Parasites cultures conditions

Promastigote forms of Leishmania (Leishmania) infantum (MHOM/BR/1974/PP75) expressing red fluorescence (tdTomato) [17] and/or constitutively expressing Cas9 (plasmid pTB007; [18]) were cultured at 26°C in M199 medium (Gibco – Thermo Fischer Scientific, Grand Island, NY, USA) supplemented with the following components: 40 mM HEPES (pH 7.4), 5 μg/mL hemin, 2 μg/mL biopterin, 1 μg/mL biotin, 2 mM L-glutamine, 500 U/mL penicillin, 50 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum. Cultures were maintained by biweekly passages, inoculating 1 × 10⁶ parasites per 5 mL of medium.

Generation of DUSP-overexpressing L. infantum mutants

DUSP-overexpressing L. infantum promastigotes expressing tdTomato were generated using the pIR1_SAT expression vector. Briefly, the DUSP coding sequence (TritrypDB: LINF_340027100) was amplified by PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Grand Island, NY, USA) and assembled into the SwaI-linearized pIR1_SAT vector using Gibson Assembly (New England Biolabs, Ipswich, MA, USA). Primers (S1 Table) were designed to generate 25–30 bp overlaps between the insert and the vector backbone using the NEBBuilder Assembly Tool. The assembly reaction was transformed into chemically competent Escherichia coli TOP10F’ cells. Positive clones were screened by colony PCR and confirmed by Sanger sequencing. Parasites were transfected with circular plasmid form (New England Biolabs, Ipswich, MA, USA) and selected with 50 μg/mL nourseothricin sulfate (Sigma-Aldrich - Merck, St. Louis, MO, USA) to establish stable overexpressing lines. Transfections were performed as previously described by [19]. All primers used for Gibson Assembly construction are listed in S1 Table.

Attempt to generate DUSP L. infantum knockout

The initial attempt to generate DUSP (Tritryp DB: LINF_340027100) knockout L. infantum parasites was carried out using the CRISPR/Cas9 system [18]. Promastigotes expressing tdTomato, Cas9, and T7 RNA polymerase were co-transfected with PCR-amplified sgRNA and donor DNA fragments, which were selected using the LeishGEdit tool. Donor DNAs were amplified by PCR with specific primer pairs using plasmids pTNEO_v1 and pTPURO_v1 as templates, which confer resistance to neomycin (NEO) (Geneticin, G418 Sulfate, Gibco – Thermo Fischer Scientific, Grand Island, NY, USA)) and puromycin (PURO) (Sigma-Aldrich - Merck, St. Louis, MO, USA), respectively. For transfections, promastigotes were electroporated with equimolar amounts of sgRNAs targeting the 5′ and 3′ untranslated regions (UTRs) along with the corresponding donor DNAs. Parasites were subsequently selected using combinations of 40 μg/mL NEO with 10, 15, or 20 μg/mL PURO. Heterozygous knockout line (Li DUSP^+/-) was obtained by transfecting parasites with sgRNAs targeting the 5′ UTR and 3′ UTR and the NEO donor DNA alone, followed by selection with 40 μg/mL NEO.

In a subsequent attempt to generate endogenous DUSP-deficient mutants, the tdTomato- and Cas9-expressing L. infantum line was first transfected with the pIR1_SAT-DUSP vector, which carries nourseothricin resistance as a selectable marker, as described above. The pIR1_SAT-DUSP vector was used in its circular form without prior digestion. Following selection with 50 μg/mL nourseothricin sulfate, parasites were transfected with donor DNA fragments and sgRNA templates designed to disrupt the chromosomal DUSP locus, aiming to obtain either complete or single-knockout lines. Donor DNAs were amplified by PCR using plasmids pTNeo v1 and pTPuro v1 as templates, conferring resistance to neomycin (NEO) and puromycin (PURO), respectively. Parasites were selected using either 15 μg/mL PURO plus 40 μg/mL NEO, or 20 μg/mL PURO plus 60 μg/mL NEO for DUSP null mutant lines selection. All transfections were performed as previously described [19]. All primers used for plasmid and DNA fragment construction are listed in S1 Table.

