INFECTION AND IMMUNITY, Nov. 2006, p. 6027–6036 Vol. 74, No. 110019-9567/06/$08.00�0 doi:10.1128/IAI.00773-05Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Spontaneous Recovery of Pathogenicity by Leishmania major hsp100�/�

Alters the Immune Response in MiceLinda Reiling,1†‡ Thomas Jacobs,2‡ Manfred Kroemer,1 Iris Gaworski,2

Sebastian Graefe,2 and Joachim Clos1*Leishmaniasis Unit 11 and Department of Medical Microbiology and Immunology,2

Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

Received 23 May 2005/Returned for modification 1 August 2005/Accepted 6 August 2006

By using repeated mouse infection cycles, we obtained an escape variant with restored infectivity andpathogenicity that originated from a single, noninfectious hsp100�/� gene (formerly known as �clpB)replacement clone of Leishmania major, the causative agent of cutaneous leishmaniasis. This isolateelicited increased infiltration of immune cells to the site of infection and altered the polarization of theimmune response in BALB/c mice from a predominantly TH2 type to a TH1 type. A clonal analysis resultedin isolation of two clones with antagonistic properties. While one clone exhibited restored infectivity inisolated macrophages but caused no persistent infection in the mouse model, the second clone was unableto infect macrophages in vitro but could establish a lasting infection and form progressive lesions inBALB/c mice. Our results add to the evidence that the TH1-TH2 dichotomy of the early immune responseagainst L. major not only depends on the genetic predisposition of the host but also depends on intrinsicproperties of the parasite.

The protozoon Leishmania major is the causative agent ofcutaneous leishmaniasis in Middle Eastern countries and AsiaMinor. Due to its robust and well-defined pathogenicity invarious inbred mouse strains, L. major has become a valuablemodel pathogen for studying the TH1-TH2 dichotomy in host-parasite interactions.

Upon transmission of the cultivated insect form, the flagel-lated promastigote, into BALB/c mice, the parasites are takenup by antigen-presenting cells, most likely neutrophilic granu-locytes (9, 10), tissue macrophages (1), and dendritic cells (13,14). After uptake, the parasites survive and proliferate insidephagolysosomes as rounded, nonmotile amastigotes. The con-comitant destruction and reinfection of macrophages and theinflux of immune cells are characteristic of the lesions thatform at the site of infection. While the infection is self-limitingin some inbred mouse strains, such as C57/BL/6, lesions inBALB/c mice are progressive and the infection becomes sys-temic (3). The difference in the course of infection is attributedto the observation that C57/BL/6 mice mount a TH1 type ofimmune response, while BALB/c mice respond with a TH2-driven humoral response that is for the most part not effectiveagainst the intracellular Leishmania parasites (2).

The transmission of Leishmania spp. from phlebotominesand flies to mammals results in a drastic change in the ambi-ent temperature, to which the parasite responds with increasedsynthesis and levels of various heat shock proteins. Among theheat shock proteins of Leishmania, the 100-kDa heat shock

protein, HSP100, which is encoded by a single-copy gene,displays particularly strong up-regulation, and the proteinbecomes abundant in the amastigote stage (5, 7). In agree-ment with this expression pattern, HSP100 is largely dis-pensable during the promastigote stage, but it is criticalfor intracellular parasite survival; hsp100�/� gene (formerlyknown as �clpB) replacement mutants of L. major fail toproliferate in macrophages and are attenuated in BALB/cmice (5). Additional experiments with hsp100�/� mutants ofLeishmania donovani showed that HSP100 has a crucialfunction in the expression of amastigote-specific genes andstabilizes the amastigote stage (8).

Here we describe the appearance of a spontaneous escapevariant of an L. major hsp100�/� mutant with a nearly wild-type rate of lesion formation but a reduced parasite load in thelymphatic tissue and altered immunological properties. Ourresults show that the selective pressure encountered by atten-uated parasite strains in mammalian hosts selects for sponta-neous emergence of parasite clones with restored infectivityand/or pathogenicity. Note that the gene encoding HSP100 isalso known as clpB; here we use the more systematic genename, HSP100, to comply with kinetoplastid gene-naming con-ventions.

