systems as well as for the normal molting of arthropods. Recently, insect chitinase activity was strongly and specifically suppressed by allosamidin, an actimomycetes-derived metabolite. In part, the defense mechanism in plants against invasion of pathogens is associated with induced chitinases. Chitin, chitosan, and their oligomers are able to act as elicitors which induce enhanced levels of chitinases in various plants. Lectins which bind to N-acetyl-D-glucosamine strongly interfere with fungal and insect chitin synthases. Plant lectins with similar properties may be involved in plant-pathogen interaction inter alia by suppressing fungal invasion. 0 1993 Wiley-Liss, Inc.

Key words: allosamidin, benzoylaryl ureas, chitin synthesis inhibitors, chitin synthase, chitinase

Received January 21, 1992; accepted March 18, 1992.

Address reprint requests to Ephraim Cohen, Department of Entomology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76 100, Israel.

0 1993 Wiley-Liss, Inc.

246 Cohen

INTRODUCTION

Broad-spectrum neuroactive pesticides have been for decades the major tool for controlling arthropod pests in agriculture, forestry, stored-products, and in public health. Toxicological and environmental problems posed by the massive and widespread use of such nonselective pest control agents are amply documented. Moreover, the increasing awareness of the public to hazards associated with toxic residues in drinking water and edible crops has become a considerable stimulus to devise alternative pest control measures including more selective pesticides. Among the selective targets available in arthropods for pesticide interference, only the hormonal balance and the synthesis of the aminopolysaccharide chitin yielded commercial products.

Chitin is peculiar among other targets by being an abundantly produced extrxellular biopolymer [ 11 deposited as orderly oriented microfibrils notably in exoskeletons of arthropods and in cell walls of most fungi species. Its main function is to contribute strength and rigidity to structural elements of the organism. Being part of suprastructures such as cuticles, peritrophic membranes, or cell walls, any interference with chitin deposition or its untimely degradation is detrimental to the organisms involved.

A number of reviews concerning the biochemistry of chitin and its relevance as a target for pesticide activity have been recently published [2-6]. The present study will examine the complexity of cellular events leading to formation of the end product of chitin, e.g., the microfibrillar crystallite and the possible sites for interference throughout the pathway of chitin formation and deposition. Chitin degradation as a target for pest control by either enhancement or inhibition of the hydrolytic enzymes involved will also be considered.

CHITIN: PROPERTIES, FUNCTION, OCCURRENCE

Chitin is a straight chain homopolymer composed of p( 1,4)-linked GlcNAc units (Fig. 1) with a three-dimensional a-helical configuration stabilized by intramolecular hydrogen bonding [7]. The chains coalesce at the cell membrane surface via intermolecular hydrogen bonding to form insoluble crystalline microfibrils. X-ray diffraction analysis of chitin crystallites revealed two major polymorphs. a-Chitin, in which the polysaccharide chains in the microfibril are arranged in an antiparallel pattern [B], is the most abundant crystallite polymorph in nature. A @-chitin polymorph, which was observed in limited cases, is characterized by a parallel arrangement of the chains [9].

0 H,?.

R: N CHJ- CHITIN

n R = NH2- R

CHITOSAN

Fig. 1. Dirner units of chitin and chitosan polymers.

Chitin Metabolism as Target for Pesticides 247

The occurrence of y-chitin [lo] where the arrangement of the crystallite alternates so that for every three polymer chains two are parallel, is still debatable. Chitin, in particular the a-polymorph, is chemically stable and is insoluble in water and most organic solvents. It is degradable in very strong acid solutions or in concentrated hot alkali.

Chitosan (Fig. 1), which is a P(1-4)-linked D-glucosamine polymer formed by enzymatic deacetylation of chitin (GlcNAc), can be found as a structural component of the cell wall of various fungi, particularly in Zygomycetes [ll] as well as in arthropod cuticles [12]. The importance of chitosan and its oligomers in plant-pathogen interactions will be discussed later.

Chitin microfibrils, except in certain centric diatom algae [13], are covalently or noncovalently associated with various proteins (amoebae, arthropods, nematodes) [12] or polysaccharides (fungi) [14]. Such associations largely compose the supramolecular structures of cuticles and peritrophic membranes of arthropods, cell walls, and septa of fungi and yeast; also cyst walls in certain amoebae or egg shells of nematodes. A unique combination between chitin and keratin was recently reported in setae of a brachiopod species [15]. The importance of chitin as an integral part of the above supportive structures can be demonstrated by specific inhibition of its formation. Lack of chitin results in weak insect exoskeletons, difficulties in molting, bleeding, and mortality due to dehydration [16]. Inhibition of chitin synthesis in fungi leads to fragile cell walls which cannot withstand ambient osmotic pressures. As a result swollen and blown-up hyphae are often observed (Fig. 2) [17].

