A chemical inhibitor of heat shock protein 78 (HSP78) from Leishmania donovani represents a potential antileishmanial drug candidate

Abstract

The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 (LdHSP78+/-) and Leishmania mexicana HSP78 (LmxHSP78+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78+/- parasites, compared with WT parasites. Virulence of the LdHSP78+/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P¹,P⁵-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.

Keywords: Ap5A; Leishmania; caseinolytic protease; drug screening; heat shock protein 78 (HSP78); high-throughput virtual screening; innate immunity; kinase signaling; macrophage; molecular modeling; transgenic parasite.

Figure 1.

LdHSP78 is an essential chaperone for infection. A, semiquantitative PCR of LdHSP78 mRNA in L. donovani-infected BMDMs. B, RT-PCR of LdHSP78 mRNA from 3-month-infected hamster (Ham) spleen, liver, and blood and VL patient peripheral blood. The C_t value of LdHSP78 was normalized against that of L. donovani GAPDH and plotted as fold change (FC) (2^−ΔΔCt). C, RT-PCR of LdHSP78 mRNA from promastigotes cultured at different temperature and pH values. The C_t value of LdHSP78 was normalized against that of L. donovani GAPDH and plotted as fold change (FC) (2^−ΔΔCt). D, partial deletion of LdHSP78 validation by urea-PAGE-based separation of PCR products, amplified from WT (control transfected) and LdHSP78+/− parasites (L. donovani actin is the loading control). E and F, Determination of LdHSP78+/− promastigote fitness under (E) normal conditions (22 °C and pH 7.4) and (F) amastigote-like conditions (37 °C and pH 4.0, 5.5, and 7.4). Growth kinetics of promastigotes were determined from 5-day cultures (to obtain maximum growth). G and H, RT-PCR-based determination of LdHSP78 mRNA loads of WT, LdHSP78+/−, and resKO promastigotes (G) and amastigotes (H) in infected BMDMs. I, Giemsa staining of BMDMs infected with WT, LdHSP78+/−, and resKO parasites for 24 and 48 h at a 1:10 ratio. J and K, Western blotting of lysates from uninfected BMDMs (negative control) and WT, LdHSP78+/−, and resKO promastigotes (loading controls were macrophage actin and L. donovani actin). Fold changes of LdHSP78 were calculated from densitometric values of the bands. Each group is represented by three samples, and each experiment was performed in triplicate. Statistical significance (**, p < 0.01; ***, p < 0.001) was calculated using one-way analysis of variance followed by Dunnett's multiple-comparison test and unpaired two-tailed t test followed by Welch correction (each dot is representative of a single sample; n = 3 samples per group). Data represent mean ± S.D. (GraphPad Prism v5.0).

Figure 2.

Cytokine profiling of BMDMs infected with LdHSP78-depleted and -overexpressing parasites indicated sustained proinflammatory cytokine responses. RT-PCR (A–D) and sandwich ELISAs (E–H) of proinflammatory (IL12A) and anti-inflammatory (IL10, IL27A, and TGFβ) cytokines from WT, LdHSP78+/−, and resKO parasite-infected BMDMs. Eight hours after infection, total RNA was isolated from BMDMs and subjected to RT-PCR. Data were normalized using mouse GAPDH as a control gene. For TGFβ and IL10 ELISAs, culture supernatants were collected 12 h after infection; for IL12A and IL27A ELISAs, 48-h postinfection supernatants were collected. FC, fold change. Each experimental set is representative of three samples, and error bars are mean ± S.D. Statistical significance was analyzed using one-way analysis of variance followed by Dunnett's multiple comparison test. **, p < 0.01; ***, p < 0.001.

Figure 3.