MATERIALS AND METHODS

Parasite culture. Promastigote stages of an L. major wild-type strain (MHOM/SU/73/5ASKH), an L. major hsp100�/� mutant, and escape strains were grownat 25°C in M199 medium supplemented with 20% heat-inactivated fetal calfserum (FCS), 20 �g/ml gentamicin, 0.2% NaHCO3, and 2 mM L-glutamine. Priorto infection experiments the parasites were grown in modified (rich) M199medium supplemented with 20% heat-inactivated FCS, 40 mM HEPES (pH 7.4),100 �M adenine, 10 �g/ml heme, 1.2 �g/ml biopterin, 0.2% NaHCO3, 20 �g/mlgentamicin, and 2 mM L-glutamine. Cells were counted using a Casy cell counter(Schaerfe System).

In vitro infection of macrophages. Peritoneal exudate cells were isolated fromNMRI mice and seeded in chamber slides at a density of 106 cells/ml in supple-mented M199 medium (see above). After allowing adhesion for 1 h at 37°C, we

* Corresponding author. Mailing address: Bernhard Nocht Institutefor Tropical Medicine, Bernhard Nocht St. 74, D-20359 Hamburg,Germany. Phone: 49-40-42818 481. Fax: 49-40-42818 512. E-mail: clos@bni-hamburg.de.

† Present address: The Walter and Eliza Hall Institute, Infectionand Immunity Division, 1G Royal Parade, Parkville, Victoria 3050,Australia.

‡ L.R. and T.J. contributed equally to this paper.

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added stationary-phase promastigote parasites at a ratio (multiplicity of infec-tion) of 1:1, and the cells were incubated for 4 h at 35°C in the presence of 5%CO2. The supernatant was then removed, and the attached macrophages werewashed once with prewarmed medium. After addition of fresh medium, the cellswere incubated for 24 or 48 h at 35°C in the presence of 5% CO2. For micro-scopic analysis, the cells were fixed for 2 min with ice-cold methanol and stainedwith Giemsa stain.

Mouse infection experiments. Stationary-phase promastigotes were washedtwice in cold phosphate-buffered saline (PBS) (10 min, 4°C, 1,000 � g) and werethen resuspended at a density of 8 � 107 promastigotes/ml in PBS. Twenty-fivemicroliters (2 � 106 promastigotes) was inoculated into the hind footpads of 6-to 8-week-old female BALB/c mice obtained from Charles River Inc. The courseof infection was monitored weekly by measuring foot swelling, using an ODITESTcaliper (Kroeplin, Schluechtern, Germany).

Air pouch experiments. Experiments were performed as described by Laufs etal. (10). Briefly, 2 ml of air was injected subcutaneously into the dorsum offemale BALB/c mice to form air pouches, and 1 � 107 stationary-phase promas-tigotes of the different strains were inoculated into the air pouches. Twenty-fourhours later the mice were sacrificed, and the infiltrate was isolated by flushing theair pouches several times with ice-cold, sterile PBS. Cells were concentrated bycentrifugation (690 � g, 4°C, 10 min) and used for fluorescence-activated cellsorting analyses.

Histological analyses of infected tissues. Whole lymph nodes were fixed in 2%paraformaldehyde overnight at 4°C prior to embedding in paraffin. Four-micrometer sections were cut, and the paraffin was removed prior to incubationwith 1% ammonium chloride (30 min) and 0.1 M glycine (30 min). The parasiteswere marked by staining the Leishmania HSP90 with polyclonal chicken anti-HSP90 antibody (4) (diluted 1:500 in 2% bovine serum albumin) and rabbitanti-chicken immunoglobulin G (Jackson Immunolab). After washing with PBS,the Leishmania HSP90 was visualized with a Super Sensitive kit (Biogenex) byfollowing the manufacturer’s instructions. Tissue cells were counterstained withhematoxylin.

Quantitative PCR. For quantification of parasites by real-time PCR, DNA wasextracted using a PureGene DNA kit (Gentra Systems, Minneapolis, MN). Inbrief, whole mouse lymph nodes were ground up in lysis buffer containing 300�g/ml proteinase K and incubated at 55°C for 120 min. Protein was removed andDNA was precipitated by following the manufacturer’s instructions. The result-ing DNA pellets were resuspended in 200 �l Tris-EDTA buffer and diluted 1:10prior to analysis. The concentration of parasites was expressed as the ratio of L.major DNA to mouse �-actin DNA. Mouse �-actin DNA was quantified by5�-nuclease PCR. The Leishmania DNA concentrations in the same sampleswere determined using fluorescence resonance energy transfer real-time PCRwith leishmanial 18S ribosomal DNA sequences. The resulting Leishmania DNAcopy number was then divided by the copy number of �-actin DNA to obtain anormalized concentration ratio for the number of parasites per unit of tissue.

