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Infection and Immunity, June 2003, p. 3190-3195, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3190-3195.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Expression of Multiple CPB Genes Encoding Cysteine Proteases Is Required for Leishmania mexicana Virulence In Vivo

Wellcome Centre for Molecular Parasitology, The Anderson College, University of Glasgow, Glasgow G11 6NU,¹ Department of Immunology, University of Strathclyde, Glasgow G4 0NR,² Division of Infection and Immunity, Institute for Biomedical and Life Science, University of Glasgow, Glasgow G12 8QQ, United Kingdom³

Received 17 October 2002/ Returned for modification 14 January 2003/ Accepted 18 March 2003

	ABSTRACT

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Leishmania mexicana mutants deficient in the multicopy CPB gene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specific CPB2.8 or metacyclic stage-specific CPB2 gene into a CPB-deficient mutant L. mexicana failed to restore either a Th2 response or sustained virulence. However, reexpression of multiple CPB genes from a cosmid significantly restored virulence. This was characterized by increased lesion and parasite growth and the acquisition of a Th2 response, as determined by measuring interleukin-4 production and immunoglobulin G1 (IgG1) and IgE levels. These studies confirm that L. mexicana cysteine proteases are important virulence factors and provide an explanation for the presence in L. mexicana of a multicopy tandem array of CPB genes.

	INTRODUCTION

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Parasite cysteine proteases have been implicated in several processes including differentiation, nutrition, host cell infection, and evasion of the host immune response (10, 17, 19). Leishmania mexicana possesses three cysteine proteases of the papain family (designated clan CA), namely, the cathepsin L-like CPA (12) and CPB (25) and the cathepsin B-like CPC (4). The CPB genes are multicopy and are located in a single locus of 19 copies arranged in a tandem repeat (13, 14) (Fig. 1a). The first two copies of CPB, CPB1 and CPB2, are expressed in the infective metacyclic stage of the parasite, while the others are expressed predominantly in the intracellular amastigote stage (6, 13, 14). Information about the roles and importance of the enzymes in host-parasite interactions was obtained by the generation of a CPB-deficient (cpb) mutant. It was shown that cpb promastigotes are less infective to macrophages than wild-type parasites in vitro and that they are able to form only small, slow-growing lesions in BALB/c mice (8, 13). In contrast, cpb amastigotes were able to infect macrophages in vitro with the same kinetics as wild-type parasites but, in a similar manner to cpb promastigotes, formed only small, slow-growing lesions in mice. Subsequent studies indicated that the absence of the CPB genes resulted in a protective Th1 immune response, contrary to the Th2 response normally observed when the CPB isoenzymes are present (1, 2). The pivotal role of interleukin-4 (IL-4) and the Th2 response in establishing nonhealing L. mexicana infections is well documented (3, 21), and significantly we have recently found that enzymatically active CPB is a potent stimulator of IL-4 and a Th2 response (15).

View larger version (13K): [in this window] [in a new window]   FIG. 1. Map of the CPB array and reintegration constructs. (a) Map of the L. mexicana CPB locus. InS, insertion sequence present in the intercistronic sequence downstream of CPB1 and CPB2; SL, CPB splice leader acceptor site present upstream of each gene; vertical arrows, polyadenylation addition sites. (b and c) Maps of the CPB2 and CPB2.8 reintegration cassettes. The CPB genes, their 3' intergenic sequences, and the sat gene were cloned between the unique 5' and 3' CPB flanking regions. Symbols are as in panel a.

	MATERIALS AND METHODS

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Leishmania cultivation and transfections. The lines studied in this work were derived from L. mexicana (MNYC/BZ/62/M379) as described elsewhere (13). L. mexicana promastigotes were routinely grown in modified Eagle’s medium (designated complete HOMEM medium), pH 7.5, containing 10% (vol/vol) heat-inactivated fetal bovine serum at 25°C as described elsewhere (14). L. mexicana parasites were transformed in vitro into axenic amastigotes as described previously (8).

All transfections were carried out as previously described (13) with 10 µg of linear DNA fragments or 5 µg of cosmid DNA. After transfection, the cells were selected in complete HOMEM medium containing the appropriate antibiotic for selection (hygromycin [50 µg/ml], nourseothricin [10 µg/ml], bleomycin [10 µg/ml], or puromycin [10 µg/ml]). Individual clones were isolated on 1% (wt/vol) agar-complete HOMEM plates and then directly transferred to complete HOMEM medium. The individual CPB genes were reintegrated into the CPB locus of L. mexicana as described previously (6).

Infectivity studies. Groups of five mice were inoculated in the footpad with 5 x 10⁵ stationary-phase L. mexicana parasites resuspended in 0.025 ml of phosphate-buffered saline, pH 7.4. The resultant lesions were monitored over an 8-week period. All experiments were carried out on at least two occasions.