Quantification of transcript levels

To assess transcript levels of the target gene, quantitative reverse-transcription PCR (RT-qPCR) analysis was performed using cDNA from mutants and L. infantum controls parasites. Promastigotes (approximately 1 × 10⁸ cells) were resuspended in 1 mL of TRIzol Reagent (Invitrogen - Thermo Fisher Scientific, Waltham, MA, USA), and total RNA was extracted using the chloroform phase-separation method. The RNA samples were treated with the Turbo DNA-free Kit (Invitrogen - Thermo Fisher Scientific, Waltham, MA, USA) to remove residual genomic DNA, and complementary DNA (cDNA) was synthesized with SuperScript II Reverse Transcriptase (Invitrogen - Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions

cDNA samples were diluted to a final concentration of 100 ng/μL and used for amplification reactions with Power SYBR Green Master Mix (Applied Biosystems - Thermo Fisher Scientific, Waltham, MA, USA). Specific primers used for DUSP quantification are listed in S1 Table. Amplifications were performed in triplicate in a 96-well plate using the QuantStudio 12 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) under the following cycling conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, and a final dissociation stage at 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s.

The constitutive DNA polymerase I gene (Tritryp DB: LINF_160021500) was used as an endogenous reference for normalization. Relative quantification of mRNA expression was calculated using the comparative Ct method (2^⁻ΔΔCt) [20]. Three independent biological replicates were performed for each parasite line, with each replicate analyzed in technical quadruplicate.

Growth curve of promastigotes forms

L. infantum promastigote forms (1 × 10⁵ parasites/mL) were cultured in M199 medium (Gibco – Thermo Fischer Scientific, Grand Island, NY, USA) at 26°C, and parasite density was monitored daily using a Z1 Coulter Particle Counter (Beckman Coulter, Brea, CA, USA). All assays were carried out in triplicate across three independent biological experiments.

Ficoll enrichment of metacyclic parasites

Promastigote cultures of Li Cas9, Li DUSP^+/−, Li WT, and Li WT + pIR1_SAT-DUSP (1 × 10⁵ parasites/mL) were maintained at 26 °C for five days. Parasites were subsequently collected, and metacyclic forms were isolated by Ficoll gradient centrifugation according to [21], with minor modifications. Briefly, Ficoll 400 (Sigma-Aldrich - Merck, St. Louis, MO, USA) was used to prepare a 20% stock solution in PBS and diluted for preparation of 10% Ficoll solution. Two ml of 20% Ficoll were overlaid by 2 ml of 10% Ficoll and 2 ml of parasite suspension were layered on top of the Ficoll cushion. Tubes were centrifuged at 2,500 rpm for 15 min at room temperature without brake. The metacyclic-enriched fractions were recovered at the interface between the 20% and 10% Ficoll layers. Parasites were washed with PBS, and cell numbers were determined using a Neubauer hemocytometer. Three independent experiments were performed in duplicate.

Morphological and morphometric analysis of L. infantum promastigotes

Promastigotes from control and mutant L. infantum lines were collected at 48 and 96 hours after the initial inoculation (1 × 10⁵ parasites/mL) and washed twice with phosphate-buffered saline (PBS, pH 7.4). Parasite suspensions were then applied to glass slides, allowed air dry, and fixed with methanol for 5 minutes. Fixed slides were stained with 10% Giemsa solution (Sigma-Aldrich - Merck, St. Louis, MO, USA) for 15–20 minutes, rinsed gently with distilled water, and air-dried. Parasite morphology was evaluated under a light microscope (Axio Observer.A1, Zeiss, Germany) at 63 × magnification using oil immersion, and images were captured using a digital camera attached to the microscope.

Morphometric analyses of promastigotes from control and mutant L. infantum lines were performed using digital images obtained by light microscopy. Flagellar and cell body lengths were measured using ImageJ software (National Institutes of Health, USA). For each experimental group, 50 individual parasites were randomly selected and analyzed. Flagellar length was measured from the flagellar pocket to the distal tip of the flagellum, and cell body length was measured along the longitudinal axis of the parasite. Measurements were obtained from two independent experiments performed in duplicate.

Cell cycle analysis

Promastigotes (1 x 10⁶ parasites) of L. infantum (control and mutants) were harvested after 48 and 96 hours of growth (initial inoculation of 1 x 10⁵ parasites/mL), washed twice with phosphate-buffered saline (PBS, pH 7.4), and fixed in 70% ethanol at 4°C overnight. Fixed cells were washed with PBS and incubated for 30 minutes at room temperature in the dark with a staining solution containing propidium iodide (PI, 50 μg/mL; Sigma-Aldrich - Merck, St. Louis, MO, USA) and RNase A (100 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) to simultaneously stain DNA and remove RNA [22]. Cell cycle distribution was analyzed using flow BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), acquiring 30,000 events per sample. Data were processed using FlowJo software (v10), and the percentage of cells in sub G0/G1, G0/G1, S, and G2/M phases was determined. Only singlet events were considered for the analysis of DNA content and cell cycle distribution. Experiments were performed in three independent biological replicates.

