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Infection and Immunity, November 2006, p. 6027-6036, Vol. 74, No. 11
0019-9567/06/$08.00+0     doi:10.1128/IAI.00773-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Spontaneous Recovery of Pathogenicity by Leishmania major hsp100^–/– Alters the Immune Response in Mice

Linda Reiling ,^1,, Thomas Jacobs,^2, Manfred Kroemer,¹ Iris Gaworski,² Sebastian Graefe,² and Joachim Clos^1*

Leishmaniasis Unit 1,¹ Department of Medical Microbiology and Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany²

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

Top Abstract Introduction Materials and Methods Results Discussion References

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
The protozoon Leishmania major is the causative agent of cutaneous leishmaniasis in Middle Eastern countries and Asia Minor. Due to its robust and well-defined pathogenicity in various inbred mouse strains, L. major has become a valuable model pathogen for studying the TH1-TH2 dichotomy in host-parasite interactions.

Upon transmission of the cultivated insect form, the flagellated promastigote, into BALB/c mice, the parasites are taken up by antigen-presenting cells, most likely neutrophilic granulocytes (9, 10), tissue macrophages (1), and dendritic cells (13, 14). After uptake, the parasites survive and proliferate inside phagolysosomes as rounded, nonmotile amastigotes. The concomitant destruction and reinfection of macrophages and the influx of immune cells are characteristic of the lesions that form at the site of infection. While the infection is self-limiting in some inbred mouse strains, such as C57/BL/6, lesions in BALB/c mice are progressive and the infection becomes systemic (3). The difference in the course of infection is attributed to the observation that C57/BL/6 mice mount a TH1 type of immune response, while BALB/c mice respond with a TH2-driven humoral response that is for the most part not effective against the intracellular Leishmania parasites (2).

The transmission of Leishmania spp. from phlebotomine sand flies to mammals results in a drastic change in the ambient temperature, to which the parasite responds with increased synthesis and levels of various heat shock proteins. Among the heat 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 protein becomes abundant in the amastigote stage (5, 7). In agreement with this expression pattern, HSP100 is largely dispensable during the promastigote stage, but it is critical for intracellular parasite survival; hsp100^–/– gene (formerly known as clpB) replacement mutants of L. major fail to proliferate in macrophages and are attenuated in BALB/c mice (5). Additional experiments with hsp100^–/– mutants of Leishmania donovani showed that HSP100 has a crucial function in the expression of amastigote-specific genes and stabilizes the amastigote stage (8).

Here we describe the appearance of a spontaneous escape variant of an L. major hsp100^–/– mutant with a nearly wild-type rate of lesion formation but a reduced parasite load in the lymphatic tissue and altered immunological properties. Our results show that the selective pressure encountered by attenuated parasite strains in mammalian hosts selects for spontaneous emergence of parasite clones with restored infectivity and/or pathogenicity. Note that the gene encoding HSP100 is also known as clpB; here we use the more systematic gene name, HSP100, to comply with kinetoplastid gene-naming conventions.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Parasite culture. Promastigote stages of an L. major wild-type strain (MHOM/SU/73/5ASKH), an L. major hsp100^–/– mutant, and escape strains were grown at 25°C in M199 medium supplemented with 20% heat-inactivated fetal calf serum (FCS), 20 µg/ml gentamicin, 0.2% NaHCO₃, and 2 mM L-glutamine. Prior to infection experiments the parasites were grown in modified (rich) M199 medium supplemented with 20% heat-inactivated FCS, 40 mM HEPES (pH 7.4), 100 µM adenine, 10 µg/ml heme, 1.2 µg/ml biopterin, 0.2% NaHCO₃, 20 µg/ml gentamicin, 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 from NMRI mice and seeded in chamber slides at a density of 10⁶ cells/ml in supplemented M199 medium (see above). After allowing adhesion for 1 h at 37°C, we added stationary-phase promastigote parasites at a ratio (multiplicity of infection) 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 were washed once with prewarmed medium. After addition of fresh medium, the cells were incubated for 24 or 48 h at 35°C in the presence of 5% CO₂. For microscopic analysis, the cells were fixed for 2 min with ice-cold methanol and stained with Giemsa stain.