Screening of the L. mexicana cosmid library. An L. mexicana M379 cosmid library (D. C. Barker, Cambridge, United Kingdom) was screened with three [³²P]dCTP-labeled probes prepared with a Prime-it random-priming kit (Stratagene). One probe recognized the unique 5' flanking region (5' flank), another recognized the CPB gene (CPB), and the third recognized the unique 3' flanking region (3' flank), as previously described (13). Hybridization took place overnight at 65°C in 1 M NaCl-1% sodium dodecyl sulfate (SDS) with 100 µg of salmon sperm DNA/ml. The filters were washed stringently with 0.2x SSC buffer (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 0.1% SDS at 60°C and exposed to photographic film (Agfa) at -70°C.

A cosmid (designated pGL263) containing 5' flank, 3' flank, and CPB sequences was isolated from a selected bacterial clone with a Wizard midiprep kit by following the manufacturer's instructions (Promega, Southampton, United Kingdom). After mouse infection, the cosmid was recovered from the induced lesions as described elsewhere (7).

Gelatin SDS-PAGE analysis. Cysteine protease activity was assayed by gelatin SDS-polyacrylamide gel electrophoresis (PAGE) as previously described (16). Coomassie blue stain was used to visualize the hydrolysis of 0.2% gelatin copolymerized in the 12% separating gel.

Protease activity assays. Lysates of stationary-phase promastigotes were prepared as for gelatin SDS-PAGE analyses and assayed for their ability to hydrolyze Z-Phe-Arg-7-amido-4-methylcoumarin (ZFR-AMC) at 30°C in 0.1 M sodium acetate, pH 5.2, with 2 mM EDTA and 1 mM dithiothreitol. Lysate samples were preincubated for 10 min at 30°C in assay buffer only or in the presence of cysteine protease inhibitor E-64 (10 µM; Sigma). The reaction rate was measured on a Perkin-Elmer LS 55 spectrofluorometer, with 380 nm as the excitation wavelength and 440 nm as the emission wavelength.

Detection of Leishmania-specific IgG1, IgG2a, and total IgE. Peripheral blood was collected from infected animals by tail bleeding into heparinized capillary tubes at 6 to 8 weeks postinfection. All plasma samples were stored at -20°C before analysis. Leishmania-specific immunoglobulin G1 (IgG1), IgG2a, and total IgE were measured by enzyme-linked immunosorbent assay and end point dilution as previously described (2, 20). The end point dilution represents the final plasma concentration that yielded an absorbance higher than that yielded by a negative-control plasma sample included in the assay.

Proliferation assays. Spleens and popliteal lymph nodes from L. mexicana-infected mice were removed aseptically, and splenocytes and lymph node suspensions (5 x 10⁵/well) were incubated in 96-well plates at 37°C in complete tissue culture medium (RPMI 1640 [Life Technologies, Paisley, United Kingdom] with 10% [vol/vol] heat-inactivated fetal bovine serum) with or without soluble Leishmania antigen for 3 days. Soluble Leishmania antigen was prepared as described previously (24). Supernatants were assayed for IL-4 as previously described (2). All assays were conducted in triplicate.

	RESULTS

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Expression of CPB genes in the cpb mutant. We have shown previously that the L. mexicana CPB locus contains 19 genes in a tandem array (Fig. 1a). To isolate cosmids containing the CPB array, a CPB gene-specific probe (13) was used to screen an L. mexicana cosmid genomic library and several independent clones were isolated. Analysis using specific probes revealed that cosmid pGL263 hybridized to the 5' and 3' flank probes, as well as the CPB gene itself, supporting evidence that it contained the CPB array. Restriction mapping, coupled to pulsed-field gel electrophoresis and sequence analysis, revealed that the cosmid contained no complete open reading frames apart from the CPB array.

The cpb mutant was generated with hyg and ble drug resistance genes as selectable markers (13). As the cosmid pGL263 contains the hyg resistance gene (18), the cpb mutant was reengineered to contain the pac gene, which confers puromycin resistance, in place of the hyg gene. Confirmation that pac had replaced hyg in the cpb^pac cell line was obtained by assessing the sensitivity of clones to hygromycin and by PCR analysis of genomic DNA with combinations of oligonucleotide primers designed to anneal to pac and CPB flanking regions outside of the integration cassette (data not shown).

The cpb^pac mutant was transfected with pGL263, and a clone resistant to hygromycin, designated the cpb^pac[pGL263] mutant, was characterized. Extracts of promastigote and amastigote stages were assessed for cysteine protease activity by gelatin SDS-PAGE (Fig. 2). The cpb^pac[pGL263] mutant was found to exhibit cysteine protease activity in both life cycle stages, with higher levels detected in the amastigote. In contrast, the cpb mutant had no cysteine protease activity that was detectable by this method.

