Chitinase: a novel target for blocking parasite transmission?

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Chitinase: a Novel Target for Blocking Parasite Transmission? M, Shahabuddin and D.C. Kaslow In many species of blood-sucking arthro pod, the internal tissues are covered by chitinous material that may hinder parasite invasion. To circumvent this potential barrier, therefore, parasites have devel oped mechanisms that involve the enzyme chitinase. In this review, Mohammed Shahabuddin and David C. Kaslow examine the relationship between chitinase and parasite transmission. Blood-sucking insects act incidentally as vectors for many human parasites, and thus play a crucial role in transmitting the resultant diseases ~, The parasite may simply be carried between vertebrate hosts, or it may undergo obligatory morphological development (as in Bruda and Wuchereria), or even replication (as in Plasmodium spp and Leishmania). By using the oral route of infection and penetrating the non-chitinous midgut, these parasites avoid the tough chitinous exoskeletal defense of the insect. However, in many species of blood-sucking arthropods, the internal tissues are covered by chitinous material that can hinder or even block parasite invasion. For instance, in mosquitoes a peritrophic matrix/membrane (PM) separates the bloodmeal from the midgut epithelium2; in sandflies a chitinous lining protects the stomodeal valve and midgut epithelium 3. For successful transmission through these arthropod vectors, parasites have clearly developed mechanisms to avoid, penetrate or even destroy these potential chitinous barriers. Indeed, chitinase (EC 3.2. I. 14) plays an essential role in the successful transmission of Plosmodium spp and Leishmania through insect vectors. Chitinases are enzymes that hydrolyse chitin, a (I ~>4)-13 homopolymer of N-acetylglucosamine 4 (see Box I). Some parasites require chitinase to degrade chitin-containing structures produced by that organism (eg. Onchocerca dbsoni s) or its host (eg. L. major 6 and Plasmodium spp7), whereas other parasites rely on the chitinase of symbionts within the insect (eg. Trypanosoma8). Although in most instances details of the molecular

mechanisms of these interactions are not known, recent work suggests that parasite chitinases are potential targets for blocking the transmission of these parasites (Table I). Plasmodium

Within the 20-24 h following a bloodmeal, non-motile Plasmodium zygotes undergo a morphologic change and develop into motile ookinetes. Ookinetes penetrate the mosquito midgut epithelium 22 30 h post bloodmeal (depending on the species). Meanwhile, within hours (l~r, depending on the species) of the bloodmeal, mosquitoes begin to form a Pbl that gradually encapsulates the bloodmeal (for a recent review on PM, see Ref~ 2). The PM remains intact for at least 48 h after the bloodmeal. Therefore, ookinetes must traverse the PM before invading the midgut epithelium. In fact, a recent ultra-structural study showed numerous ookinetes 'trapped' in the endoperitrophic space during invasion, suggesting that the PM acts as a barrier in malaria transmission9 and accounts for the lower number of oocysts on the midgut compared to the number of ookinetes in the endoperitrophic space. Along with concentrated elec tron-dense materials at the anterior end of the penetrating ookinetes, the laminated structure of PM is focally damaged, indicating that the pen etration of PM could be an enzymatic process9. Huber et al.7 subsequently found that the PM could be degraded by chitinase, and that ookinetes synthesize and secrete chitinase in the culture supematant. The coincidence of chitinase secretion with the penetration of the PM implied the involvement of this enzyme in the parasite's egress from the bloodmeal. We have recently shown that allosamidin (see Box I) is a potent inhibitor of Pfasmodium chitinase, which is not toxic to the mosquitoes and which does not interfere with normal egg development I°. When fed with infected bloodmeal, allosamidin cam-

pletely blocked the sporogonic development of P. falciparum and P. gallinaceum parasites in the mosquito midgut I° (Fig. I). Feeding mosquitoes Streptomyces ~riseus chitinase in bloodmeal disrupted the PM formation, in viva, and completely reversed the transmission-blocking effect of allosamidin (Fig. I). Therefore, the blocking of the sporogonic development of Plasma dium spp in the mosquito midgut was not due to non-specific killing of the parasite, but was due to the specific inhibition of chitinase in mosquito midgut. Because ookinetes produce chitinase within 24 h after bloodmeal while the degradation of the PM by the mosquito does not start until 48 h after bloodmeal, we suspected that the transmission-blocking effect of allosamidin was due to the inhibition of parasite chitinase rather than mosquito chitinases. Although the exact mechan ism of PP1 penetration by Plasmodium ookinetes is not yet clear, there is little doubt that this enzyme is essential for the successful transmission of this parasite. Leishmania