Infection of THP-1 cell-derived macrophages

THP-1 cells (a human monocyte lineage) were cultured in RPMI-1640 (Sigma-Aldrich - Merck, St. Louis, MO, USA) medium supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum, and maintained at 37°C in a 5% CO₂ atmosphere. To induce differentiation into macrophages, cells were treated with 20 ng/mL phorbol myristate acetate (PMA; Sigma-Aldrich - Merck, St. Louis, MO, USA) and incubated in black 96-well plates under the same conditions. After 72 hours, differentiated macrophages were infected with Leishmania promastigotes from the second day of the stationary phase at a ratio of 20 parasites per macrophage for 4 hours. Non-internalized parasites were removed by washing, and the infected macrophages were then maintained in RPMI-1640 medium for 72 hours. The intracellular amastigote development was assessed at 4- and 72-hours post-infection by measuring fluorescence intensity using a microplate reader (Variokan Lux, Thermo Fisher Scientific, Waltham, MA, USA) (excitation at 554 nm and emission at 581 nm) [17].

Effective concentration (EC₅₀) assays in promastigotes

Parasite susceptibility to different compounds was assessed using L. infantum promastigotes from both control and mutant lines (2 × 10⁶ parasites/mL) cultured in 1 mL of M199 medium containing increasing concentrations of each compound: 25–350 μM potassium antimony tartrate (Sigma-Aldrich, St. Louis, MO, USA), 0.05-0.4 μM amphotericin B (Sigma-Aldrich, St. Louis, MO, USA), 5–40 μM miltefosine (Cayman Chemical, Ann Arbor, MI USA), and 30–210 μM hydrogen peroxide (Sigma-Aldrich - Merck, St. Louis, MO, USA). After 48 hours of incubation at 26°C, parasite viability was evaluated by measuring the fluorescence intensity of tdTomato-expressing parasites using a microplate fluorescence reader (Variokan Lux, Thermo Fisher Scientific, Waltham, MA, USA) with excitation and emission wavelengths of 554 nm and 581 nm, respectively [17]. The half-maximal effective concentration (EC₅₀) values were calculated by non-linear regression using GraphPad Prism 8.2.0 from data obtained in three independent biological replicates, each performed in triplicate.

Effective concentration (EC₅₀) assays in intracellular amastigotes

Following infection as described above, THP-1 cell–derived macrophages were incubated in RPMI-1640 medium containing increasing concentrations of trivalent antimony (2.3 – 300 µM) or hydrogen peroxide (1.2 – 600 µM) for 72 hours. After treatment, parasite viability was assessed by measuring the decrease in fluorescence intensity of tdTomato-expressing parasites using a fluorescence microplate reader (Variokan Lux, Thermo Fisher Scientific, Waltham, MA, USA) with excitation and emission wavelengths of 554 nm and 581 nm, respectively [17]. The half-maximal effective concentration (EC₅₀) values were calculated by non-linear regression using GraphPad Prism 8.2.0 from data obtained in three independent biological experiments, each performed in triplicate.

Axenic amastigote-like culture for quantification of gene transcript levels

L. infantum promastigotes were differentiated into axenic amastigote-like forms following an established protocol [23,24] with minor modifications. Briefly, promastigotes in the stationary growth phase were harvested, washed twice with PBS, and resuspended in RPMI medium (Sigma-Aldrich - Merck, St. Louis, MO, USA) supplemented with 20% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The pH was adjusted to 5.5 to mimic the phagolysosomal environment. Parasites were incubated at 37°C in a 5% CO₂ atmosphere. Axenic amastigote-like morphology and viability were monitored daily by light microscopy. Parasites were allowed to differentiate for 3–5 days, and the resulting axenic amastigote-like forms were used for subsequent analyses.