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

Air pouch experiments. Experiments were performed as described by Laufs et al. (10). Briefly, 2 ml of air was injected subcutaneously into the dorsum of female BALB/c mice to form air pouches, and 1 x 10⁷ stationary-phase promastigotes of the different strains were inoculated into the air pouches. Twenty-four hours later the mice were sacrificed, and the infiltrate was isolated by flushing the air pouches several times with ice-cold, sterile PBS. Cells were concentrated by centrifugation (690 x g, 4°C, 10 min) and used for fluorescence-activated cell sorting 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 incubation with 1% ammonium chloride (30 min) and 0.1 M glycine (30 min). The parasites were marked by staining the Leishmania HSP90 with polyclonal chicken anti-HSP90 antibody (4) (diluted 1:500 in 2% bovine serum albumin) and rabbit anti-chicken immunoglobulin G (Jackson Immunolab). After washing with PBS, the Leishmania HSP90 was visualized with a Super Sensitive kit (Biogenex) by following the manufacturer's instructions. Tissue cells were counterstained with hematoxylin.

Quantitative PCR. For quantification of parasites by real-time PCR, DNA was extracted using a PureGene DNA kit (Gentra Systems, Minneapolis, MN). In brief, whole mouse lymph nodes were ground up in lysis buffer containing 300 µg/ml proteinase K and incubated at 55°C for 120 min. Protein was removed and DNA was precipitated by following the manufacturer's instructions. The resulting DNA pellets were resuspended in 200 µl Tris-EDTA buffer and diluted 1:10 prior to analysis. The concentration of parasites was expressed as the ratio of L. major DNA to mouse ß-actin DNA. Mouse ß-actin DNA was quantified by 5'-nuclease PCR. The Leishmania DNA concentrations in the same samples were determined using fluorescence resonance energy transfer real-time PCR with leishmanial 18S ribosomal DNA sequences. The resulting Leishmania DNA copy number was then divided by the copy number of ß-actin DNA to obtain a normalized concentration ratio for the number of parasites per unit of tissue.

Infection of bone marrow-derived dendritic cells. The bone marrow of the femur and tibia of BALB/c mice was isolated by injecting RPMI medium with a syringe. Cells were washed and counted. A total of 2 x 10⁶ cells were cultivated in 10 ml RPMI medium containing 10% FCS and granulocyte-macrophage colony-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. On day 6, 10 ml of medium was removed and replaced by fresh GM-CSF-containing medium. On day 7, the differentiated cells were washed. Then 1 x 10⁶ cells per well were seeded into a 24-well plate and infected with stationary-phase promastigote parasites at a ratio of 1:1. After 48 h the supernatant was harvested and analyzed to determine the presence of interleukin-12p40 (IL-12p40) by an enzyme-linked immunosorbent assay (ELISA) performed according to the manufacturer's instructions (Becton Dickinson, Heidelberg, Germany).

Analysis of cytokine production. Draining lymph nodes were removed, and single-cell suspensions were seeded in 96-well plates at a concentration of 1 x 10⁵ cells per well, using RPMI medium supplemented with 10% heat-inactivated fetal-calf serum. Cells were stimulated either with 3 µg/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 specific two-sided ELISA using supernatants of stimulated lymph node cells. Antibody pairs and cytokine standards were purchased from Becton Dickinson (Heidelberg, Germany).