Like Plasmodium, there is now good evidence that chitinase is involved in the transmission of L. major by Phlebotomus papatasi 3. However, unlike Plasmodium, Leishmania does not cross the midgut epithelium of the insect, but rather develops from amastigotes to promastigotes exclusively in the endoperitrophic space of the midgut lumen. Two to three days after bloodmeal, promastigotes move towards the anterior part of the midgut, escape through the disrupted PM, and colonize the cardia/stomodeal valve region. The parasite is then transmitted to a new host through the food channel in the proboscis. In most Leishmaniainfected sandflies, the stomocleal valve (the main valve that maintains the unidirectional blood flow during normal feed) is damaged, causing infected blood from the gut to be regurgitated into the new host. @ 1993, Elsevier Science Pubhshers I td, (UK)

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Box I. Structure and Properties of Chitin, AIIosamidin and Chitinase Chitin Chitin is one of the most abundant polysaccharides in nature. It is an insoluble, linear (I-->4)-{3-1inked homopolymer of H-acetylglucosamine. In some cases, ocher aminated sugar may also be present, for example, the peritrophic membrane (PM) of tsetse contains D-glucosamine in addition to N-acetylglucosamine. Chitin is commonly found in insect exoskeletons, shells of crustaceans and fungal cell walls. Chitinous outer structures in these organisms act primarily as the first-line defence against adverse surroundings.

••oHOH

O~

,/OH OH H

NH Ac

01

Chitin

chitinase by hemoglobin may account for the lower infectivity of the flies maintained on repeated blood feeding compared to those flies maintained on sugar-only diets 3. Because hemoglobin inhibits Leishmania chitinase, consecutive bloodmeals may protect the PM and the stomodeal valve from being damaged by this enzyme. Further studies involving (for instance) feeding exogenous chitinase to sandflies with a sugar meal (which should lead to damage of the stomodeal valve) and using allosamidin to inhibit chitinase activity, in vivo, should reveal the role of chitinase in the transmission process and establish whether chitinase is a target for blocking transmission of Leishrnania. Filaria

-OH

OH

o

HO

HC~

~HA-~"v

H

~

_

~ N~

Allosamidin

N/CH3

I CH3

AIIosamidin AIIosamidin was initially isolated from fungus, Streptomyces spp 18. It is a potent inhibitor of insect chitinases. Structurally it comprises a (I-~4)-J3-1inked dimer of ~-N-acetylallosamine bound with a dimethylaminocyclitol moiety. N-acetylallosamine is a C-3 epimer (see diagram of structure of N-acetylglucosamine, which is the building block of chitin. Therefore, allosamidin resembles chitin structurally and may bind to chitinase competitively and affect its activity. Chitinase Chitinases, a family of enzymes that catalyse the hydrolysis of the 13-linkage of N-acetylglucosamine polymer of chitin, are found in a wide range of organisms, including bacteria, fungi, higher plants, crustaceans and insects. The role of chitinases can be divided into several categories. For example, a major role of chitinases found in fungi, crustaceans and insects is modification of the organism's structural constituent chitin. The production of chitinase by plants is considered to be a part of their defence mechanism against fungal pathogens, which contains chitin as a structural constituent. Bacteria produce chitinase to digest chitin primarily to utilize it as a carbon and energy source. According to their mode of action on chitin, chitinases can be divided into exo- and endochitinases. Endochitinases act on chitin randomly along the chain and cause random chain cleavage. In contrast~ exochitinases act processively and release monomers or dimers of the sugar unit from the non-reducing end of chitin. Chitinases are also classified as either basic or acidic chitinases (also called class I or class II, respectively). The diversity of chitinases is reflected in the high degree of divergence in their primary amino acid sequences.