Transcriptional analysis of MAPKs in promastigotes and axenic amastigote-like forms

The transcriptional levels of MAPK1 (Tritryp DB: LINF_360076200), MAPK3 (Tritryp DB: LINF_100011600), and MAPK10 (Tritryp DB: LINF_100007100) were assessed in L. infantum promastigote and axenic amastigote-like forms of mutants using RT-qPCR. cDNA was prepared as described above, and RT-qPCR reactions were carried out under the same conditions, with the only difference being the use of gene-specific primers for each MAPK (S1 Table). Relative transcript levels were calculated using the comparative Ct (2^⁻ΔΔCt) method, with the constitutive DNA polymerase I gene serving as an endogenous reference. All reactions were performed in triplicate across three independent biological replicates.

Statistical analysis

Data were analyzed using GraphPad Prism version 8.2.0 (San Diego, CA, USA). Comparisons between Li Cas9 and Li DUSP^+/- or Li WT and Li WT + pIR1_SAT-DUSP parasites were performed using ordinary one-way or two-way ANOVA followed by Bonferroni’s post hoc test. Analysis between two groups was performed using t-test. Differences were considered statistically significant at p < 0.05.

Genomic Localization and Predicted Structure of the DUSP/Kinatase LINF_340027100

A search using the TriTrypDB resource [25] for DUSP LINF_340027100 reveals that it corresponds to LmjF.34.2190, a protein identified as an atypical DUSP “Kinatase” [9]. A protein-level comparison of LINF_340027100 from L. infantum and LmjF.34.2190 from L. major is presented in S1 Fig, which shows a CLUSTAL O (1.2.3) multiple sequence alignment. The genome of the L. infantum JPCM5 strain encodes a single copy of the atypical DUSP/Kinatase gene. This gene spans 4,149 base pairs and encodes a protein of 1,382 amino acids.

Analysis of the LINF_340027100 protein sequence using PFAM data retrieved from TriTrypDB (release 68, May 2024) revealed a multi-domain architecture characteristic of atypical kinatases (S2A Fig). The protein contains a single Protein Kinase domain (PF00069; Pkinase), a Dual-Specificity Phosphatase catalytic domain (PF00782; DSPc), and a Leucine-Rich Repeat domain (PF13855; LRR_8). Consistent with this annotation, the protein is assigned to the KEGG orthologous group K14165, which corresponds to atypical DUSPs with predicted phosphatase activity against phosphotyrosine, phosphoserine, and phosphothreonine substrates (EC 3.1.3.16; EC 3.1.3.48).

Structural predictions derived from the AlphaFold database (entry A4I9Z1) reveal a multi-domain architecture comprising seven distinct structural regions, with large portions of the model displaying high or very high confidence scores (pLDDT) (S2B and S2C Fig).

Modulation of DUSP/Kinatase LINF_340027100 expression in Leishmania infantum: Generation of loss- and gain-of-function lines

An initial attempt to generate DUSP knockout L. infantum lines using the CRISPR/Cas9 system was unsuccessful, as no viable parasites were recovered after antibiotic selection, suggesting that DUSP is essential for promastigote survival. A second strategy was then employed, in which DUSP was first expressed episomally, followed by CRISPR/Cas9-mediated disruption of the chromosomal copy. Correct integration of the NEO and PURO selection cassettes was confirmed by PCR using primer pairs targeting the 5′ UTR region adjacent to the donor DNA and the coding sequences of NEO or PURO (S3A and S3B Fig). However, PCR analysis revealed that the endogenous DUSP locus was still present in these transfectants (S3C Fig). This outcome suggests the retention of at least one copy of DUSP, likely through aneuploidy or gene amplification, despite successful integration of both selectable markers. Collectively, these findings suggest that DUSP is indispensable for the viability of L. infantum promastigotes.

In contrast, viable parasites were successfully obtained by deleting a single DUSP allele, resulting in the heterozygous knockout mutant L. infantum line Li DUSP^+/-. This line was obtained by transfecting L. infantum Cas9-expressing parasites with sgRNAs targeting the 5′ and 3′ untranslated regions (UTRs) of DUSP, together with a donor cassette containing the neomycin resistance (NEO) gene for selection. Homologous recombination following sgRNA-directed cleavage at the target locus replaced one DUSP allele with the NEO cassette. Correct integration of the NEO cassette into the L. infantum genome was confirmed by PCR using primer P1, which anneals to the 5′ UTR of DUSP, and primer P2, specific to the NEO sequence (Fig 1A). Additional PCR using DUSP-specific primers confirmed the presence of a single intact allele in Li DUSP^+/-parasites, consistent with the replacement of only one DUSP copy (Fig 1B). Quantitative analysis of transcript expression level of the DUSP in the mutant Li DUSP^+/- line is shown in Fig 1C. Deletion of one DUSP allele resulted in a 2.1-fold decrease in transcript expression levels.