Western blotting. Sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, semidry blotting, and immune detection with anti-HSP100 immunoglobulin Y 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 performed using the built-in Student's t test or the U test (12).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Parasites of L. major hsp100^–/– strain 1 caused lesions in BALB/c mice with a pronounced delay of 3 to 20 weeks compared with the lesions caused by wild-type parasites (5). During routine passage using infection of BALB/c mice with one mutant clone, we observed rapid lesion development in one mouse that was roughly comparable to the lesion growth caused by wild-type L. major (data not shown). Parasites were reisolated from the footpad lesion of this mouse and grown in vitro as promastigotes. The isolates exhibited markedly delayed outgrowth from the host tissue. After we confirmed by immunoblot analysis (see Fig. 5E) that the isolated parasites in fact did not express HSP100, we compared the isolated population with the L. major wild type and another hsp100^–/– strain, strain 2.2 (5). A total of 2 x 10⁶ promastigotes of these strains were inoculated into the footpads of BALB/c mice, and foot swelling was monitored at weekly intervals. As shown in Fig. 1A, the escape population was comparable to the wild-type L. major population, causing swelling after a 2 weeks of incubation, albeit at a lower rate. By contrast, hsp100^–/– control strain 2.2 caused no lesion formation during the observation period and for up to 25 weeks postinfection.

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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 and stained with Giemsa stain. Note the elongated flagellum of esc2 promastigotes. (B) Infection experiment. Two 8-week-old BALB/c mice were inoculated with the L. major wild type (squares), L. major hsp100–/– clone esc1 (circles), and L. major hsp100–/– clone esc2 (diamonds). Footpad swelling was monitored weekly. (C) Relative parasite loads in draining lymph nodes of mice infected with wild-type and esc2 parasites were determined by real-time PCR (see Fig. 1). The horizontal lines indicate the medians for two mice. (D) In vitro infection. Mouse peritoneal exudate cells were infected with equal numbers of promastigotes of the L. major wild type, the L. major hsp100–/– mutant, and clones esc1 and esc2. After 48 h, cells were fixed, stained with Giemsa stain, and analyzed microscopically. The number of infected macrophage cells was counted. The horizontal 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 x 107 promastigotes, after incubation for 24 h at 35°C, were lysed in SDS sample buffer and loaded on a 7% SDS-polyacrylamide gel. After Western blotting, the membrane was developed with anti-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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FIG. 1. (A) Lesion formation in BALB/c mice. A total of 2 x 106 stationary-phase promastigotes of the L. major wild type, the L. major hsp100–/– mutant, or the escape isolate were inoculated subcutaneously into the footpads of BALB/c mice. Footpad swelling was monitored at weekly 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 draining lymph nodes of infected BALB/c mice (four mice per strain) were isolated and weighed. The horizontal lines indicate the medians for four lymph nodes. The experiment was performed in quadruplicate. The asterisk indicates that the P value is <0.05, as determined by the U test. (C) Parasite loads of draining lymph nodes after infection with the L. major wild type or the escape strain. After isolation of the draining lymph nodes from infected BALB/c mice (four mice per strain), total DNA was prepared and subjected to real-time PCR. Leishmania DNA was quantified relative to the mouse gene for ß-actin. The horizontal lines indicate the medians for four independent lymph nodes. The experiment was performed in quadruplicate. The asterisk indicates that the P value is <0.05, as determined by the U test. (D) Analysis of flagellar length. The flagella of 110 parasites of the L. major wild type, the freshly isolated L. major hsp100–/– mutant, and the escape variant were measured. The median values for the strains are indicated by horizontal lines. Three asterisks indicate that the P value is <0.001, as determined by the U test.

To further assess the pathogenic effects of the strains, the draining lymph nodes were removed from the infected mice after 7 weeks and weighed (Fig. 1B). The lymph node mass in mice infected with the escape population was slightly, but significantly (P = 0.028, as determined by the U test), larger than the lymph node mass in mice infected with the wild type. Complete DNA was extracted from the lymph node tissues. By using quantitative PCR, the relative parasite load was determined for each animal. As shown in Fig. 1C, the relative parasite loads due to the L. major wild type were larger than the relative parasite loads due to the escape population (P = 0.028, as determined by the U test). The mice infected with hsp100^–/– clone 2.2 of L. major contained no detectable parasite DNA. We concluded that the escape isolate caused exacerbated lymph node swelling, while the actual parasite load was reduced compared with that of the wild type.