Again, like Plasmodium spp, Leishmania parasites secrete chitinase into the culture supematant, in vitro (Table I), suggesting that the parasite interacts with chitinous matrices of the sandfly during transmission. The interaction may occur at several sites. The enzyme may damage the PM (particularly at the anterior end) to facilitate the escape of the parasites. This is consistent with the observations that the mass of mature

parasites concentrate in the anterior part of the PM prior to the damage 6. In contrast, the degradation of PM in uninfected blood-fed sandflies starts from the posterior end 6. The enzyme may also damage the stomodeal valve by destroying the chitinous cuticular lining of the valve3. There is no direct evidence that inhibition of Leishmania chitinase, in vivo, blocks transmission. However, the inhibition of Leishrnania

Within an hour of a bloodmeal (which is well before the formation of a PM in the mosquito), microfilaria invade the midgut epithelium. Therefore, these parasites do not need chitinase to cross the PM. Nevertheless, a monoclonal antibody, MFI, to Brug~a malayi chitinase blocks transmission It,t2. How MFI blocks transmission is not known. However, it is known that the MFI antigen is expressed as microfilaria develop in the mosquito 13. Chitinase may be involved in microfilaria transmission by way of its role in the exsheathment process and/or N-acetylglucosamine production. Exsheathment starts the subsequent molting and development of the filarial larvae in mosquito vector. The sheath of the microfilaria contains chitin, and perhaps the parasite chitinase plays an active role in the exsheathment process. Lectins in mosquito block microfilaria invasion of midgut epithelium, which can be reversed by adding N-acetylglucosamine in the feed 14. It is likely that N-acetylglucosamine released by the action of chitinase may increase the infectivity of microfilaria by inhibiting specific lectins that block the parasites' invasion of the midgut. Chitinase has also been detected in O. gibsoni s, the causative agent of cattle filariasis. Although the microfilaria of these parasites do not have sheaths (and thus have no exsheathment process in the arthropod vector), they may use chitinases in the remodelling of chitin during egg development in vertebrate hosts. If the exsheathment of Brugia malayi and remodelling of chitin in the egg shells of Onchocerca are essential for the development of these parasites, blocking chitinase activity may disrupt their transmission.

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Trypanosoma

Table I. Parasites for which chitinase has been reported, and their insect hosts Parasite

Insect v e c t o r

Refs

Plasmodium falciparum Plasmodium gallinaceum Leishmania major I_ braziliensis L. donovani I_ infantum Trypanosoma lewisi T. b. brucei Leptomonas seymouri Herpitomonas muscarum Crithidia fasciculata Brugia malayi Onchocerca gibsoni

Anopheles spp Aedes aegypti Phlebotomus spp Lutzomyia spp Phlebotomus spp Phebotomus spp Ceratophyllus fasciatus Glossina spp Dysdercus surucellus Musca spp Culicine and Anophelene spp Aedes spp, Anopheles spp Culicoides pungens

II 7 6 6 6 6 6 19 6 6 6 13 5

a

~ l ] ~okinete PM

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^

b

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^

,

~

c & I

Inactivated chitinase

~ , - ~ = Intact peritrophic ~)~)~ matrix

^

^

^

.

d

I ~

!~i

I

I

.,~,~:.-^-, = Active .-^-.~.-.^.- chifinase

.

^

^

^

~

^

,,,,-,,,,,, •,,,,,,,,,,,

Disruptedpefitrophic matrix

Fig. I. Experimental design to study the role of chitinase in the transmission of malaria. Four groups of mosquitoes were fed Plasmodium-infected blood. Eight days after the bloodmeal, mosquitoes were dissected to check for the development of oocysts, as the index of successful transmission• Untreated control (a): malaria ookinetes can disrupt mosquito peritrophic matrix (PM). The number of oocysts observed in this group of mosquitoes gives an indication of the quality of the parasites used in the feeding. MV = microvilli. Chitinase-fed control (b): this group of mosquitoes were fed exogenous chitinase mixed with infected blood. Because exogenous chitinase disrupts mosquito PM formation, the parasite's chitinase activity is not needed for transmission. No significant effect was observed on oocyst number in this group of mosquitoes. AIIosamidin-fed (c): this group of mosquitoes were fed allosamidin with the infected blood. Due to the inhibition of the parasite chitinase by allosomidin, the ookinetes were unable to cross the PM and the oocysts' development was completely blocked. AIIosamidin- and chitinase-fed (d): this group of mosquitoes were fed both allosamidin and a fungal chitinase. The exogenous chitinase disrupts the mosquito PM, obviating the need for the parasite to produce its own chitinase. Addition of allosamidin to inhibit the parasite's chitinase had no effect on the normal oocyst development, indicating that the blocking effect of allosamidin is not due to non-specific killing of the parasite.