Chitin has become an attractive target for pest control since it occurs in invertebrates, notably arthropods, and in cell walls of most fungi except the Oomycetes where cellulose functions as the supportive component. How- ever, all vertebrates and vascular plants use alternative polymers (cellulose, collagen) as supportive components. The differential distribution of chitin among organisms (Table 1) gives the basis for the selectivity of pest control agents acting by interference with chitin metabolism as their primary mode of action. It should be stressed, nevertheless, that the above selectivity is limited since some nontarget aquatic arthropods [18-201 or beneficial preda- tors [21,22] were reported to be affected by commercial pesticides which act as inhibitors of chitin synthesis.

TARGETS FOR INHIBITION

Chitin Synthesis From Sugar Precursors to Polymer

The pathways of chitin biosynthesis and degradation, which consist of catalytic units, precursors, biotransformations, polymerization, fibrillogenesis, and hydrolysis, are depicted in Figure 3 . The unequivocal commitment to chitin synthesis occurs at the membrane-bound polymerizing system (chitin synthase) site. Apparently, catalytic units synthesized at the endoplasmic reticulum compartment are packaged in clusters which involve the Golgi apparatus. Subsequently, the assembled multiunits are translocated and integrated into the plasma membrane at the apical compartments of epidermal cells of arthropods or at tips of fungal mycelium. Electron microscopy studies

248 Cohen

Fig. 2. Electron micrographs of Sclerotiurn rolfsii hyphae. A: A control fungus culture (X2700); B: The culture was exposed to polyoxin-D ( x 4300). The hyphae of the polyoxin-treated culture, which due to lack of chitin in the cell wall cannot withstand ambient osmotic pressures, swell to considerable proportions.

revealed cytoplasmic organelles (chitosomes) believed to be packaged chitin polymerase units in a variety of fungal species [23] and in arthropods [24,25]. The chitin synthase in many fungi, and most likely also in certain insect systems, is initially in an inactive zymogenic state. Proteolytic cleavage is needed to convert the enzyme into a functional entity [ 5 ] . An electron microscopy study by Locke and Huie [26] using the insect Calpodes efhZius, showed areas of plasma membrane plaques in epidermal cells. These plaques,


TABLE 1. Phylogenetic Occurrence of Chitina

Fungi (cell wall) Amoebae (cyst wall) Diatom algae Rotifers (egg shell) Nematodes (egg shell) Anthropods (cuticle, peritrophic membrane) Molluscs (radula, shell) Sauids

aEstimated annual production of chitin: 10'o-lO" tons.

which presumably represent membrane-embedded clusters of chitin polymerase units, underwent hormonally-controlled cyclic turnovers.

To provide a flow of substrate molecules for the functional catalytic units, a cascade of cytoplasmic biotransformations of sugar precursors takes place. Trehalose, glucose, or the polysaccharide glycogen could be the starting points for a chain of bioconversions which include major steps such as phosphorylation, amination, and acetylation (Fig. 3). This metabolic pathway culminates in the formation of UDP-GlcNAc, the ultimate substrate of chitin synthase. Transformation of fructose-6-phosphate to glucosamine-6-phosphate by glutamine-fructose-6-phosphate aminotransferase can be considered as the first turning point on the pathway to chitin synthesis. Indeed, the

CHITIN SYNTHASE PRECURSORS synthesis biotransfomation: translocation phosphorylation insertion amination activation acetylation

turnover +

UDP-GlcNAc formation

POLYMERIZATION + +

HYDROGEN BOND FORMATION (crystallization, fibrillogenesis)

ORIENTATION OF MICROFIBRILS (cytoskeleton components?)

t SUPRAMOLECULAR ORGANIZATION

(covalent and noncovalent interactions) (with proteins, polysaccharides)

DEGRADATION (chitinase, chitobiase, chitodextrinase)

RECYCLING MINERALIZATION

Fig. 3 . Chitin metabolism.

