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Kinetoplastid Biology and Disease

Kinetoplastid Biology and Disease 2002, 1 xReviewMolecular determinants and regulation of Leishmania virulenceKwang-Poo Chang*1 and Bradford S McGwire2

Address: 1Department of Microbiology/Immunology, University of Health Sciences/Chicago Medical School, North Chicago, IL. USA and 2Department of Pathology, The Feinberg School of Medicine, Northwestern University and Section of Infectious Diseases, College of Medicine, University of Illinois-Chicago, Chicago, IL. USA

E-mail: Kwang-Poo Chang* - changk@finchcms.edu; Bradford S McGwire - bmcgwire@uic.edu

*Corresponding author

AbstractA Leishmania model to explain microbial virulence in chronic infectious diseases is proposed. Allthese diseases progress from infection to symptomatic phase to host death or recovery. Theoutcome of each phase is depicted to result from the interactions of a distinct group of parasitemolecules with a specific host immune compartment. The first group consists of invasive/evasivedeterminants, which are largely parasite cell surface and secreted molecules. Their activities helpparasites establish infection by overcoming host immunologic and non-immunologic barriers. Thesedeterminants do not cause disease per se, but are indispensable for infection necessary for thedevelopment of a disease-state. The second group of parasite molecules consists of"pathoantigenic" determinants – unique parasite epitopes present often within otherwise highlyconserved cytoplasmic molecules. Immune response against these determinants is thought to resultin immunopathology manifested as clinical signs or symptoms, namely the virulent phenotype. Thethird group of parasite molecules is hypothetically perceived as vaccine determinants. Theirinteractions with the host immune system lead to the elimination or reduction of parasites to effecta clinical cure. Differential expression of these determinants alone by parasites may alter theirinteractions with the hosts. Virulent phenotype is consequently presented as a spectrum ofmanifestations from asymptomatic infection to fatality. A secondary level of regulation lies in hostgenetic and environmental factors. The model suggests that different parasite determinants may betargeted by different strategies to achieve more effective control of leishmaniasis and other similardiseases.

IntroductionSome Leishmania species are inherently endowed as path-ogens, as they cause diseases collectively known as leish-maniasis. The degree of their pathogenicity, traditionallydefined as "virulence", is manifested as a spectrum of clin-ical symptoms in human diseases [1]. Those causing aself-healing cutaneous leishmaniasis are considered asless virulent than those causing the potentially fatal kala-azar or visceral leishmaniasis. Disease entities with graded

clinical severity exist between these two extremes. Al-though Leishmania virulence may be modulated by envi-ronmental and genetic factors of their mammalian hosts[2], and sand fly vectors [3], molecular determinants ofLeishmania spp. are the key elements. This is considered tobe the case, since there is no leishmaniasis without infec-tion by intact living Leishmania. That is, leishmaniasisdoes not occur, or at least has not been successfully dupli-cated experimentally, by injection or manipulation of an-

Published: 20 May 2002

Kinetoplastid Biology and Disease 2002, 1:1

Received: 21 March 2002Accepted: 20 May 2002

This article is available from: http://www.kinetoplastids.com/content/1/1/1

© 2002 Chang and McGwire; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any purpose, provided this notice is preserved along with the article's original URL.

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imals with anything other than viable Leishmaniaparasites. There is no evidence in the literature that Leish-mania spp. produce "toxins" in the conventional sense todirectly cause the clinical symptoms of leishmaniasis.How Leishmania cause leishmaniasis is thus a complicatedissue, involving apparently multiple factors of Leishmaniaorigin.