Infection of bone marrow-derived dendritic cells. The bone marrow of thefemur and tibia of BALB/c mice was isolated by injecting RPMI medium with asyringe. Cells were washed and counted. A total of 2 � 106 cells were cultivatedin 10 ml RPMI medium containing 10% FCS and granulocyte-macrophagecolony-stimulating factor (GM-CSF) (20 ng/ml; Biocarta, Hamburg, Germany)in a petri dish. On day 3, 10 ml of GM-CSF-containing medium was added. Onday 6, 10 ml of medium was removed and replaced by fresh GM-CSF-containingmedium. On day 7, the differentiated cells were washed. Then 1 � 106 cells perwell were seeded into a 24-well plate and infected with stationary-phase promas-tigote parasites at a ratio of 1:1. After 48 h the supernatant was harvested andanalyzed to determine the presence of interleukin-12p40 (IL-12p40) by an en-zyme-linked immunosorbent assay (ELISA) performed according to the manu-facturer’s instructions (Becton Dickinson, Heidelberg, Germany).

Analysis of cytokine production. Draining lymph nodes were removed, andsingle-cell suspensions were seeded in 96-well plates at a concentration of 1 �105 cells per well, using RPMI medium supplemented with 10% heat-inactivatedfetal-calf serum. Cells were stimulated either with 3 �g/ml anti-CD3 or with L.major lysate. After 48 h, supernatants were removed and frozen at �20°C.Production of gamma interferon (IFN-�) and IL-4 was analyzed by a specifictwo-sided ELISA using supernatants of stimulated lymph node cells. Antibodypairs and cytokine standards were purchased from Becton Dickinson (Heidel-berg, Germany).

Western blotting. Sodium dodecyl sulfate (SDS)-polyacrylamide electrophore-sis, semidry blotting, and immune detection with anti-HSP100 immunoglobulinY were performed by using established protocols (4, 5, 7).

Statistical analysis. Sets of data were analyzed using Prism 4 for Macintosh(GraphPad Software Inc.), version 4.0a. Statistical analyses were performedusing the built-in Student’s t test or the U test (12).

RESULTS

Parasites of L. major hsp100�/� strain 1 caused lesions inBALB/c mice with a pronounced delay of 3 to 20 weeks com-pared with the lesions caused by wild-type parasites (5). Duringroutine passage using infection of BALB/c mice with onemutant clone, we observed rapid lesion development in onemouse that was roughly comparable to the lesion growthcaused by wild-type L. major (data not shown). Parasites werereisolated from the footpad lesion of this mouse and grown invitro as promastigotes. The isolates exhibited markedly de-layed outgrowth from the host tissue. After we confirmed byimmunoblot analysis (see Fig. 5E) that the isolated parasites infact did not express HSP100, we compared the isolated popu-lation with the L. major wild type and another hsp100�/�

strain, strain 2.2 (5). A total of 2 � 106 promastigotes of thesestrains were inoculated into the footpads of BALB/c mice, andfoot swelling was monitored at weekly intervals. As shown inFig. 1A, the escape population was comparable to the wild-type L. major population, causing swelling after a 2 weeks ofincubation, albeit at a lower rate. By contrast, hsp100�/� con-trol strain 2.2 caused no lesion formation during the observa-tion period and for up to 25 weeks postinfection.

To further assess the pathogenic effects of the strains, thedraining lymph nodes were removed from the infected miceafter 7 weeks and weighed (Fig. 1B). The lymph node mass inmice infected with the escape population was slightly, but sig-nificantly (P 0.028, as determined by the U test), larger thanthe lymph node mass in mice infected with the wild type.Complete DNA was extracted from the lymph node tissues. Byusing quantitative PCR, the relative parasite load was deter-mined for each animal. As shown in Fig. 1C, the relativeparasite loads due to the L. major wild type were larger thanthe relative parasite loads due to the escape population (P 0.028, as determined by the U test). The mice infected withhsp100�/� clone 2.2 of L. major contained no detectable par-asite DNA. We concluded that the escape isolate caused ex-acerbated lymph node swelling, while the actual parasite loadwas reduced compared with that of the wild type.