A histological analysis of draining lymph nodes from animals infected with wild-type L. major, the L. major hsp100^–/– clone, and the escape variant further highlighted the differences in the course of infection. Compared with the L. major wild type and the hsp100^–/– parent mutant (Fig. 2A and C), the escape variant caused exacerbated infiltration of granulocytes into the lymph nodes and a reduction in the amount of connective tissue lining the lymph nodes (Fig. 2E). However, with the escape isolate there was an intermediate parasite load (Fig. 2F) compared with the parasite loads of the wild type (Fig. 2B) and the L. major hsp100^–/– clone (Fig. 2D). This finding confirms the data obtained by real-time PCR.

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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 and D), 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 with hematoxylin. (A, C, and E) Magnification, x10. (B, D, and F) Magnification, x63. The arrow in panel D indicates a single L. major hsp100–/– mutant amastigote.

Interestingly, histological analysis of footpad lesion tissue did not reveal overt differences between the wild type and the escape isolate. Skin tissue from the perimeter of footpad lesions 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 terms of their overall appearance (Fig. 3A and D). Closer inspection of intact (Fig. 3B and E) or degraded (Fig. 3C and F) tissue also did not reveal overt differences between wild-type strain-infected and escape isolate-infected lesions. Footpad tissue from mice infected with the hsp100^–/^– clone could not be analyzed due to the lack of lesions. We concluded that parasite density and parasite-induced pathogenicity did not differ at the lesion site but parasite dissemination into the lymphoid tissue was suppressed with the escape variant.

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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 antibody and Fast Red stain to highlight the leishmaniae and were counterstained with hematoxylin. (A and D) Magnification, x10. (B, C, E, and F) Magnification, x63. The arrows indicate the origins of the magnified areas.

The increased lymph node swelling and the effects on lymph node histology suggested that the escape variant may elicit an altered immune response compared with the immune response elicited by wild-type L. major or the hsp100^–/– parent mutant. Since it is well established that the early IL-4 production by BALB/c mice is responsible for their susceptibility by inducing a TH2 type of immune response, we analyzed the production of two cytokines, IFN- and IL-4, in culture supernatants from lymph node cells isolated 9 days postinfection. Upon stimulation with anti-CD3 or L. major lysate, cells from mice infected with wild-type L. major or the hsp100^–/– mutant produced large amounts of IL-4, indicating that both strains induced the typical TH2-like cytokine profile upon infection (Fig. 4). In contrast, infection with the escape isolate induced higher expression of IFN- and lower production of IL-4, indicating that the mice developed a TH1 type of immune response similar to that of resistant C57BL/6 mice.

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FIG. 4. Cytokine production by lymph node cells after infection with different L. major strains. Mice were infected with 1 x 106 promastigotes of the L. major strains indicated. On day 9 postinfection lymph node cells were isolated and stimulated with anti-CD3 or leishmania antigen. After 48 h supernatant was collected and analyzed for either IFN- production (A) or IL-4 production (B). The data are the results of one experiment in which there were three mice per group and are means and standard deviations. The experiment was repeated, and similar results were obtained. An asterisk indicates that the P value is <0.05, as determined by Student's t test. wt, wild type; clp, L. major hsp100–/– mutant; esc, escape clone.

Upon reisolation of the escape variant, there was a striking morphological difference between the initial population of promastigotes and wild-type L. major. The flagellum was, at first approximation, twice as long as that observed for wild-type controls. The parent hsp100^–/– clones did not have this characteristic (5). This impression was confirmed by measurement of the flagellar length for 110 individual cells belonging to each group (Fig. 1D). The morphological difference was highly significant (P < 0.0001, as determined by the U test), but it vanished with prolonged in vitro passage along with the slow in vitro growth phenotype (not shown). This finding led us to suspect that the escape isolate is a polyclonal population, with individual clones under antagonistic selection in vitro and in the animal host. To test this hypothesis and to increase the stability of the escape phenotype, we attempted to raise individual clones from a freshly reisolated escape population. Promastigotes were diluted to obtain a concentration of 0.5 cell/100 µl and distributed into microtiter plate wells. Clones that showed early outgrowth were discarded as they would have been dominant in the in vitro grown population. Three clones, however, displayed markedly delayed outgrowth 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 culture and was lost.