View larger version (62K): [in this window] [in a new window]   FIG. 2. Gelatin SDS-PAGE analysis of the cpbpac[pGL263] mutant. Lysates of 107 wild-type, cpbpac, and cpbpac[pGL263] promastigotes and 4 x 106 axenic amastigotes were analyzed by gelatin SDS-PAGE. Molecular mass standards are indicated.

Infectivity of promastigotes of the mutant parasites. As previously observed (2), the cpb mutants produced small and slow-growing lesions in mice compared with the rapidly growing lesions produced by wild-type L. mexicana (Fig. 3). The cpb::CPB2.8 mutant induced early lesion growth (2 weeks), similar to wild-type parasites. Lesions were significantly larger at 2 weeks than those produced by cpb mutants (P < 0.01), but this was not sustained, and lesion size thereafter was similar to that for cpb mutant-infected animals (Fig. 3a) and significantly less than that of wild-type L. mexicana lesions. In contrast, the increase in footpad thickness induced by the cpb::CPB2 mutant line was at no stage greater than that produced by cpb mutants (data not shown). However, cpb^pac[pGL263] promastigotes induced lesions which were significantly larger than those produced by cpb^pac parasites at 4, 6, and 8 weeks (P < 0.05) postinfection and approximately 50% of the size of wild-type parasite-induced lesions (Fig. 3b). The total parasite counts at 8 weeks reflected differences in lesion size between mutants and wild-type parasites (wild-type, [8.0 ± 1.2] x 10⁸; cpb^pac[pGL263] mutant, [1.1 ± 0.6] x 10⁸; cpb^pac mutant, [5.0 ± 1.3] x 10⁶). These results show that the presence of multiple CPB genes facilitates the establishment and sustained development of promastigote-initiated infection by L. mexicana.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Lesion development in BALB/c mice infected with L. mexicana mutants. (a) BALB/c mice were inoculated with 5 x 105 wild-type, cpb, and cpb::CPB2.8 parasites. Footpad thickness was monitored for 8 weeks. Values represent means ± standard errors of the means (n = 5). (b) Same as panel a except that mice were infected with cpbpac and cpbpac[pGL263] mutants.

Expression of multicopy CPB restores the Th2 response, as determined by measuring IL-4 production and IgG1 and IgE levels. We have previously shown that when BALB/c mice are infected with the cpb mutant, they are able to mount a protective Th1 response, rather than the nonprotective Th2 response normally observed in wild-type infections (2). The latter response is characterized by elevated IL-4 production and comparatively high titers of IgG1 and IgE. To determine the type of immune response induced by the different mutant parasite lines, we measured the Ig titers in the plasma of infected mice.

As previously demonstrated, mice infected with wild-type parasites exhibited a high level of IgG1 at 8 weeks postinfection, characteristic of a Th2 response, while cpb mutant infections led to a relatively low titer, indicative of a Th1 response (Fig. 4a). Infection with the cpb::CPB2.8 mutant was similar to infection with the cpb mutant in terms of IgG1 and IgG2a antibody production. While BALB/c mice infected with wild-type parasites had significant IgE production, again indicative of IL-4 production and a Th2 response, IgE levels were significantly lower (P < 0.001) in mice infected with cpb and cpb::CPB2.8 parasites (Fig. 4b). Conversely, following infection with the cpb^pac[pGL263] mutant, BALB/c mice produced significantly more IgG1 (P < 0.01) than mice infected with cpb^pac parasites but significantly less (P < 0.001) than mice infected with the wild type (Fig. 4c). Importantly, infection with the cpb^pac[pGL263] mutant increased IgE levels so that they were equivalent to that produced by wild-type infection and significantly greater (P < 0.001) than that produced by infection with the cpb^pac mutant (Fig. 4d).

View larger version (35K): [in this window] [in a new window]   FIG. 4. Analysis of parasite-specific IgG1 and IgG2a levels (a and c) and total IgE levels (b and d) in BALB/c mice infected with L. mexicana mutants. The antibody titers were determined in the plasma of mice infected with wild-type, cpb, cpb::CPB2.8, cpbpac, and cpbpac[pGL263] strains at 8 weeks postinfection. Values represent the mean end point dilutions ± standard errors of the means (n = 5).

View larger version (12K): [in this window] [in a new window]   FIG. 5. Lymphocyte IL-4 responses of mice infected with L. mexicana mutants. IL-4 production was measured after stimulation of isolated splenocytes with 107 cell equivalents (c.e.) of soluble Leishmania antigen/ml. Error bars, standard errors of the means (n = 5). (a) cpb::CPB2.8 mutant. (b) cpbpac[pGL263] mutant.