A histological analysis of draining lymph nodes from animalsinfected with wild-type L. major, the L. major hsp100�/� clone,and the escape variant further highlighted the differences inthe course of infection. Compared with the L. major wild typeand the hsp100�/� parent mutant (Fig. 2A and C), theescape variant caused exacerbated infiltration of granulo-cytes into the lymph nodes and a reduction in the amount ofconnective tissue lining the lymph nodes (Fig. 2E). How-ever, with the escape isolate there was an intermediate par-asite load (Fig. 2F) compared with the parasite loads of thewild type (Fig. 2B) and the L. major hsp100�/� clone (Fig.2D). This finding confirms the data obtained by real-timePCR.

Interestingly, histological analysis of footpad lesion tissuedid not reveal overt differences between the wild type and theescape isolate. Skin tissue from the perimeter of footpad le-sions was embedded in paraffin and stained as described above.The lesion tissues of mice infected with L. major and the L.major hsp100�/� escape isolate were indistinguishable in termsof their overall appearance (Fig. 3A and D). Closer inspectionof intact (Fig. 3B and E) or degraded (Fig. 3C and F) tissue

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also did not reveal overt differences between wild-type strain-infected and escape isolate-infected lesions. Footpad tissue frommice infected with the hsp100�/� clone could not be analyzed dueto the lack of lesions. We concluded that parasite density andparasite-induced pathogenicity did not differ at the lesion site butparasite dissemination into the lymphoid tissue was suppressedwith the escape variant.

The increased lymph node swelling and the effects on lymphnode histology suggested that the escape variant may elicit analtered immune response compared with the immune responseelicited by wild-type L. major or the hsp100�/� parent mutant.Since it is well established that the early IL-4 production byBALB/c mice is responsible for their susceptibility by inducinga TH2 type of immune response, we analyzed the productionof two cytokines, IFN-� and IL-4, in culture supernatants fromlymph node cells isolated 9 days postinfection. Upon stimula-tion with anti-CD3 or L. major lysate, cells from mice infected

with wild-type L. major or the hsp100�/� mutant producedlarge amounts of IL-4, indicating that both strains induced thetypical TH2-like cytokine profile upon infection (Fig. 4). Incontrast, infection with the escape isolate induced higher ex-pression of IFN-� and lower production of IL-4, indicating thatthe mice developed a TH1 type of immune response similar tothat of resistant C57BL/6 mice.

Upon reisolation of the escape variant, there was a strikingmorphological difference between the initial population of pro-mastigotes and wild-type L. major. The flagellum was, at firstapproximation, twice as long as that observed for wild-typecontrols. The parent hsp100�/� clones did not have this char-acteristic (5). This impression was confirmed by measurementof the flagellar length for 110 individual cells belonging to eachgroup (Fig. 1D). The morphological difference was highly sig-nificant (P 0.0001, as determined by the U test), but itvanished with prolonged in vitro passage along with the slow in

FIG. 1. (A) Lesion formation in BALB/c mice. A total of 2 � 106 stationary-phase promastigotes of the L. major wild type, the L. majorhsp100�/� mutant, or the escape isolate were inoculated subcutaneously into the footpads of BALB/c mice. Footpad swelling was monitored atweekly intervals. The values are the means for four animals per parasite strain. The error bars indicate standard deviations. wt, wild type; clp–,hsp100�/�; p.i., postinfection. (B) Comparison of lymph node masses after infection with the L. major wild type or the escape strain. The draininglymph nodes of infected BALB/c mice (four mice per strain) were isolated and weighed. The horizontal lines indicate the medians for four lymphnodes. The experiment was performed in quadruplicate. The asterisk indicates that the P value is 0.05, as determined by the U test. (C) Parasiteloads of draining lymph nodes after infection with the L. major wild type or the escape strain. After isolation of the draining lymph nodes frominfected BALB/c mice (four mice per strain), total DNA was prepared and subjected to real-time PCR. Leishmania DNA was quantified relativeto the mouse gene for �-actin. The horizontal lines indicate the medians for four independent lymph nodes. The experiment was performed inquadruplicate. The asterisk indicates that the P value is 0.05, as determined by the U test. (D) Analysis of flagellar length. The flagella of 110parasites of the L. major wild type, the freshly isolated L. major hsp100�/� mutant, and the escape variant were measured. The median values forthe strains are indicated by horizontal lines. Three asterisks indicate that the P value is 0.001, as determined by the U test.