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Fig 1. Generation of L. infantum Lines with altered DUSP/Kinatase LINF_340027100 expression.

(A) The correct integration of the resistance marker neomycin in Li DUSP^+/- parasites (944 bp) was evaluated by PCR by annealing a primer in a 5′UTR region adjacent to the cassette (primer P1) and another primer annealed within resistance marker sequence (primer P2). (B) Fragment DUSP-coding sequence in Li DUSP^+/- parasites (532 bp) was amplified using PCR with primers P3 and P4. (C) Relative DUSP transcript level in Li DUSP^+/- parasites. (D) Relative DUSP transcript level in Li WT + pIR1_SAT-DUSP parasites. Relative DUSP transcript levels were determined by RT-qPCR. Relative quantification was performed using the 2^⁻ΔΔCt method, with the DNA polymerase gene used as an internal reference for normalization. T test was used to compare control parasite Li Cas9 and Li DUSP^+/- or Li WT and Li WT + pIR1_SAT-DUSP parasites. Data represents the mean ± standard deviation of three independent experiments performed in triplicates. * Represents significant differences in relation to the control parasite (* p < 0.05 and ** p < 0.01). MW: molecular weight; NC: negative control; bp: base pair.

https://doi.org/10.1371/journal.pntd.0014330.g001

Episomal overexpression of DUSP yielded viable L. infantum lines, designated Li WT + pIR1_SAT-DUSP, which exhibited a 2.3-fold upregulation of DUSP transcript levels (Fig 1D).

DUSP/Kinatase LINF_340027100 modulation alters morphology, cell cycle, and metacyclogenesis in L. infantum promastigotes

The growth of Li Cas9 and Li DUSP^+/- L. infantum mutants, as well as Li WT and Li WT + pIR1_SAT-DUSP promastigotes, was assessed by counting parasite numbers at 24-hour intervals. As shown in Fig 2A, Li DUSP^+/− parasites exhibited growth kinetics comparable to those of the Li Cas9 control line, indicating that loss of a single DUSP allele does not significantly impair parasite proliferation. In contrast, DUSP-overexpressing parasites showed a modest reduction in growth relative to Li WT controls, with statistically significant differences observed at two time points along the growth curve (Fig 2B). At 48 h, Li WT parasites reached an average density of 24.1 × 10⁵ parasites/mL, whereas DUSP-overexpressing parasites reached 16.2 × 10⁵ parasites/mL, corresponding to an approximate 32.7% reduction. A more pronounced difference was observed at 72 h, when DUSP-overexpressing parasites (~62.2 × 10⁵ parasites/mL) exhibited approximately 49.5% lower density than Li WT controls (~123.1 × 10⁵ parasites/mL). The complete dataset is presented in S2 Table.

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Fig 2. Growth curve, morphological and cell cycle analysis of promastigote forms of DUSP/Kinatase LINF_340027100 mutant parasites.

An initial inoculum of 1 x 10⁵ parasites per mL was prepared for the Li Cas9, Li DUSP^+/-, Li WT and Li WT + pIR1_SAT-DUSP parasites, which were counted every 24 h using the Z1 Coulter Counter. (A) Growth curve of Li Cas9 and Li DUSP^+/-. (B) Growth curve of Li WT and Li WT + pIR1_SAT-DUSP. Morphological analysis of (C) DUSP heterozygous knockout and (D) overexpressing parasites after 48 hours and 96 hours of growth stained with Giemsa. (E) and (F) cell cycle analysis of DUSP heterozygous knockout at 48 hours and 96 hours, respectively. (G) and (H) cell cycle analysis of Li WT and Li WT + pIR1_SAT-DUSP parasites at 48 hours and 96 hours, respectively. The parasites were stained with propidium iodide and evaluated by flow cytometry. Quantification of metacyclic forms in DUSP heterozygous knockout (I) and DUSP-overexpressing parasites (J). Metacyclic promastigotes were enriched using Ficoll gradient centrifugation. Data represents the mean ± standard deviation of three independent experiments performed in triplicates. Two-way ANOVA with Bonferroni’s post hoc test or T test was used to compare Li Cas9 and Li DUSP^+/-, and Li WT and Li WT + pIR1_SAT-DUSP parasites at each time point. * Represents significant differences in relation to the control parasite (* p < 0.05 and *** p < 0.001). Image bars: 2 µm.