	DISCUSSION

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The finding that cpb mutants reexpressing CPB2, an isoenzyme expressed primarily in the metacyclic form, failed to promote lesion development was not unexpected. While it is conceivable that expression of metacyclic stage-specific CPB2 may support initial infection, the low levels of CPB activity expressed following transformation into the amastigote stage (6) would afford these cpb::CPB2 mutants no advantage over cpb parasites. It is interesting, however, that the parasites with amastigote-specific CPB2.8 induced increased initial lesion growth compared with cpb parasites, even though this was not sustained. Presumably, for prolonged virulence in mice the parasites require higher levels of expression of CPB than was mediated by the single gene or, alternatively, expression of other CPB isoenzymes with different substrate specificities (6, 14). Both of these possibilities are consistent with the results obtained with the mutant expressing multiple CPB genes, the cpb^pac[pGL263] mutant, which produced lesions that were significantly larger throughout the course of the experiment than those induced by the cpb mutant. These data suggest that the expression of multiple CPB isoenzymes greatly facilitates virulence. However, they do not indicate whether the effect is mediated simply by enhanced CPB activity or requires several isoenzymes with subtly different substrate specificities (14). Previous studies where single CPB genes were reexpressed from episomes which were present in multiple copies in the parasite but did not restore virulence (8) could indicate that higher expression alone is insufficient to induce higher infectivity in vivo. Nevertheless this question requires further investigation.

The mean lesion size produced by the cpb^pac[pGL263] mutant was only one-half that produced by using equivalent numbers of wild-type parasites. This could have been due to differing expression of isoenzymes from the cosmid compared with that for the natural CPB array or different overall CPB activity. The regulation of gene expression from the cosmid is likely to differ from the regulation of chromosomally located-gene expression, and indeed the gelatin SDS-PAGE data (Fig. 2) suggest that the two parasite lines are not equivalent and express different numbers or different arrays of isoenzymes. Moreover, some parasites in the population in the lesions may have lost the cosmid, which clearly would make them less virulent. Further studies, involving reexpression of all different combinations of CPB genes, will be necessary to understand whether all of the isoenzymes are required for full effect and whether any in particular are playing the major role in the polarization of the immune response.

Infection with L. mexicana in the BALB/c mouse is associated with a polarized Th2 response, and endogenous IL-4 has been demonstrated to be necessary for early lesion development (3, 22). Moreover, susceptibility of BALB/c mice to Leishmania major has been shown to be the result of IL-4 production by Vß4 V8CD4⁺ T cells in response to a single T-cell epitope derived from the parasite LACK antigen (the Leishmania homologue of receptors for activated C kinase) (11). However, recent studies have demonstrated that LACK-tolerant BALB/c mice remain susceptible to L. mexicana despite resistance to L. major (26). As L. mexicana expresses LACK and as LACK is recognized by BALB/c CD4⁺ T cells that produce IL-4, these observations clearly demonstrate that there are alternative or additional mechanisms for inducing the susceptibility of mice to L. mexicana. Our data suggest that the CPB enzymes may be crucially involved. Significantly, L. mexicana cpb mutants have reduced infectivity for BALB/c mice and induce a polarized type 1 response (2). Of relevance to this is the report that during wild-type infections CPB occurs in large quantities in the extracellular milieu (9). Subsequently we have demonstrated that recombinant, enzymatically active CPB2.8 is a potent stimulator of IL-4 and IgE production in BALB/c mice (15), like the DerP1 antigen, which is also a cysteine protease (23). Our present findings, that reexpression of multiple CPB genes, but not single genes, in cpb mutants restored a significantly increased IgE production as well as significantly elevated lymph node and splenocyte IL-4 production, are consistent with this hypothesis of a key role for CPB in the host-L. mexicana interactions and particularly in the modulation of the immune response.

The findings of this study confirm that the level of CPB expression or the diversity in CPB isoenzymes or both are important for parasite virulence. Moreover, this study has demonstrated a role for the multiple CPB isoenzymes in the modulation of the immune response to L. mexicana infection, although the mechanisms underlying this process await further characterization.

	ACKNOWLEDGMENTS

 
This investigation received financial support from the UNDP/World Bank/WHO special program for research and training in tropical diseases and the Wellcome Trust. J. C. Mottram is an MRC Senior Research Fellow.

	FOOTNOTES

 
* Corresponding author. Mailing address: Wellcome Centre for Molecular Parasitology, The Anderson College, University of Glasgow, Glasgow G11 6NU, United Kingdom. Phone: 44-141-330-3745. Fax: 44-141-330-5422. E-mail: j.mottram{at}udcf.gla.ac.uk.

Editor: J. M. Mansfield

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