Long-term hematopoietic stem cells trigger quiescence in Leishmania parasites

Abstract

Addressing the challenges of quiescence and post-treatment relapse is of utmost importance in the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence after an estimated 2-3 divisions in both human and mouse bone marrow stem cells. Interestingly, this behavior is not observed in macrophages, which are the primary host cells of the Leishmania parasite. Transcriptional comparison of the quiescent and non-quiescent metabolic states confirmed the overall decrease of gene expression as a hallmark of quiescence. Quiescent amastigotes display a reduced size and signs of a rapid evolutionary adaptation response with genetic alterations. Our study provides further evidence that this quiescent state significantly enhances resistance to treatment. Moreover, transitioning through quiescence is highly compatible with sand fly transmission and increases the potential of parasites to infect cells. Collectively, this work identified stem cells in the bone marrow as a niche where Leishmania quiescence occurs, with important implications for antiparasitic treatment and acquisition of virulence traits.

Fig 1. Leishmania infection of mouse LT-HSC and human HSPC triggers amastigote quiescence.

(a) Amastigotes recovered from infected mouse LT-HSC and measured via flow cytometry. Cells in the left panel were infected with L. infantum LEM3323 WT^PpyRE9/DsRed, middle panel with L. infantum clinical isolate LLM2346 WT^PpyRE9/DsRed, right panel with L. donovani Ldl82 WT^PpyRE9/DsRed. (b) Amastigotes recovered from L. infantum LLM2346 WT^PpyRE9/DsRed infected human HSPC. (c) BM derived macrophages were infected with L. infantum (LEM3323 WT^PpyRE9/DsRed) and intracellular amastigotes were isolated and measured by flow cytometry. (d) DsRed expression measured by flow cytometry after promastigote back-transformation of DsRed^hi and DsRed^lo (i.e. quiescent) amastigotes recovered from LT-HSC. (e) L. infantum LEM3323 WT^PpyRE9/DsRed amastigotes recovered from infected mouse LT-HSC and measured via flow cytometry, back-gated on SSC versus FSC. Mann-Whitney test, *p < 0.05, six independent repeats. (f) Sorted mouse LT-HSC were infected with L. infantum (LEM3323 WT^PpyRE9/DsRed) and processed for microscopy. DAPI (blue), amastigotes (red). Scale bar = 10 μm. (g) Analysis of microscopy images of (f) in the FIJI software, comparing the expression level of DsRed to its respective size.

Fig 2. Number of divisions associated with quiescence in amastigotes from mouse LT-HSC and human HSPC.

(a) Number of divisions as calculated by CFSE staining and defined by curve-fitting of the cellular CFSE-intensity using the Proliferation Analysis tool of FlowLogic (left panel). Controls are unstained and CFSE L. infantum (LEM3323 WT^PpyRE9/DsRed) promastigote cultures. Sorted mouse LT-HSC were infected with CFSE labelled L. infantum promastigotes and amastigotes were recovered after 6 hours of co-incubation (right panel). (b) Percentage of cells in each division range from (a). Results are based on three independent repeats. (c) Number of divisions as calculated by CFSE staining using the Proliferation Analysis tool (left panel). Controls include unstained and CFSE-stained L. infantum (LLM2346 WT^PpyRE9/DsRed) promastigote cultures. Sorted human HSPC were infected with CFSE labelled L. infantum promastigotes and amastigotes were recovered after 24 hours of co-incubation (right panel). (d) Percentage of cells in each division range from (c). Results are based on three independent repeats. (e) Early infection of L. infantum (LEM3323 WT^PpyRE9/DsRed) in mouse LT-HSC at 1, 3, 6, and 24 hours post infection visualised by Giemsa staining. Amastigotes (arrow), promastigotes (dotted arrow), promastigotes transitioning to amastigotes, already without flagellum (asterisk), dividing amastigotes (red asterisk).

Fig 3. Phenotypic characteristics of quiescent amastigotes from LT-HSC.