250 Cohen

antibiotic bacilysin (tetaine), which is a specific inhibitor of the above enzyme, interfered with chitin biosynthesis in the fungus Candida ulbicans [27]. How- ever, since glucosamine-6-phosphate can still be diverted to and used by other metabolic pathways, inhibition of the amination step by the antibiotic compound is not specific with respect to chitin synthesis.

Chitin Synthase Catalytic Activity and Inhibition

The most extensively investigated target in chitin metabolism is the polymerizing enzyme chitin synthase. Using the fungus Neurosporu crassu Glaser and Brown [28] back in 1957 were the first to demonstrate chitin polymerization in a cell-free system. Later, the enzyme was obtained from numerous filamentous fungi and yeast species [5,14]. Due to methodological difficulties, cell-free preparations from insects were hard to obtain for a long time. The first reports describing insect chitin synthases from the Coleopteran TriboZium custaneum [29] and the Dipteran Stomoxys calcitruns [30] were published in 1980, Since then, polymerizing enzyme activity has been demonstrated in one crustacean [31] and 5 additional insect species [32-351. In insect and fungal systems the polymerization reaction for in vitro chitin synthesis is usually carried out in simple buffer solutions containing divalent cations like magne- sium and the (radioactive) substrate, UDP-GlcNAc [3,5]. Largely, both enzyme systems share similar properties such as the stimulatory response to the allosteric effector glucosamine, inhibition by nucleoside peptides and UDP, and lack of requirement for a primer or a lipid intermediate for their catalytic activity [3,5]. Unlike the insect chitin synthase, most of the fungal enzyme preparations, being zymogenic in nature, require proteolytic activation and are apparently insensitive to the fungicide captan [36].

Chitin synthase from the yeast Succharomyces cerevisiue was isolated and purified using the detergent digitonin [37]. At present, three different yeast chitin synthase enzymes (Chs 1,2,3) with different functions and their corresponding genes (CHS 1,2,3) were reported [3842]. Chitin synthase genes of the zoopathogen C. albicans [43] and of the filamentous fungus N . crussa [44], were recently cloned and sequenced. At present, no insect chitin synthase gene has been cloned. Based on differences in properties of chitin synthases [29,32,36], the existence of at least separated genes coding for integumental and peritrophic membrane enzymes can be anticipated.

The first documented inhibition of chitin biosynthesis involved nucleoside peptide metabolites (Fig. 4) isolated from actinomycetes. Resembling the structure of the chitin synthase substrate, UDP-GlcNAc, they act as potent competitive inhibitors of the enzyme. These structurally-related antibiotics are the polyoxins (A-M) [45] produced by cultures of Streptomyces cacaoi var. asoensis and nikkomycins ( Z , X, J, I [46] and KZ, K,, Q, 0, [47]) which are metabolites of Streptomyces tendue. Recently, two additional nucleosides related to nikkomycins (Wz, W,) were obtained from the culture broth of a fluorotyrosine-resistant strain of S. tendue [48]. It was suggested that the uridyl moiety, which is shared by the nucleoside antibiotics and the UDP-GlcNAc substrate, attaches to the binding site of the polymerizing enzyme [49,50]. The catalytic site of the enzyme, suggested to be hydrophobic in nature, apparently binds to the dipeptidyl (nikkomycins) or the tripeptidyl (polyoxins) part


I H

I H2N-CH

POLYOXIN - b NIKKOMYCIN- Z

Fig. 4. Chemical structures of nucleoside peptide inhibitors.

of the corresponding inhibitors. Most likely, the nucleoside peptides, being of relatively high molecular weight, enter the fungal cells via a dipeptidyl or tripeptidyl transport system [52,53]. Polyoxins have been commercialized and reported to be used in Japan for the control of a limited number of phy- topathogens causing diseases in fruit trees and vegetables [53] . Developing the nikkomycins as pesticides has been discontinued by Bayer company. The Streptomyces antibiotics are potent inhibitors of insect chitin synthase [3], yet they display poor insecticidal activity due to polar properties, relatively high molecular weights, and perhaps lack of appropriate transport systems in insect cells.

Inhibition of arthropod chitin synthases by several toxicants such as plum- bagin [54], buprofezin [55], avermectin [56], and terpenoyl benzimidazoles [57] was reported. Since relatively high levels of the above compounds are required for inhibitory effects, their action was regarded as nonspecific, A large number of pesticides and other toxicants were reported to inhibit the polymerizing enzyme extracted from the fungus Phycomyces [58]. Their actions were attributed to a general disruption of the plasma membrane lipid bilayer, nonspecifically affecting the membrane-bound chitin synthase [59].