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vitro growth phenotype (not shown). This finding led us tosuspect that the escape isolate is a polyclonal population, withindividual clones under antagonistic selection in vitro and inthe animal host. To test this hypothesis and to increase thestability of the escape phenotype, we attempted to raiseindividual clones from a freshly reisolated escape popula-tion. Promastigotes were diluted to obtain a concentrationof 0.5 cell/100 �l and distributed into microtiter plate wells.Clones that showed early outgrowth were discarded as theywould have been dominant in the in vitro grown population.

Three clones, however, displayed markedly delayed out-growth and slow proliferation kinetics compared with wild-type L. major and were thus compatible with our hypothesis.One of these clones could not be maintained in liquid cul-ture and was lost.

A comparison of the morphologies of the wild type and theremaining escape clones, esc1 and esc2, revealed that onlyclone esc2 had the elongated flagellum characteristic of theoriginal escape isolate (Fig. 5A). Clone esc1, if anything, had aslightly reduced flagellum length compared with that of wild-

FIG. 2. Histological analysis of lymph nodes from mice infected with the L. major wild type (A and B), the L. major hsp100�/� mutant (C andD), or the L. major hsp100�/� escape isolate (E and F). The draining lymph nodes were isolated from infected mice and embedded in paraffin.Ultrathin sections were stained with anti-HSP90 antibody and Fast Red stain to highlight the leishmaniae and were counterstained withhematoxylin. (A, C, and E) Magnification, �10. (B, D, and F) Magnification, �63. The arrow in panel D indicates a single L. major hsp100�/�

mutant amastigote.

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FIG. 3. Histological analysis of lesion tissue from mice infected with the L. major wild type (A to C) or the L. major hsp100�/� escape isolate(D to F). Tissue was isolated from the perimeter of lesions and embedded in paraffin. Ultrathin sections were stained with anti-HSP90 antibodyand Fast Red stain to highlight the leishmaniae and were counterstained with hematoxylin. (A and D) Magnification, �10. (B, C, E, and F)Magnification, �63. The arrows indicate the origins of the magnified areas.

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type L. major. These data suggest that clone esc2 was respon-sible for the observed restoration of virulence.

The two remaining clones, along with wild-type L. major,were used to inoculate two BALB/c mice each. Foot swellingwas monitored weekly (Fig. 5B). During the observation time(11 weeks), only wild-type L. major and escape clone esc2caused notable footpad swelling. All mice were sacrificed after11 weeks, and footpad tissue and lymph nodes were placed inliquid cultures to allow parasite outgrowth. As expected, wild-type L. major and escape clone esc2 parasites could be grownfrom host tissue. The esc1 clone, however, could not be recov-ered from the tissues. Parasite loads in infected lymph nodeswere determined by real-time PCR. As expected, the esc2clone exhibited reduced parasite density compared with thewild type (Fig. 5C). No parasite DNA was detected in esc1-infected mouse tissue (not shown).

We also compared the abilities of the two escape clones toinfect peritoneal exudate cells with the abilities of wild-type L.major and the hsp100�/� mutant to do this. Surprisingly, weobserved restored infectivity in vitro with the esc1 clone (Fig.5D). By contrast, the esc2 clone showed no improved infectiv-ity compared with the background value for the hsp100�/�

mutant. We concluded that the restored pathogenicity of theesc2 clone was not due to restored infectivity for macrophages.On the other hand, clone esc1 was not able to cause a persis-

tent infection in mice in spite of its ability to become estab-lished in macrophages in vitro.

To exclude the possibility that there was contamination ofthe escape variant cultures with wild-type L. major, we per-formed a Western blot analysis with the escape isolate andclones esc1 and esc2 (Fig. 5E). Neither of the latter parasitesexpressed HSP100 after exposure to 35°C.

It is known that dendritic cells play a central role in initiatinga specific immune response by presenting antigen to T cells.Furthermore, they produce cytokines that influence the polar-ization of T cells in either TH1 or TH2. One of the most potentTH1 inducers produced by dendritic cells is IL-12. Therefore,we analyzed the abilities of the two escape clones to induceIL-12 after infection of bone marrow-derived dendritic cells invitro (Fig. 6). Whereas wild-type L. major and the hsp100�/�

mutant induced comparable levels of IL-12 expression, thevirulent esc2 clone induced significantly lower levels of IL-12production. By contrast, the esc1 clone, which was not able toelicit lesion development, induced significantly increased ex-pression of IL-12 compared with the expression induced bywild-type parasites.