https://doi.org/10.1371/journal.pntd.0014330.g002

Morphological analysis of promastigotes at 48 and 96 hours in culture, after Giemsa staining, revealed distinct phenotypic alterations associated with DUSP modulation. The heterozygous knockout line Li DUSP^+/− displayed an elongated and slender morphology at both points compared with the Li Cas9 control (Fig 2C). An increase in flagellar length was also observed at 48 hours. Quantitative morphometric analysis confirmed these observations. At 48 hours, Li DUSP^+/− parasites exhibited significantly longer flagella (15.9 ± 1.8 µm) than Li Cas9 parasites (8.2 ± 0.2 µm), as well as increased cell body length (14.9 ± 1.4 µm vs 8.6 ± 0.3 µm, respectively). Similar differences were observed at 96 hours, when Li DUSP^+/− parasites showed significantly greater cell body length than controls (13.9 ± 0.2 µm vs 11.8 ± 0.1 µm). No statistically significant difference in flagellar length was detected at 96 hours (9.0 ± 0.3 µm for Li DUSP^+/− vs 10.4 ± 0.5 µm for Li Cas9).

In contrast, DUSP-overexpressing parasites displayed a rounded cell body and reduced overall size, with significantly shorter flagella at both time points compared with Li WT controls (Fig 2D). Morphometric analysis confirmed this phenotype. At 48 hours, DUSP-overexpressing parasites exhibited significantly shorter flagella (6.4 ± 0.4 µm) than Li WT parasites (9.6 ± 0.2 µm), along with reduced cell body length (7.2 ± 0.6 µm vs 10.6 ± 0.5 µm). A similar pattern was observed at 96 hours, when overexpressing parasites showed significantly shorter flagella (8.2 ± 0.6 µm vs 12.7 ± 0.6 µm) and reduced cell body length (6.5 ± 0.4 µm vs 9.2 ± 0.4 µm) compared with Li WT controls. Together, these findings indicate that DUSP expression levels play a key role in maintaining promastigote morphology and flagellar architecture, likely through effects on cytoskeletal organization and dynamics.

Cell-cycle distribution of L. infantum promastigote lines was analyzed after 48 and 96 hours of cultivation in medium using propidium iodide staining. The heterozygous knockout Li DUSP^+/− line exhibited a reduced proportion of cells in the G0/G1 phase compared with Li Cas9 controls at 48 h (Fig 2E). This reduction persisted at 96 h and was accompanied by a corresponding accumulation of cells in S phase (Fig 2F). DUSP-overexpressing parasites also displayed an increased proportion of cells in S phase at both time points (48 and 96 h) compared with Li WT controls (Fig 2G-2H). Representative histograms are shown in S4 Fig. Collectively, these findings indicate that precise regulation of DUSP expression is required to maintain normal promastigote proliferation and proper cell-cycle progression.

Additionally, we evaluated whether modulation of DUSP expression affects metacyclogenesis. Metacyclic forms were enriched and quantified by Ficoll gradient centrifugation. Our results revealed a significant 33.5% reduction in the number of metacyclic forms in Li DUSP^+/− cultures compared with the Li Cas9 control (10.54 ± 0.96 × 10⁶ vs 15.85 ± 2.77 × 10⁶ parasites, respectively) (Fig 2I). Similarly, DUSP-overexpressing parasites exhibited a significant 18.7% reduction in metacyclic forms relative to Li WT controls (18.11 ± 0.69 × 10⁶ vs 22.27 ± 2.14 × 10⁶ parasites, respectively) (Fig 2J). These results indicate that both decreased and increased DUSP expression impair metacyclogenesis.