Chitin From Polymer to Microfibril

Based on studies with fungal chitin synthase, the membrane-bound enzyme is most likely exposed to the cytoplasm [60-62]. Apparently, the insect polymerase obeys the same topography. However, studying cultured insect integuments, Mitsui and coworkers [63,64] claimed that the catalytic site faces the outer cell surface. Consequently, such a location invokes a transport system for the high molecular weight UDP-GlcNAc substrate which cannot diffuse through the plasma membrane. No evidence for such sidedness or proof for a transport system was provided. Electron microscopic studies of cell membranes using freeze-fracture techniques showed structures of rosettes and globules, which were suggested to be part of the cellulose biosynthesis system in algae and higher plants [65] . The molecular organization of the chitin synthase units in the cell membrane and the intricate processes which involve the translocation of chitin polymers across this barrier are mysterious. The energy required for the movement or translocation of the chitin polymers may be provided by the formation of 1,4-P-glycoside bonds or by the crystal-

252 Cohen

DIFLUBENZURON (Dimilin)

Fig. 5 . Chemical structure of a representative benzoylaryl urea compound.

lization process. The implication of different membrane proteins in translocation of chitin polymers has been suggested [59]. The involvement of auxiliary membrane protein or proteins having lectin-like properties in the translocation of chitin polymers is at present speculative. It is nevertheless tempting to speculate that the mode of action of the insecticidal benzoylaryl ureas (Fig. 5) is related to inhibition of such proteins. This group of commercial insecticides, which are highly potent inhibitors of chitin synthesis in insects, but not in fungi, does not inhibit the chitin polymerase. The exact biochemical lesion inflicted by these inhibitors is not known. Among the suggestions forwarded were 1) enhanced activities of phenoloxidase and chitinase [66]; 2) inhibition of proteases which convert the inactive zymogenic chitin synthase into an active enzyme [67]; 3) inhibition of DNA synthesis [68]; 4) inhibition of influx of nucleosides [69]; 5) interference with the insect hormonal balance [70]; and 6) inhibition of the speculative GlcNAc transporter [64]. Some of the suggested mechanisms of action such as 1, 3, and 4 have been regarded as secondary effects [3]. Hypotheses 2 and 5 were refuted since an on-going chitin synthesis is rapidly blocked [71,72] and the insect endocrine system is not affected by benzoylaryl ureas [72-741.

The last step in chitin biotransformation involves the formation of microfibrils at the cell surface, following the coalescence of adjacent nascent chitin chains via extensive hydrogen bonding. Disrupting these hydrogen bonds by dyes such as Calcofluor white, Congo red, and Primulin inhibited chitin synthesis in a number of fungal systems [75-791, insects [59,80], and a diatom alga [HI.

Albeit chitin polymerization and crystallization are coupled, they are con- secutive processes, evidence of which emerged from experiments using dye compounds such as Calcofluor white and Congo red [38,79]. It has been reported that chitin synthase activity was stimulated in the yeast S. cerevisiue [38] and the flour beetle T. castaneum [82] by Calcofluor white and chito- oligosaccharide-binding lectins, respectively. In the case of GlcNAc-binding lectins, it was suggested [82] that disruption of polymerization and detach- ment of polymers from the catalytic sites were followed by renewed biosynthesis of chitin polymers. Presumably, the observed stimulatory effect was due to a more efficient and rapid incorporation of the radioactive substrate into newly-synthesized shorter chitin polymers.

The characteristic helicoidal pattern of the arthropod cuticle suprastructure may indicate some sort of association between the cytoskeletal components of the epidermal cells and the membrane chitin synthase units. This probable connection also invokes an appreciable fluidity of the plasma membrane to


facilitate mobility of the embedded chitin synthase clusters. Such mobility may account for changes in orientation of the chitin microfibrils and, thus, for the helicoidal appearance. It is worthy to note that the fungicide benomyl as well as colchicine and vinblastine, known to disrupt cytoskeletal microtubular proteins, inhibited chitin synthesis in yeast [77] and in imaginal disks of an insect species [83].

In summary, throughout the pathway of chitin synthesis from simple precursors to microfibrils, a number of target sites for pest control can be exploited. Practically, the catalytic event is susceptible to strong inhibition by structural analogs of the substrate. There is no experimental proof, but it is plausible that post-catalytic events associated with translocation of chitin polymers across the cell membrane are perturbed by insecticidal benzoylaryl ureas.