We also tested the IFN-� production in mice infected withthe two escape clones 9 days postinfection. Supernatants oflymph node cells from mice infected with L. major, the L.major hsp100�/� mutant, and clones esc1 and esc2 were ana-lyzed by ELISA after stimulation with medium, anti-CD3 an-tibody, or L. major lysate (Fig. 6B). We observed a strongincrease in inducible IFN-� production by the cells isolatedafter infection with esc2, indicating that the observed inductionof a TH1-type immune response in mice infected with theescape isolate was indeed due to the esc2 subpopulation. Weconcluded that the restored virulence observed with the escapeisolate and the altered immune response were both caused bya single clone, esc2.

Previous work suggested that the immediate early phase ofinfection determines the dichotomy of the pathogen-specificT-cell response and thus drives the response in either the TH1direction or the TH2 direction. To determine the early, innateimmune response to infection with wild-type or escape isolateparasites, we performed an air pouch infection experiment(10). A total of 1 � 107 promastigotes were inoculated into asubcutaneous air pouch. Twenty-four hours postinfection, thecellular infiltrate was isolated by flushing the air pouch withDulbecco’s PBS. Parasites or parasite-containing cells couldnot be detected in the exudate after Giemsa staining and mi-croscopy (data not shown). The exudate was analyzed by flowcytometry, using antibodies against immune cell surface mark-ers (Fig. 7). As determined previously, polymorphonuclearleukocytes (PMN) appeared to be a major constituent of thecellular infiltrate. However, a variety of other cell types werefound in the air pouch that could be clearly differentiated fromPMN using a combination of morphological criteria and stain-ing with anti-Gr-1, as analyzed using flow cytometry. Interest-ingly, infection with the esc2 strain led to increased infiltrationof PMN and also CD11b� macrophages. In addition, thisstrain induced infiltration of CD11c� dendritic cells. By con-trast, esc1 induced significantly less infiltration of cells thanesc2 or wild-type Leishmania induced. To exclude the possi-bility that the cellular infiltrate in the air pouch was due to aninjury during infection, we also stained CD19� B cells since

FIG. 4. Cytokine production by lymph node cells after infectionwith different L. major strains. Mice were infected with 1 � 106 pro-mastigotes of the L. major strains indicated. On day 9 postinfectionlymph node cells were isolated and stimulated with anti-CD3 or leish-mania antigen. After 48 h supernatant was collected and analyzed foreither IFN-� production (A) or IL-4 production (B). The data are theresults of one experiment in which there were three mice per groupand are means and standard deviations. The experiment was repeated,and similar results were obtained. An asterisk indicates that the Pvalue is 0.05, as determined by Student’s t test. wt, wild type; �clp, L.major hsp100�/� mutant; esc, escape clone.

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FIG. 5. (A) Promastigotes of the L. major wild type and L. major hsp100�/� escape clones esc1 and esc2 were fixed on microscope slides andstained with Giemsa stain. Note the elongated flagellum of esc2 promastigotes. (B) Infection experiment. Two 8-week-old BALB/c mice wereinoculated with the L. major wild type (squares), L. major hsp100�/� clone esc1 (circles), and L. major hsp100�/� clone esc2 (diamonds). Footpadswelling was monitored weekly. (C) Relative parasite loads in draining lymph nodes of mice infected with wild-type and esc2 parasites weredetermined by real-time PCR (see Fig. 1). The horizontal lines indicate the medians for two mice. (D) In vitro infection. Mouse peritoneal exudatecells were infected with equal numbers of promastigotes of the L. major wild type, the L. major hsp100�/� mutant, and clones esc1 and esc2. After48 h, cells were fixed, stained with Giemsa stain, and analyzed microscopically. The number of infected macrophage cells was counted. Thehorizontal lines indicate the medians of four independent experiments. (E) Western blot analysis of wild-type L. major (lane 1), the hsp100�/�

mutant (lane 2), the escape isolate (lane 3), and clones esc1 (lane 4) and esc2 (lane 5). A total of 1 � 107 promastigotes, after incubation for 24 hat 35°C, were lysed in SDS sample buffer and loaded on a 7% SDS–polyacrylamide gel. After Western blotting, the membrane was developed withanti-Hsp100 antibody (4). The lower panel shows an identical SDS-polyacrylamide gel after staining with Coomassie brilliant blue. wt wild type;p.i., postinfection.