The role of chitinase appears to be more complicated in the transmission of Trypanosoma than in those parasites mentioned above. In the life cycle of T brucei, which has a midgut stage in the tsetse, the PM may play a major role. How procyclic stage t~/panosoma cross the PM and escape to the exoperitrophic space to become mature metacyclic stage is debatable8. It is likely that a chitinolytic enzyme plays a crucial role in this process. Evidence is mounting that susceptibility of Trypanosoma to tsetse flies is at least in part controlled by chitinase. Chitinase has been detected in the culture supematant of T. lewisi6 and T brucei brucei ~9. In addition, a similar enzyme secreted by a symbiotic rickettsia-like organism (RLO) in tsetse appears to be involved in the enhancement of transmission of this parasite 8. A positive correlation between the RLO load and the susceptibility to Trypanosoma infection has been described ~s. How can chitinase from a different organism help in the transmission of trypanosomes? Lectins secreted in the tsetse midgut in response to bloodmeal are toxic to this parasite 16. This effect can be reversed by feeding D-glucosamine, which also inhibits the midgut lectin in v~tro. However, the parasite needs a small amount of lectin for maturation and development w. Complete inhibition of this lectin by D-glucosamine blocks the parasite development in the midgut. It therefore appears that for successful development and transmission Trypanosoma require a balanced concentration of midgut lectin. Chitinase from RLO may act on the tsetse PM to release D-glucosamine (because chitin in the tsetse contains D-glucosamine along with N-acetylglucosamine), which helps to maintain the optimum concentration of the active lectin for the development and transmission of the parasite 8, Further studies of RLO chitinase and trypanosome chitinase will clanfy their exact role in the transmission of this parasite. Conclusions and Perspectives

Because vertebrate hosts do not contain chitin, the enzymes involved in chitin metabolism could be suitable candidates for vaccine development. It is now clear that Plasmodium uses a chitindegrading enzyme for successful transmission through their insect hosts. It also seems likely that Leishmania, filaria and

Parasitology Today, vol. 9, no. 7, 1993

Trypanosoma use this enzyme for a similar purpose. The exact mechanism of chitinase action in parasite transmission is not clear. Thus, further molecular biological and biochemical studies are needed to develop chitinases into novel candidates for transmission-blocking vaccines. Detailed analysis of the molecular mechanisms of chitinase action in insects may also identify new candidates for transmission-blocking vaccines. For example, we have recently found that purified mosquito midgut protease activates Plasmodium chitinase, in vitro. If Plasmodium chitinase activity relies wholly or mainly on such activation, the corresponding protease would also be a target for transmission-blocking antibodies. However, because proteases are essential for digestion of food, alteration of protease activity may be incompatible with normal mosquito physiology. Certainly, more information on the molecular basis of protease action in midgut will be needed to determine if these proteases are feasible target antigens of transmission-blocking immunity. Establishment of the molecular basis of para-

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site-vector interaction, such as the role of chitinase and protease in parasite transmission, could be an important addition in the development of vaccine against parasites transmitted by bloodsucking arthropods.

Acknowledgements The authors thank Louis H. Miller, Enrico Cabib and lan Maudlin for insightful discussions. The authors received financial support from the UNDP/WorldBank/WHO Programme for Research and Training in Tropical Diseases and John D. and Catherine T. MacArthur Foundation. References

I Lehane, M,J. (I 99 I) in Biology of Blood-sucking Insects, pp 143--192, Harper Collins Academic 2 Peters, W. (1992) Pentrophic membranes: Zoophysiology (Vol. 30), Springer Verlag 3 Schlein, Y., Jacobson, R.L. and Messer, G. (1992) Proc. Natl Acad Sci. USA 89, 9944-9948 4 Flach, j., Pilet, P.E and Jolles, P. (1992) Experientio 48, 701 716 5 Gooday, G.W., Brydon, L.J. and Chappell, L.H. (1988) Mol. Biochem. Parasitol. 29, 223-225 6 Schlein, Y., Jacobson, R.L. and Shlomai, J. (1991) Proc. R. Sac. London: Set. B: 245, 121 126