Effect of DUSP/Kinatase LINF_340027100 modulation on L. infantum infectivity in macrophages

The effect of DUSP modulation on parasite infectivity was assessed using THP-1–derived macrophages infected with L. infantum promastigotes, with intracellular parasite burden quantified by fluorescence intensity. After 4 hours of infection, Li DUSP^+/- parasites exhibited an infection profile comparable to that of the Li Cas9 control (Fig 3A). However, at 72 hours post-infection, a significant decrease in fluorescence was observed, indicating reduced intracellular replication of the DUSP heterozygous knockout mutant Li DUSP^+/- parasites (Fig 3A). In contrast, DUSP-overexpressing parasites displayed higher fluorescence at 4 hours post-infection (Fig 3B), suggesting enhanced macrophage entry, but markedly lower fluorescence after 72 hours, consistent with impaired intracellular replication relative to Li WT controls (Fig 3B). Collectively, these results suggest that both downregulation and overexpression of DUSP disturb the balance required for optimal L. infantum entry and replication within host macrophages.

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Fig 3. Analysis of the infectivity of DUSP/Kinatase LINF_340027100 heterozygous knockout and overexpressing parasites in THP-1 derived-macrophages.

The fluorescence emission (R.F.U) of parasites (A) Li Cas9 and Li DUSP^+/- and (B) Li WT and Li WT + pIR1_SAT-DUSP after 4 and 72 hours of infection using THP-1 derived-macrophages. Two-way ANOVA with Bonferroni’s post hoc test was used to compare Li Cas9 and Li DUSP^+/-, and Li WT and Li WT + pIR1_SAT-DUSP parasites at each time point. Data represents the mean ± standard deviation of three independent experiments performed in triplicates. * Represents significant differences in relation to the control (* p < 0.05).

https://doi.org/10.1371/journal.pntd.0014330.g003

DUSP/Kinatase LINF_340027100 expression alters susceptibility to oxidative stress and trivalent antimony in L. infantum promastigotes

We evaluated the susceptibility of L. infantum promastigotes from Li Cas9 and Li DUSP^+/- mutants, as well as Li WT and Li WT + pIR1_SAT-DUSP parasites, to antileishmanial drugs, including trivalent antimony, amphotericin B, and miltefosine. In addition, we assessed the tolerance of these parasite lines to hydrogen peroxide, an inducer of oxidative stress (Fig 4).

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Fig 4. In vitro susceptibility analysis of DUSP/Kinatase LINF_340027100 mutant L. infantum promastigotes.

Promastigotes parasites were cultured in the presence of different concentrations of (A) and (E) potassium antimony tartrate (Sb^III) (25 to 350 μM), (B) and (F) amphotericin B (0.05 to 0.4 μM), (C) and (G) miltefosine (5 to 40 μM), and (D) and (H) hydrogen peroxide (30 to 210 μM). Parasite growth was determined after 48 h of incubation in the presence or absence of drugs. Data was plotted on dose-response curves in which the mean ± standard deviation of three independent experiments performed in triplicate are represented. The EC₅₀ was determined using the linear interpolation method. Two-way ANOVA with Bonferroni’s post hoc test was applied to compare Li Cas9 and Li DUSP^+/-, and Li WT and Li WT + pIR1_SAT-DUSP parasites for each drug concentration. * Represents significant differences in relation to the control (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).

https://doi.org/10.1371/journal.pntd.0014330.g004

Reduction of DUSP expression rendered the heterozygous knockout Li DUSP^+/- parasites approximately 1.5-fold more sensitive to hydrogen peroxide compared with the L. infantum Cas9-expressing control (EC₅₀: 135.30 μM for Li Cas9 vs. 92.07 μM for Li DUSP^+/-) (Fig 4D). In contrast, no significant differences in susceptibility were observed for trivalent antimony (EC₅₀: 47.72 vs. 55.07 μM) (Fig 4A), amphotericin B (EC₅₀: 0.07 μM vs. 0.08 μM) (Fig 4B), or miltefosine (EC₅₀: 24.61 μM vs. 22.11 μM) (Fig 4C).

Conversely, DUSP-overexpressing parasites exhibited a 2.6-fold increase in resistance to trivalent antimony compared with Li WT parasites (EC₅₀: 86.64 μM for Li WT vs. 226.40 μM for Li WT + pIR1_SAT-DUSP) (Fig 4E). No significant changes were detected in susceptibility to amphotericin B (EC₅₀: 0.11 μM vs. 0.10 μM) (Fig 4F), miltefosine (EC₅₀: 10.56 μM vs. 9.58 μM) (Fig 4G), or hydrogen peroxide (EC₅₀: 64.13 μM vs. 72.20 μM) (Fig 4H).