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they are found at high frequencies in the blood. However, Bcells appeared to be absent, suggesting that cells traveled spe-cifically to the air pouch in response to infection with theparasite.

DISCUSSION

During routine passage in susceptible BALB/c mice, apathogenic variant of the attenuated L. major hsp100�/� mu-tant (5) was isolated. This isolate’s pathogenicity was unstableduring in vitro cultivation, as were its peculiar morphology andits reduced in vitro growth rate.

The exact function of Leishmania HSP100 is not known.Orthologous proteins in yeast (Saccharomyces cerevisiae) andEscherichia coli are known to be involved in inducible thermo-tolerance and general stress tolerance. This effect is less pro-nounced in Leishmania spp., as the hsp100�/� mutants showonly limited effects on thermotolerance (5, 8). HSP100 familymembers in several model organisms have been shown to formhomohexameric rings, not unlike members of the closely re-lated family of Clp protease regulatory subunits, ClpA andClpX. The major function of the hexameric HSP100 or ClpBproteins appears to be disassembly of protein aggregates, aidedby the HSP70/HSP40 chaperone complexes (16). Whether thisfunction is conserved in Leishmania HSP100 is not known yet.In contrast to the other members of the family, LeishmaniaHSP100 appears to be associated in homotrimers rather thanhexamers (7). This abrogates the formation of a ring-shapedcomplex. Recent evidence argues in favor of the hypothesisthat the central cavity in the hexameric ring of ClpB performsthe deaggregation reactions. We therefore have to take intoaccount the possibility that HSP100 in Leishmania has a dif-ferent function. For instance, in yeast, HSP104 has a clusteredappearance throughout the cytoplasm (6). In L. donovani,HSP100 is also clustered; however, the majority of the mole-cules localize close to the cytoplasmic membrane (7).

In yeast, the loss of HSP104 and the concomitant loss of in-ducible thermotolerance can be compensated for, in part, byoverexpression of the major heat shock protein, HSP70 (11, 15).In the escape variants of the L. major hsp100�/� mutant, how-ever, we found no reproducible evidence of HSP70 overexpres-sion (Kroemer, unpublished). Also, we performed a functionalcomplementation screen using an L. major hsp100�/� genomicDNA cosmid library transfected into the L. major hsp100�/�

mutant and subsequent selection in BALB/c mice. No heat shockgenes were found on the recovered cosmids (Reiling, unpub-lished). Evidently, intracellular survival of L. major requires ahighly specialized function of HSP100.

This spontaneous recovery of virulence in an attenuatedmutant of L. major is not unexpected. Spath et al. reportedspontaneous recovery of virulence in an lpg2 mutant of L.major (17). This mutant does not synthesize a major surfacemolecule, lipophosphoglycan. For L. major, loss of LPG abro-gates virulence in the mouse model. A partially revertant pop-ulation of the lpg2 mutants was identified that exhibited de-layed lesion formation and was able to survive and proliferatewithin macrophages. No mechanism for this recovery of viru-lence has been established yet. However, it is interesting thatfull reversion to wild-type virulence did not occur. In this re-gard, this revertant phenotype is comparable to the hsp100�/�

escape phenotype. The major difference is that restoration ofpathogenicity in hsp100�/� clone esc2 is accompanied by an al-tered immune reaction of the host organism.

We have compared the escape isolate with wild-type L. ma-jor and the hsp100�/� mutant, using comparative proteomicsand comparative immunoblot analysis. To date, no reproduc-ible qualitative or quantitative differences have been observed,except for an increase in paraflagellar rod proteins in theescape isolate, which can be explained by the exceedingly longflagellum observed (data not shown).

When BALB/c mice were infected with the escape strain, weobserved lesion development that was comparable to that of

FIG. 6. (A) IL-12p40 production by dendritic cells after infectionwith L. major. A total of 1 � 105 bone marrow-derived dendritic cellswere infected with different numbers of L. major parasites, as indi-cated. After 48 h the supernatant was collected and analyzed forIL-12p40 production by ELISA. The values are means � standarddeviations. The experiment was repeated twice, and similar resultswere obtained. (B) Cytokine production by lymph node cells afterinfection with different L. major mutants and clones. Mice were in-fected with 1 � 106 promastigotes of the L. major isolates and clonesindicated. On day 9 postinfection lymph node cells were isolated andstimulated with anti-CD3 or leishmania antigen. After 48 h superna-tant was collected and analyzed for IFN-� production. The values wereobtained from one experiment in which there were three mice pergroup and are means and standard deviations. The experiment wasrepeated, and equivalent results were obtained. wt, wild type; MOI,multiplicity of infection.