7 Huber, M., Cabib, E. and Miller, L.H. (1991) Proc. Natl Acad. Sci. USA 88, 2807-2810 8 Maudlin, I. (1991) in Advances in Disease Vector Research (Vat. 7) (Hams, K.F., ed.), pp I 17 148, Springe~Verlag 9 Sieber, K-P, et al. (1991) Exp. Porasitol. 72, 145-156 I 0 Shahabuddin, M, et al. (I 993) Proc. Natl Acad Sci. USA 90, 4266-4270 II Fuhrman, J.A. et ol. (1987) Am. J. Trap. Med. Hyg. 36, 70-74 12 Fuhrman, J.A. et al. (I 992) Proc. Natl Acad Sci USA, 89, 1548 1552 13 Fuhrman, J.A. and Piessens, W.F, (1989) Mol. Biochem. Parasitol. 35, 249-258 14 Phin, J. and Ham, P.J. (1990) Trans. R. Sac. Trap. Meal. Hyg. 84, 462 15 Welbum, S.C. and Mauldin, I. (1991) Parasib ology 102, 201 206 16 Welburn, S.C., Mauldin, I. and Ellis, D.S. (I 989) Meal Vet. Entomot. 3, 77 82 17 Welbum, S.C. and Mauldin, I. (1990) Med. Vet. Entomol. 4, 4 3 4 8 18 Sakuda, S. eta/. (1986) Tetrohedron Lett. 27, 2475-2478 19 Schlein, Y. and Jacobson, R. (1992) Parasit ology Today 8, 367

Mohammed Shahabuddin and David Kaslow are at the Molecular Vaccine Section, Laboratory of Malaria Research, National Institute of Allergy and Infectious Diseases, National Insti tutes of Health, Bethesda, MD 20892, USA.

Leishmania and Sandflies: Interactions in the Life Cycle and Transmission Y. Schlein Leishmania infections in the vector sandfly are limited to the gut, where contact with tissues, secretions and the medium of sandfly food influence their cycle of development. The parasites cope with and exploit their habitat by generating products that impair the function and damage the tissues of the gut. In this review, Yosef Schlein concentrates an some of the recent advances in the limited knowledge of these parasite-vector interactions. Details of parasite development and morphology, as well as the adaptations to life in the vectors, have previously been summarized and discussed extensively 1,2. Many studies, in vitro, have characterized developmentally regulated changes in the expression of the major surface glycoconjugate of Leishmania, the lipophosphoglycan (LPG) 3,4 and the glycoprotein protease gp63 (Ref. 5). During the growth of parasites, in vitro, there is a progressive increase in the infectivity of promastig© 1993,ElsevierSciencePublishersLtd,(UK)

otes that is characterized by altered glycosylation of the LPG. This results in masking of the specific binding sites for peanut agglutinin (Gal~l-~3GaINAc). The loss of ability to bind this lectin distinguishes between these types of LPG and enables separation of procyclic from metacyclic promastigotes 6. The role of LPG in the sandfly was recently investigated using both types of LPG, and phosphoglycan (PG), which is released from the parasites 7. Phosphoglycan is also the major component of the excreted factor (EF)8,9 isolated from medium that was called dried culture overlay ~° or released glycoconjugate ~l in previous studies with sandflies. Monoclonal antibodies (mAbs) recognizing both common epitopes and metacyclic specific LPG have provided markers for parasites in the sandfly7,12.t3. Other products of Leishmania that have recently been defined and function in the sandfly are the chitinolytic enzymes t
Life Cycle in the Sandfly The summarized life cycle in Fig. I is that of the Leishmania spp (suprapylarian) without a hindgut phase, based on the thorough study of L. chagasi in Lutzomyia Iongipalpis~6. Amastigotes in the abdominal midgut undergo several divisions and progressively change to long slender nectomonads. This takes place within the sac-like peritrophic 'membrane', comprising chitin lattice embedded in protein-carbohydrate matrix, which is secreted by gut cells around the bloodmeal. Before the blood is completely digested the anterior part of the membrane disintegrates and exiting parasites migrate to the thoracic midgut and cardiac valve. Other nectomonads remain in the abdominal midgut after the excretion of residues of the membrane and blood. The continuously dividing nectomonads attach to the microvilli of the midgut, particularly in the thoracic