Work undertaken in the past two decades to elucidatehost-parasite cellular interactions has made this point ap-parent. It is clear that Leishmania possess infection-relatedmolecules (=invasive/evasive determinants) [1], which al-low them to establish successful intracellular parasitism inphagolysosomes or parasitophorous vacuoles of macro-phages [4]. In human leishmaniasis, these mononuclearphagocytes are invariably seen as the only parasitizedcells. They are thus undoubtedly the principal host cells ofLeishmania, although experimental evidence exists forleishmanial infection of other cell types, e.g. dendriticcells [5] and fibroblasts [6,7]. The association of Leishma-nia with macrophages at the cellular level is characterizedas akin to "symbiosis" [8]. This notion was proposed fromobservations of host-parasite cellular interactions in the L.amazonensis-J774 macrophage in vitro system [9]. In thatcase, the infection produces no acute cytopathology orrapid cytolysis of the host cells. It is essentially a self-re-newable or self-sustainable host-parasite in vitro system.Since human disease does occur with infection of macro-phages in vivo, it is likely that the pathology and clinicalsymptoms may originate from interactions of these infect-ed macrophages with other elements in the host. We pro-pose here that some parasite-specific antigens, derivedfrom the infected cells, interact with the host immune sys-tem in a negative way, directly responsible for the immu-nopathology manifested as the clinical symptoms seen inleishmaniasis. Consistent with this notion are findingsthat kala-azar patients produce large amounts of Leishma-nia-specific, but non-protective antibodies against certainintracellular antigens of these organisms [10].

A hypothetical model is thus proposed accordingly, de-picting that Leishmania virulence results from interactionsof differentLeishmania determinants with separate com-partments of host immune system, namely those for in-fection and those involved in immunopathology. If thehypothetical model proves correct, the two groups of de-terminants may be targeted differently by molecular ge-netic approaches relevant to the control of leishmaniasis.

Invasive/evasive determinants of Leishmania are crucial for infection, but produce no pathology in the hostThese determinants include most, if not all Leishmaniamolecules that have been studied as "virulence factors" inthe literature (see Fig. 1). All these molecules appear toplay certain roles in Leishmania infection of macrophages

[1]. They are referred to here as invasive/evasive determi-nants because they help Leishmania successfully establishintracellular parasitism in the following sequential events:(A) evasion of humoral lytic factors; (B) attachment ofparasites to macrophages followed by their intracellularentry into these phagocytes; (C) the intracellular survivalof the endocytized parasites; (D) promastigote-to-amas-tigote differentiation; and (E) replication of the amastig-otes. The categorization of the host-parasite cellularinteractions into sequential events pertains to the primaryinfection of macrophages in the mammalian hosts by pro-mastigotes. Events (A) – (C) and (E) are relevant as well tothe secondary infection of macrophages by amastigotesfrom already infected cells. The spreading of amastigotesto infect additional cells must be considered as crucial forthe development of leishmaniasis. However, it may bemechanistically a rather simple event in considering nor-mal functions of macrophages. One of their functions isto scavenge damaged or dying cells and their cellular de-bris, which may include degenerating cells (due to heavyparasitization), parasitophorous vacuoles and even re-leased amastigotes with adherent host molecules in leish-maniasis.

Much attention has been thus devoted to the infection ofmacrophages by promastigotes, although the manner, bywhich their molecules (listed in Fig. 1) actually functionin infection remains to be elucidated. Data obtained fromdifferent host-parasite systems are also not always consist-ent even for the molecules more extensively studied, likegp63 and LPG. Gp63 is an ecto-metalloprotease that is es-pecially abundant in the surface of promastigotes and alsoreleased by this stage of Leishmania[11]. Gp63 is known tohelp promastigotes in event (A) by rendering them resist-ant to complement-mediated cytolysis [12]. It also ap-pears to act, (perhaps, together with LPG) in event (B),namely infection of macrophages by promastigotes via re-ceptor-mediated endocytosis [13–16]. Both may be im-

Figure 1Some invasive/evasive determinants of Leishmania proposedto play important roles in their infection of mammalian hosts.

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portant in event (C) for their intraphagolysosomalsurvival [16–18]. Some of the other molecules listed inFig. 1 may be involved directly or indirectly in these and/or the remaining events. Parasite molecules involved inevents (D) and (E) for differentiation and replication ofintracellular Leishmania may have additional functionsbeyond infection, especially in the latter case. Regardlessof the functional and definitional ambiguity associatedwith these determinants, there is no evidence that they di-rectly cause the clinical symptoms seen in leishmaniasis.For example, repeated injections of susceptible animalswith gp63 or LPG do not reproduce the typical symptomsof leishmaniasis, e.g. various types of cutaneous lesions orhepatosplenomegaly and other clinical signs related tokala-azar.