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the L. major wild-type strain. The number of intracellular par-asites seen in histological sections from the perimeter of thelesions was indistinguishable from the number in lesions in-duced by the L. major wild type. However, at 7 weeks postin-fection, we observed a reduced parasite load in the draininglymph nodes, indicating that the escape population showedreduced dissemination. Moreover, the escape variant exhibitedan altered immune response. In contrast to wild-type L. major,the escape isolate induced rapid induction of the TH1 cytokineIFN-�, which is associated with the protective immune re-sponse in resistant C57BL/6 mice. Furthermore, we found thatin the very early phase of the infection, the escape isolateattracted a higher number of phagocytic cells than the wild-type parasites and the parent hsp100�/� mutant attracted. Thissuggests that the bias of the subsequent immune response isdependent on the quality or quantity of the infiltrating antigen-presenting cells. Interestingly, the hsp100�/� mutant, which iscompletely avirulent and unable to persist, does not triggerinfiltration of inflammatory cells, indicating that a minimallevel of pathogen-derived “danger signals” must be exceededto induce inflammation. After subcloning of the escape isolate,we established two stable clones which exhibited very differentphenotypes with regard to both morphology and the immuneresponse upon infection. Our results indicate that an escapevariant of the L. major hsp100�/� mutant, clone esc2, persistsin the face of a TH1 type of immune response that has been

found to induce immunity when wild-type L. major is used.Although this mutant shows poor infectivity in vitro usingisolated macrophages, it is able to survive and proliferate inmice. It is interesting that esc2 induces less IL-12 upon infec-tion of bone marrow-derived dendritic cells in vitro than otherstrains induce, even though it was found to be the cause ofelevated IFN-� production at 9 days postinfection in BALB/cmice. One explanation for the persistence of esc2 despite itspoor infectivity in vitro may be recruitment of safe host cells.Indeed, this strain induces the highest number of infiltratingLy1� neutrophilic granulocytes, which were previously shownto be a safe vehicle for dissemination. Efforts will be made toanalyze whether this increased recruitment of granulocytes isresponsible for the persistence of esc2. On the other hand, wemust consider the fact that in vitro infection of peritonealmacrophages with promastigotes is highly artificial and reflectsonly primary infection and amastigote differentiation, whilemacrophages in a lesion are infected by fully differentiatedamastigotes.

Clone esc1, by contrast, can survive in the mouse only inassociation with clone esc2. A single infection with clone esc1does not lead to a patent infection, and the parasite is rapidlycleared from different tissues, as judged by real-time PCR.However, esc1 has restored ability to infect macrophages withwild-type-like efficiency. The lack of pathogenicity in mice sug-gests that the restored infectivity may be offset by the increased

FIG. 7. Analysis of the cellular infiltrate in air pouches after L. major infection. A total of 1 � 106 parasites were inoculated into each air pouchcavity. The cellular infiltrate was isolated 24 h postinfection and was subsequently stained with the antibodies indicated and analyzed by flowcytometry. The results of a representative analysis of three independent experiments in which there were two mice per group are shown.

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activation of IL-12 production very early in infection and theresulting cell-mediated immune reaction.

The genetic variation discussed above occurred within amaximum of four mouse infection cycles, starting with a singleL. major hsp100�/� clone. This finding underscores the under-lying genetic flexibility of Leishmania species. Different im-mune responses were induced by different clones derived froma single original L. major clone. These data imply that not onlythe genetic background of the host, which was studied in detailusing BALB/c or C57BL/6 mice as examples of a polarizedhost, but also the highly dynamic expression of parasite pro-teins that are triggered by environmental factors influence thedeveloping immune response. The observed clonal variabilityof L. major parasites and their different abilities to influencethe host immune system might explain why parasites are ableto adapt very quickly to different immunological niches. Ourfindings also bring into question efforts to generate safe atten-uated vaccines using targeted replacement of single genes re-quired for parasite pathogenicity.

ACKNOWLEDGMENTS

We thank Sabine Becker and Andrea Macdonald for technical as-sistance.

This work was supported in part by DFG grant Cl120/5.1. L.R. wasa fellow of the Fonds der Chemischen Industrie.

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Editor: J. F. Urban, Jr.

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