A comparison of the morphologies of the wild type and the remaining escape clones, esc1 and esc2, revealed that only clone esc2 had the elongated flagellum characteristic of the original escape isolate (Fig. 5A). Clone esc1, if anything, had a slightly reduced flagellum length compared with that of wild-type L. major. These data suggest that clone esc2 was responsible 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 swelling was monitored weekly (Fig. 5B). During the observation time (11 weeks), only wild-type L. major and escape clone esc2 caused notable footpad swelling. All mice were sacrificed after 11 weeks, and footpad tissue and lymph nodes were placed in liquid cultures to allow parasite outgrowth. As expected, wild-type L. major and escape clone esc2 parasites could be grown from host tissue. The esc1 clone, however, could not be recovered from the tissues. Parasite loads in infected lymph nodes were determined by real-time PCR. As expected, the esc2 clone exhibited reduced parasite density compared with the wild 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 to infect peritoneal exudate cells with the abilities of wild-type L. major and the hsp100^–/– mutant to do this. Surprisingly, we observed restored infectivity in vitro with the esc1 clone (Fig. 5D). By contrast, the esc2 clone showed no improved infectivity compared with the background value for the hsp100^–/– mutant. We concluded that the restored pathogenicity of the esc2 clone was not due to restored infectivity for macrophages. On the other hand, clone esc1 was not able to cause a persistent infection in mice in spite of its ability to become established in macrophages in vitro.

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

It is known that dendritic cells play a central role in initiating a specific immune response by presenting antigen to T cells. Furthermore, they produce cytokines that influence the polarization of T cells in either TH1 or TH2. One of the most potent TH1 inducers produced by dendritic cells is IL-12. Therefore, we analyzed the abilities of the two escape clones to induce IL-12 after infection of bone marrow-derived dendritic cells in vitro (Fig. 6). Whereas wild-type L. major and the hsp100^–/– mutant induced comparable levels of IL-12 expression, the virulent esc2 clone induced significantly lower levels of IL-12 production. By contrast, the esc1 clone, which was not able to elicit lesion development, induced significantly increased expression of IL-12 compared with the expression induced by wild-type parasites.

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FIG. 6. (A) IL-12p40 production by dendritic cells after infection with L. major. A total of 1 x 105 bone marrow-derived dendritic cells were infected with different numbers of L. major parasites, as indicated. After 48 h the supernatant was collected and analyzed for IL-12p40 production by ELISA. The values are means ± standard deviations. The experiment was repeated twice, and similar results were obtained. (B) Cytokine production by lymph node cells after infection with different L. major mutants and clones. Mice were infected with 1 x 106 promastigotes of the L. major isolates and clones indicated. On day 9 postinfection lymph node cells were isolated and stimulated with anti-CD3 or leishmania antigen. After 48 h supernatant was collected and analyzed for IFN- production. The values were obtained from one experiment in which there were three mice per group and are means and standard deviations. The experiment was repeated, and equivalent results were obtained. wt, wild type; MOI, multiplicity of infection.

We also tested the IFN- production in mice infected with the two escape clones 9 days postinfection. Supernatants of lymph node cells from mice infected with L. major, the L. major hsp100^–/– mutant, and clones esc1 and esc2 were analyzed by ELISA after stimulation with medium, anti-CD3 antibody, or L. major lysate (Fig. 6B). We observed a strong increase in inducible IFN- production by the cells isolated after infection with esc2, indicating that the observed induction of a TH1-type immune response in mice infected with the escape isolate was indeed due to the esc2 subpopulation. We concluded that the restored virulence observed with the escape isolate and the altered immune response were both caused by a single clone, esc2.