Leishmania pathoantigenic determinants elicit host immu-nopathology as the principal cause of clinical symptomsFig. 2 lists a number of Leishmania antigens found to elicitantibodies often at high titers in kala-azar patients [10].The data presented were summarized from articles pub-lished by others and some are proposed hypothetically.These Leishmania antigens are identified by Western blotanalysis and/or by immunoscreening of Leishmania ex-pression libraries with patients' sera.

Notably, Fig. 2 does not include the invasive/evasive de-terminants discussed in the foregoing paragraph. This isnot to say that patients of leishmaniasis do not produceany antibodies against these determinants. There havebeen a number of publications, which demonstrated thepresence of, for example, anti-LPG and anti-gp63 antibod-ies in these patients. However, the titers against these in-vasive/evasive determinants are insignificant incomparison to those listed in Fig. 2. Perhaps, this is not

unexpected for a successful parasite, like Leishmania. Sincethe invasive/evasive determinants often exist on the cellsurface and/or are secreted, they bear the brunt of expo-sure to and confrontation with the host immune system.Evolutionary pressure may well select these invasive/eva-sive determinants for low antigenicity, thereby escapingfrom neutralization by specific antibodies and/or otherlytic factors to ensure successful parasitism.

Another striking feature of those listed in Fig. 2 is that theyare all conserved structural or soluble cytoplasmic pro-teins, which are often complexed with other molecules toform subcellular particles. Although some of them, e.g.histones and heat shock proteins, are seemingly sharedwith those found in autoimmune diseases, they are notcross-reactive. Epitope-mapping reveals unique Leishma-nia sequences, which are recognized only by sera from pa-tients with kala-azar [10]. One example worthy ofmention is the unique 117 bp repeats in the Leishmania ki-nesin-like gene [19]. It is expressed by the amastigotes ofvisceralizing Leishmania, but not by cutaneous species.Sera from human kala-azar patients contain antibodiesspecific to the 39 amino acid peptides encoded by the 117bp repeats (recombinant products =rK39). Anti-rK39 anti-bodies reach exceedingly high titers as much as 10-6 in In-dian kala-azar patients [20]. Although this antigen hasbeen used successfully for serodiagnosis of active kala-azar cases, it is difficult to assign any protective functionfor the specific antibodies at such high titers producedagainst this and other similar antigens. The antigens ofconcern are in the cytoplasm of intracellular amastigotesand thus beyond the reach of these antibodies. Their po-tential contributions to immunopathology are apparent,e. g. albumin-IgG ratio reversal in the plasma and hyper-plasia of plasma cells in the lymphoid organs in kala-azar.Similarly, some Leishmania-specific T-cell epitopes mayalso exist and cause additional immunopathology, al-though these epitopes have not been extensively studiedin human leishmaniasis. Recent work in the direction ofelucidating protective immunity has identified T-cellepitopes, which exist also in Leishmania cytoplasmic mol-ecules [21].

A hypothetical model for Leishmania virulence involving sequential actions of invasive/evasive- and pathoantigenic determinantsFig. 3 depicts schematically Leishmania invasive/evasive-and pathoantigenic determinants as different groups ofparasite virulence-related molecules. Presumably, they in-teract sequentially with different compartments of thehost immune system to cause leishmaniasis. The invasive/evasive determinants of Leishmania help them overcomethe host immune and non-immune barriers to establishintracellular infection of macrophages. This infection byitself does not cause the disease, but it is a prerequisite for

Figure 2Some Leishmania pathoantigenic determinants proposed tocause immunopathology manifested as the clinical symptomsin leishmaniasis. The molecules listed have been found tocontain immunogenic B-cell epitopes.