Expression of phospho-p38, iNOS, and NO increased during LdHSP78+/− parasite infection in BMDMs. A–H, lysates were collected from BMDMs infected for 24 h with WT, LdHSP78+/−, and resKO parasites and Western blotted for phospho-ERK1/2 and total ERK1/2 (phospho-ERK1/2 bands were normalized to ERK1/2 bands) (A and E), phospho-p38 and total p38 (phospho-p38 bands were normalized to p38 bands) (B and F), arginase 1 (Arg1) (C and G), and iNOS (D and H) (arginase 1 and iNOS bands were normalized to β-actin bands). Lipopolysaccharide (LPS) (10 μg/ml) was the positive control and mouse β-actin was the loading control. Scattered plots adjacent to each blot represented mean densitometric values of three blots performed for each protein. I, the Griess assay was performed with culture supernatants of WT, LdHSP78+/−, and resKO parasite-infected BMDMs collected 24 and 48 h postinfection. Each experimental set is representative of three samples, and error bars are mean ± S.D. Statistical significance was analyzed using one-way analysis of variance followed by Dunnett's multiple-comparison test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4.

LdHSP78+/− parasites showed compromised infectivity in hamsters. A, comparative pathophysiology of WT, LdHSP78+/−, and resKO parasite-infected spleens procured from hamsters 3 months postinfection (p.i.), showing splenomegaly during WT and resKO parasite infection. B, Leishman-Donovan units (LDU) from phase-contrast microscopy of Giemsa-stained images (scale bar, 10 μm). C, LDA results (log₁₀ parasite numbers/ml) for WT, LdHSP78+/−, and resKO parasite-infected spleen and liver. D, genomic DNA isolated from WT parasites was serially diluted, and RT-PCR for kDNA and L. donovani GAPDH was performed. C_t values obtained were used to prepare the standard curve of kDNA versus parasite count in log₁₀ count/ml. E, C_t values of kDNA from hamster whole blood, spleen, and liver were obtained and plotted in the standard curve of kDNA to calculate the parasite burden of each organ, and results were plotted in this scatter graph. F, RT-PCR of LdHSP78 mRNA from infected hamster organs and whole blood was performed. FC, fold change. Each experimental set is representative of three samples, and error bars are mean ± S.D. Statistical significance was analyzed using one-way analysis of variance followed by Dunnett's multiple-comparison test. **, p < 0.01; ***, p < 0.001.

Figure 5.

Protective cytokine responses and NO generation were augmented in LdHSP78+/− parasite-infected hamsters. A–C, IL10 (A), TGFβ (B), and IFNγ (C) levels in whole blood, spleen, and liver of WT, LdHSP78+/−, and resKO parasite-infected hamsters from three groups were measured by RT-PCR. D and E, sandwich ELISAs for IL10 (D) and IL12 (E) were performed with blood samples collected at regular time intervals up to 3 months. F and G, enrichment of iNOS (F) and NO (G) in whole blood, liver, and spleen of hamsters was determined by RT-PCR and Griess assays, respectively. Hamster GAPDH was used as the control for all RT-PCR assays. FC, fold change. Each experimental set is representative of three animals, and error bars are mean ± S.D. Statistical significance was analyzed using one-way analysis of variance followed by Dunnett's multiple-comparison test. *, p < 0.05, **, p < 0.01; ***, p < 0.001.

Figure 6.

Structural features of L. donovani HSP78 protein. A, 2D and 3D domain organizations of L. donovani HSP78 protein and their relative conservation. Blue, red, and green bars show conservation of individual domain calculated using the representative sequences from all phyla, Euglenozoa, and Ascomycota, respectively. B, binding mode and probable interactions of the selected inhibitor Ap5A, which shows significant binding potential for NBD1 and NBD2 shown in upper and lower panel, respectively.

Figure 7.

Ap5A showed comparable antileishmanial efficacy with AmB. A, E, F, and H, the viability of axenic promastigotes (A and F) and healthy BMDMs (E and H) treated with Ap5A and AmB was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. B, pH sensitivity and temperature sensitivity of Ap5A (15 μm)-treated WT and LdHSP78+/−promastigotes were compared by determination of promastigote growth. C and G, Giemsa staining was performed to calculate the amastigote burden of BMDMs, infected with L. donovani for 24 h and treated with either Ap5A or AmB. A and D, the comparative sensitivity of WT, LdHSP78+/−, and resKO promastigotes and amastigotes for Ap5A treatment was analyzed to understand the specificity of Ap5A for LdHSP78. Each experimental set is representative of three samples, and error bars are mean ± S.D. Statistical significance was analyzed using one-way analysis of variance followed by Dunnett's multiple-comparison test.