Previous work suggested that the immediate early phase of infection determines the dichotomy of the pathogen-specific T-cell response and thus drives the response in either the TH1 direction or the TH2 direction. To determine the early, innate immune response to infection with wild-type or escape isolate parasites, we performed an air pouch infection experiment (10). A total of 1 x 10⁷ promastigotes were inoculated into a subcutaneous air pouch. Twenty-four hours postinfection, the cellular infiltrate was isolated by flushing the air pouch with Dulbecco's PBS. Parasites or parasite-containing cells could not be detected in the exudate after Giemsa staining and microscopy (data not shown). The exudate was analyzed by flow cytometry, using antibodies against immune cell surface markers (Fig. 7). As determined previously, polymorphonuclear leukocytes (PMN) appeared to be a major constituent of the cellular infiltrate. However, a variety of other cell types were found in the air pouch that could be clearly differentiated from PMN using a combination of morphological criteria and staining with anti-Gr-1, as analyzed using flow cytometry. Interestingly, infection with the esc2 strain led to increased infiltration of PMN and also CD11b⁺ macrophages. In addition, this strain induced infiltration of CD11c⁺ dendritic cells. By contrast, esc1 induced significantly less infiltration of cells than esc2 or wild-type Leishmania induced. To exclude the possibility that the cellular infiltrate in the air pouch was due to an injury during infection, we also stained CD19⁺ B cells since they are found at high frequencies in the blood. However, B cells appeared to be absent, suggesting that cells traveled specifically to the air pouch in response to infection with the parasite.

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
During routine passage in susceptible BALB/c mice, a pathogenic variant of the attenuated L. major hsp100^–/– mutant (5) was isolated. This isolate's pathogenicity was unstable during in vitro cultivation, as were its peculiar morphology and its reduced in vitro growth rate.

The exact function of Leishmania HSP100 is not known. Orthologous proteins in yeast (Saccharomyces cerevisiae) and Escherichia coli are known to be involved in inducible thermotolerance and general stress tolerance. This effect is less pronounced in Leishmania spp., as the hsp100^–/– mutants show only limited effects on thermotolerance (5, 8). HSP100 family members in several model organisms have been shown to form homohexameric rings, not unlike members of the closely related family of Clp protease regulatory subunits, ClpA and ClpX. The major function of the hexameric HSP100 or ClpB proteins appears to be disassembly of protein aggregates, aided by the HSP70/HSP40 chaperone complexes (16). Whether this function is conserved in Leishmania HSP100 is not known yet. In contrast to the other members of the family, Leishmania HSP100 appears to be associated in homotrimers rather than hexamers (7). This abrogates the formation of a ring-shaped complex. Recent evidence argues in favor of the hypothesis that the central cavity in the hexameric ring of ClpB performs the deaggregation reactions. We therefore have to take into account the possibility that HSP100 in Leishmania has a different function. For instance, in yeast, HSP104 has a clustered appearance throughout the cytoplasm (6). In L. donovani, HSP100 is also clustered; however, the majority of the molecules localize close to the cytoplasmic membrane (7).

In yeast, the loss of HSP104 and the concomitant loss of inducible thermotolerance can be compensated for, in part, by overexpression of the major heat shock protein, HSP70 (11, 15). In the escape variants of the L. major hsp100^–/– mutant, however, we found no reproducible evidence of HSP70 overexpression (Kroemer, unpublished). Also, we performed a functional complementation screen using an L. major hsp100^–/– genomic DNA cosmid library transfected into the L. major hsp100^–/– mutant and subsequent selection in BALB/c mice. No heat shock genes were found on the recovered cosmids (Reiling, unpublished). Evidently, intracellular survival of L. major requires a highly specialized function of HSP100.

This spontaneous recovery of virulence in an attenuated mutant of L. major is not unexpected. Späth et al. reported spontaneous recovery of virulence in an lpg2 mutant of L. major (17). This mutant does not synthesize a major surface molecule, lipophosphoglycan. For L. major, loss of LPG abrogates virulence in the mouse model. A partially revertant population of the lpg2 mutants was identified that exhibited delayed lesion formation and was able to survive and proliferate within macrophages. No mechanism for this recovery of virulence has been established yet. However, it is interesting that full reversion to wild-type virulence did not occur. In this regard, this revertant phenotype is comparable to the hsp100^–/– escape phenotype. The major difference is that restoration of pathogenicity in hsp100^–/– clone esc2 is accompanied by an altered immune reaction of the host organism.