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this state. Infection must be maintained in order for thetransition from asymptomatic phase to symptomaticphase, especially when host immunity becomes down-regulated. The latter event alone produces no leishmania-sis without persistence of the infection. The invasive/eva-sive determinants are thus virulence factors in that sense,even though they contribute only indirectly to the virulentphenotype. During the subsequent chronic course of in-fection, it appears that some intracellular amastigotes arekilled or lysed inadvertently due, perhaps, to the incom-plete protection by their invasive/evasive determinants. Asa result of this, some cytoplasmic molecules of amastig-otes are exposed to the host immune system. The resultingimmune response to these unique epitopes does not con-tribute to the anti-Leishmania immunity, but to the clini-cal symptoms observed in leishmaniasis. Thus,Leishmania determinants of infection and immunopa-thology are considered here as different, but sequentiallynecessary components for the expression of virulence.

Regulation of Leishmania virulenceAccording to the hypothetical model, it is possible to en-vision the regulation of Leishmania virulence via differen-tial expression or qualitative differences of the invasive/evasive and pathoantigenic determinants. Virulence ismanifested in a clinical setting as a spectrum of humandiseases with different severity, ranging from asympto-matic infection to the appearance of self-healing nodulesto necrotic, diffuse mucocutaneous lesions to fatal viscer-

alizing disease. Up- or down-regulation of either group ofindividual determinants quantitatively or qualitativelyalone can conceivably result in the spectrum of the clinicaloutcomes seen. The extreme scenarios of asymptomaticinfection and fatal outcome thus may be due to a patternof varying expression of these determinants. In this view,Leishmania determinants are considered as the drivingforce of virulent phenotype. Host and vector determinantsare undoubtedly involved, but they play a secondary orpassive role in natural conditions.

Fig. 4 considers Leishmania virulence at the gene levelbased on hypothetical grounds and on some gene knock-out experimental data in the literature. There appear to bethree functional groups of genes involved: (A) Positivecontributors for the multiple determinants already dis-cussed (Fig. 4., Blue, Green and Yellow squares with "+");(B) negative contributors (pink square with "-") codingfor products, which reduce virulence; and (C) facultativecontributors (Red square blank), which normally do notparticipate in virulence, but do so in the absence of a pos-itive contributor. Normally, the virulent phenotype seenin a given condition represents a balanced presence ofpositive and negative genes with no involvement of thefacultative ones (Fig. 4, 1st row, double-headed horizon-tal arrow). When one of the positive genes is silenced orlost, virulence may decrease, as expected (Fig. 4, 2nd row,downward arrow). Alternatively, it may remain un-changed when the gene of concern is functionally "backed

Figure 3A hypothetical model to explain virulent phenotype in leishmaniasis. The three groups of determinants are thought to interactwith host immune system independently, but may progress sequentially to produce the spectrum of subclinical and clinicalmanifestions as the basis of virulent phenotypes seen.

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up" or replaced with another positive contributor via up-regulation of its expression (Fig. 4, 3rd Row, duplicationof the green squares with "++") or with that of a facultativegene (Fig. 4, 4th row, red square with "+"). This scenariomay explain the experimental findings that knockouts of"virulence genes" do not always result in changes in thephenotype expected. Silencing or loss of the negative geneis accompanied by an increase in virulence (Fig. 4, 6thRow, upward arrow). There is at least one example in theliterature for the presence of negative genes [22].

The hypothesis proposed is speculative, especially for thepresence of the "back-up" genes. Although they have notbeen demonstrated experimentally in Leishmania, func-tionally overlapping genes with very different sequencesare expected to exist, in keeping with the proposed contri-bution of such genes to genetic flexibility in biological sys-tems [23]. If such a strategy should exist in Leishmania andcontribute to the regulation of their virulence, the evolu-tionary pressure of maintaining successful parasitismmight favor this mechanisim for the invasive/evasive de-terminants, but not for the pathoantigenic determinants.