Chitin Degradation

Chitin degradation involves a joint action of an endo enzyme, chitinase, which yields oligomers, and an exo enzyme, P-N-acetyl-glucosaminidase, which hydrolyzes dimers or terminal (GlcNAc), polymers [84]. A third enzyme (chitodextrinase), which hydrolyzes small chitooligosaccharides, has been recently reported in a chitinolytic marine bacterium [85]. Since chitin microfibrils are tightly associated with various proteins (arthropods) or polysaccharides such as glucan (fungi), proteolytic or glucanase activities often accompany and facilitate chitin hydrolysis.

Degradation of chitin is physiologically necessary to maintain normal hyphal growth and branching in filamentous fungi, and septum formation in yeast [86]. It has been shown that the dual action of chitin synthesis and degradation exists at the hyphal tips. Hydrolysis of chitin at specific sites on the hypha is a prerequisite for branching. Chitin degradation also facilitates spore release and germination [86]. The cyclic phenomenon of molting in arthropods is highly complex and involves a concerted and coordinated flow of hormonally-controlled biochemical events [84]. One such major event is the degradation of the endocuticular chitin, followed by the subsequent recycling of the aminosugar monomers into newly-formed polymers. The normal chitin degradation, be it in insects or fungi, could serve as a prime target site for interfering with essential biochemical and physiological processes. Indeed, in addition to organisms which form chitin (Table 2), chitin degradation systems have been evolved in nonchitinous organisms such as in prokaryotic microflora, certain vertebrates, and vascular plants [12,86]. Chitin is a nutritional source for chitinolytic microorganisms [12], crustacean-

TABLE 2. Phylogenetic Occurrence of Chitin Metabolic Svstems

Chitin synthesis Chitin degradation

Fungi Fungi Protozoa Protozoa Nematodes Nematodes Invertebrates Invertebrates

Bacteria Vertebrates Vascular ulants

254 Cohen

N CH3 OH

ALLOSAMIDIN

Fig. 6. Chemical structure of a chitinase inhibitor.

eating fish [87], and in insectivorous plants [88]. Mycopathogens [89,90] and entomopathogens [91] use their respective chitinolytic systems to penetrate arthropod cuticules and fungal cell walls.

Plants contain inducible systems which react to invasion of pathogens [92] inter alia by de novo synthesis of pathogenesis-related (PR) proteins. Among these proteins are glucanases, chitinases, and chitosanases, believed to act as a defense mechanism against phytopathogenic fungi [93-961. There is no direct evidence for the protective role of chitinase. However, recent studies in which the expression of the chitinase genes in the plant was amplified, should confirm the above role. It is noteworthy that externally applied chitosan oIigomers functioned as elicitors of chitinase formation in pea [97]. It was also suggested that chitosan acts as a transducing signal in the interaction between a gall-producing mite and a plant [98]. Moreover, externally applied polycationic chitosan or chitosan oligomers, which have high affinity for DNA, were detected within plant cell nuclei [99]. This observation is a clear indication that the above polysaccharides act at the genomic level to induce PR proteins.

Only recently allosamidin (Fig. 6) and desmethylallosamidin, two metabolites isolated from a Streptomyces sp., were reported to strongly and specifically inhibit insect chitinase and disrupt insect ecdysis [loo, 1011, These competitive inhibitors of chitinase are dimers of the aminosugar P-N-acetylallosamin attached to an aminocyclitol moiety. It was observed that while allosamidin is not effective against bacterial and plant chitinases, it dispIays appreciable inhibitory effects against chitinases of a nematode species [lo21 and the fungus N. CYUSSU [103]. Currently, the prospects for developing the allosamidins or similar effective products as commercial pesticides are unclear. However, the possibility of using chitin degradation as a new target for pest control is attractive.

CONCLUSIONS

In principle, the elaborate processes which involve the biosynthesis of chitin and its degradation should offer a number of interesting selective targets for the control of insect pests as well as phyto and zoopathogens. So far in practice, only the chitin polymerization step was proven to be a useful target and has been exploited. The polyoxins, which have been developed in Japan, reached the status of commercial fungicides, and are being applied on a limited scale. Since the introduction of diflubenzuron, a number of potent


analogs of the insecticidal benzoylaryl urea group have been developed and registered for commercial application. The use of these insect growth regulators is now widespread for the control of a variety of pests. Being selective to an appreciable degree, they are excellent candidates to be incorporated in integrated pest management (IPM) programs [104,105].

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