We have compared the escape isolate with wild-type L. major and the hsp100^–/– mutant, using comparative proteomics and comparative immunoblot analysis. To date, no reproducible qualitative or quantitative differences have been observed, except for an increase in paraflagellar rod proteins in the escape isolate, which can be explained by the exceedingly long flagellum observed (data not shown).

When BALB/c mice were infected with the escape strain, we observed lesion development that was comparable to that of the L. major wild-type strain. The number of intracellular parasites seen in histological sections from the perimeter of the lesions was indistinguishable from the number in lesions induced by the L. major wild type. However, at 7 weeks postinfection, we observed a reduced parasite load in the draining lymph nodes, indicating that the escape population showed reduced dissemination. Moreover, the escape variant exhibited an altered immune response. In contrast to wild-type L. major, the escape isolate induced rapid induction of the TH1 cytokine IFN-, which is associated with the protective immune response in resistant C57BL/6 mice. Furthermore, we found that in the very early phase of the infection, the escape isolate attracted a higher number of phagocytic cells than the wild-type parasites and the parent hsp100^–/– mutant attracted. This suggests that the bias of the subsequent immune response is dependent on the quality or quantity of the infiltrating antigen-presenting cells. Interestingly, the hsp100^–/– mutant, which is completely avirulent and unable to persist, does not trigger infiltration of inflammatory cells, indicating that a minimal level of pathogen-derived "danger signals" must be exceeded to induce inflammation. After subcloning of the escape isolate, we established two stable clones which exhibited very different phenotypes with regard to both morphology and the immune response upon infection. Our results indicate that an escape variant of the L. major hsp100^–/– mutant, clone esc2, persists in 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 using isolated macrophages, it is able to survive and proliferate in mice. It is interesting that esc2 induces less IL-12 upon infection of bone marrow-derived dendritic cells in vitro than other strains induce, even though it was found to be the cause of elevated IFN- production at 9 days postinfection in BALB/c mice. One explanation for the persistence of esc2 despite its poor infectivity in vitro may be recruitment of safe host cells. Indeed, this strain induces the highest number of infiltrating Ly1⁺ neutrophilic granulocytes, which were previously shown to be a safe vehicle for dissemination. Efforts will be made to analyze whether this increased recruitment of granulocytes is responsible for the persistence of esc2. On the other hand, we must consider the fact that in vitro infection of peritoneal macrophages with promastigotes is highly artificial and reflects only primary infection and amastigote differentiation, while macrophages in a lesion are infected by fully differentiated amastigotes.

Clone esc1, by contrast, can survive in the mouse only in association with clone esc2. A single infection with clone esc1 does not lead to a patent infection, and the parasite is rapidly cleared from different tissues, as judged by real-time PCR. However, esc1 has restored ability to infect macrophages with wild-type-like efficiency. The lack of pathogenicity in mice suggests that the restored infectivity may be offset by the increased activation of IL-12 production very early in infection and the resulting cell-mediated immune reaction.

The genetic variation discussed above occurred within a maximum of four mouse infection cycles, starting with a single L. major hsp100^–/– clone. This finding underscores the underlying genetic flexibility of Leishmania species. Different immune responses were induced by different clones derived from a single original L. major clone. These data imply that not only the genetic background of the host, which was studied in detail using BALB/c or C57BL/6 mice as examples of a polarized host, but also the highly dynamic expression of parasite proteins that are triggered by environmental factors influence the developing immune response. The observed clonal variability of L. major parasites and their different abilities to influence the host immune system might explain why parasites are able to adapt very quickly to different immunological niches. Our findings also bring into question efforts to generate safe attenuated vaccines using targeted replacement of single genes required for parasite pathogenicity.

 
We thank Sabine Becker and Andrea Macdonald for technical assistance.

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

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

Editor: J. F. Urban, Jr.

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

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

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, November 2006, p. 6027-6036, Vol. 74, No. 11
0019-9567/06/$08.00+0     doi:10.1128/IAI.00773-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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