Can we temper with Leishmania virulence genes to deal with leishmaniasis?More genetic and functional data for the various determi-nants discussed are needed to consider this question morethoroughly. On hypothetical grounds, it is possible toconsider that: (A) the invasive/evasive determinants maybe targeted for developing specific inhibitors to preventinfection; and (B) the pathoantigenic determinants maybe suitable targets for considering "molecular attenua-tion" of virulence by gene deletions or modifications,thereby producing infective, but non-pathogenic mutantsfor vaccination. The schemes related to the second pro-posal may be more interesting for further discussion.

It is feasible to begin the discussion with the self-curingsimple cutaneous leishmaniasis. In this case, "host protec-tive determinants" of Leishmania origin are depicted toemerge with the progression of the infection from "dis-ease" to spontaneous "cure" (see Fig. 3). These hypotheti-cal determinants may be the "natural vaccines", whichinteract with the host immune system in a positive way tocure the disease. Although they have not been identifiedand isolated, there are ongoing research efforts towardthis end [21]. On the other hand, "Leishmanization" hasbeen practiced for centuries by inoculating or immunizingindividuals with live Leishmania. Although this process

Figure 4Regulation of Leishmania virulence at genetic levels. Blue, Green and Yellow squares with "+", Positive contributors for the mul-tiple determinants discussed; pink square with "-", negative contributors coding for products, which reduce virulence; Redsquare blank, Facultative contributors, which do not contribute to virulence under the normal conditions.

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leads to development of cutaneous lesions, it provideslife-long protection to re-infection by the same species af-ter cure. It is theoretically possible to improve "Leishman-ization" by several potential schemes. One is togenetically engineer parasites with "suicidal cassettes"[24] for self-destruction as a strategy for vaccination, ashas been already carried out in experimental animals [25].It would be of further interest to improve this by "immu-nizing" susceptible hosts with such transgenic Leishmania,which are responsive to external signals for their destruc-tion [26,27] at different times after inoculation. Perhaps,effective immunity could be activated by such manipula-tion before the manifestation of extensive clinical pathol-ogy. "Leishmanization" thus may become more effectiveor rendered more innocuous. Another possibility is to de-lete or modify the gene(s) encoding pathoantigenic deter-minants. Although this is a more desirable approach, itwill require a substantial investment to identify and selectsuitable targets. This is the case, since these determinantsappear to be encoded apparently by conserved and thusfunctionally important genes. Candidates may includeantigens empirically identified for vaccination againstkala-azar. There may be sufficient incentives to producesuch attenuated mutants in considering the potential ben-efits that they may be used as vehicle for delivery of anti-gens as vaccines for other diseases. This may be consideredin view of the "homing" of Leishmania to macrophages(and possibly dendritic cells). These "vaccines" could betransgenetically expressed by Leishmania for targeted de-livery and accumulation in these cells. Antigen processingand presentation may be rendered more effective toachieve desirable immune response.

DiscussionLeishmania virulence has been discussed often in the con-text of infection-related molecules or invasive/evasive de-terminants, independent of the pathoantigenicdeterminants. A hypothetical model is presented to depictinfection, virulence and cure as sequential, but independ-ent events. The actions of both groups of determinants intandem are proposed here to cause leishmaniasis. This hy-pothetical model is probably applicable to other chronicinfections caused by nontoxigenic pathogens. If so, theproposed differential targeting strategies could havebroader applications. The actual translation of these ideasfor fighting leishmaniasis and other infectious diseasesinto practical use requires additional studies. Howeverpreliminary the schemes may seem, they deserve consid-eration and discussion for the sake of millions who sufferfrom these diseases in the developing world. It is desirableto develop field deployable products for preventing thesediseases, but this has been hampered by the lack of invest-ment by the pharmaceutical industry. Hopefully, this ob-stacle will be rendered easier to overcome under the

coattails of our renewed interests in vaccines in the waragainst bioterrorism.

AcknowledgementsThe authors are supported by the NIH (AI-20486 to KPC) and the Ameri-can Heart Association (Physician-Scientist Postdoctoral Fellowship to BSM).

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