(a-b) Sorted LT-HSC were infected with L. infantum (LEM3323 WT^PpyRE9/DsRed) for 24 hours followed by treatment with 250 μM PMM or 7.5 μM MIL for 72 hours. To compare pre- and post-treatment distribution of quiescent parasites, amastigotes were isolated and remeasured on the FACSMelody. (c) Sand flies were infected by L. infantum LEM3323 promastigotes recovered from DsRed^hi and quiescent amastigotes in mouse LT-HSC. The parasite load in the gut was assessed at days 5, 7, 9 and 12 after infection (blood meal). Sand fly infections were repeated three independent times. Unpaired t test, 10 < n < 30, *p <0.05. (d) L. infantum LEM3323 promastigotes of a control culture and promastigotes recovered from DsRed^hi and quiescent parasites in mouse LT-HSC, pre- and post-sand fly passage (pre- and post-SF) were co-incubated with peritoneal macrophages for 96 hours and infectivity was assessed with Giemsa staining. Cultures were visually confirmed to contain > 90% metacyclics and normalized by counting to expose macrophages at a MOI of 5 (S6 Fig). The original promastigote culture was used as a control and infectivity was found to be significantly lower than those of pre-SF DsRed^hi (p <0.01), post-SF DsRed^hi (p <0.05), and pre- and post-SF quiescent parasites (p <0.0001). Ordinary one-way ANOVA, 30 < n < 130, ****p <0.0001. % infected cells and number of parasites per 100 macrophages are included in S1 Table. (e) Sand flies were infected with L. infantum LLM2346 promastigotes, recovered from DsRed^hi and quiescent amastigotes in human HSPC. The parasite load in the gut was assessed at days 5, 7, 9 and 12 after the infectious blood meal. Sand fly infections were repeated three independent times. 10 < n < 34. (f) L. infantum LLM2346 promastigotes of DsRed^hi and quiescent recovered promastigotes from human HSPC were co-incubated with peritoneal macrophages for 96 hours and infectivity was assessed with Giemsa. Mann-Whitney test, n = 100, ****p <0.0001. (g) DsRed expression of L. infantum LEM3323 DsRed^hi and quiescent promastigotes recovered from mouse LT-HSC before or after passage through the sand fly, and L. infantum LLM2346 DsRed^hi and quiescent promastigotes recovered from human HSPC. Mann-Whitney test, **p <0.01. All experiments are expressed as mean ± SEM.

Fig 4. Phenotypic characteristics of promastigotes from relapsed BM.

(a) BALB/c mice were infected with 10⁸ stationary phase promastigotes of L. infantum LEM3323 WT^PpyRE9/DsRed. One group was treated with PMM 350 mg/kg per day (IP) for 5 consecutive days. Infection was followed up by BLI, BM was collected at 6 weeks post infection (wpi) from untreated and relapsed mice. (b) Stationary phase promastigotes recovered from relapsed BM (relapse) and untreated BM (infection) were co-incubated at a MOI of 5 with peritoneal macrophages for 96 hours and infectivity was assessed with Giemsa. Parasite cultures were counted and visually confirmed to contain > 90% metacyclics to exclude an infectivity bias based on the parasite culture (S6 Fig). Mann-Whitney test, n = 100, ***p <0.001. % infected cells and number of parasites per 100 macrophages are included in S1 Table. (c) L. infantum LEM3323 promastigotes recovered from relapsed BM (relapse) and untreated BM (infection) were co-incubated with peritoneal macrophages for 24 hours and treated with 120 μM, 250 μM or 350 μM PMM for 72 hours, infectivity was assessed with Giemsa. (d) Sand flies were infected by L. infantum LEM3323 promastigotes recovered from relapsed BM (relapse) and untreated BM (infection) and the parasite load in the gut of infected flies was assessed at days 5, 7, 9 and 12 after infection (blood meal). Sand fly infections were repeated three independent times. 10 < n < 32.