Together, these findings suggest that DUSP acts as a key modulator of both redox balance and antimony susceptibility in L. infantum, with its overexpression promoting antimony resistance and its depletion increasing oxidative stress sensitivity.

DUSP/Kinatase LINF_340027100 modulation affects oxidative stress response and antimony susceptibility in L. infantum intracellular amastigotes

Given that Li DUSP^+/- promastigotes exhibited increased sensitivity to hydrogen peroxide and Li WT + pIR1_SAT-DUSP parasites displayed enhanced resistance to trivalent antimony, we next examined whether these phenotypes were maintained in the intracellular amastigote stage. THP-1-derived macrophages were infected and subsequently exposed to serial concentrations of the respective compounds to determine EC₅₀ values. Our results revealed that heterozygous knockout Li DUSP^+/- intracellular amastigotes were 5.7-fold more sensitive to hydrogen peroxide than Li Cas9 controls (EC₅₀: 106.80 μM for Li Cas9 vs. 18.73 μM for Li DUSP^+/-) (Fig 5A). Conversely, DUSP-overexpressing intracellular amastigotes exhibited a 3.1-fold increase in resistance to trivalent antimony compared with Li WT parasites (EC₅₀: 3.61 μM for Li WT vs. 11.08 μM for Li WT + pIR1_SAT-DUSP) (Fig 5B). These findings suggest that DUSP expression has an even greater impact on parasite susceptibility during the intracellular amastigote stage, reinforcing its potential role in modulating redox balance and antimony tolerance within the host environment.

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Fig 5. In vitro susceptibility analysis of DUSP/Kinatase LINF_340027100 mutant L. infantum intracellular amastigotes and quantification of DUSP transcript levels in axenic amastigote-like forms.

The THP-1-derived macrophages were infected with the (A) Li Cas9 and Li DUSP^+/- and treated with hydrogen peroxide (1.2 to 600 µM) for 72 h; and (B) Li WT and Li WT + pIR1_SAT-DUSP and treated with trivalent antimony (2.3 to 300 µM) for 72 h. Data were plotted on dose-response curves in which the mean with standard deviations of three independent experiments performed in triplicate are represented. The EC₅₀ was determined using the linear interpolation method. (C) Relative DUSP transcript level in Li DUSP^+/- axenic amastigotes-like forms. (D) Relative DUSP transcript level in Li WT + pIR1_SAT-DUSP axenic amastigotes-like. Relative DUSP gene transcript levels were determined by RT-qPCR. Relative quantification was performed using the 2^⁻ΔΔCt method, with the DNA polymerase gene used as an internal reference for normalization. Data represents the mean ± standard deviation of three independent experiments performed in triplicates. The T test was used to compare control parasite Li Cas9 and Li DUSP^+/- or Li WT and Li WT + pIR1_SAT-DUSP parasites. * Represents significant differences in relation to the control (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).

https://doi.org/10.1371/journal.pntd.0014330.g005

Given the pronounced phenotypes observed in intracellular amastigotes, we next examined DUSP expression in axenic amastigote-like forms derived from the L. infantum lines. cDNA was prepared from axenic amastigote-like parasites to quantify DUSP transcript levels. The heterozygous knockout Li DUSP^+/− line exhibited a 71.4-fold reduction in transcript abundance (Fig 5C) compared with Li Cas9 controls. Conversely, DUSP-overexpressing amastigote-like forms displayed a 2.8-fold increase in transcript levels (Fig 5D) relative to Li WT parasites. These results indicate that DUSP expression is stage-regulated and support an important role for this phosphatase in maintaining amastigote physiology and adaptation to the intracellular environment.

Modulation of DUSP/Kinatase LINF_340027100 affects the transcriptional regulation of MAPK1, MAPK3, and MAPK10 in L. infantum

To investigate whether modulation of DUSP expression affects MAPK-mediated signaling in L. infantum, we analyzed the transcript levels of three representative members of this kinase family — MAPK1, MAPK3, and MAPK10 (Fig 6). These isoforms were selected because they represent distinct, yet complementary biological roles previously characterized in Leishmania: MAPK1 is essential for parasite survival and trivalent antimony resistance [26–28]; MAPK3 regulates flagellar biogenesis and infectivity [29] and MAPK10 contributes intracellular adaptation [30,31].

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Fig 6. Analysis of MAPK1, MAPK3, and MAPK10 transcript levels in promastigotes and axenic amastigote-like forms of Li Cas9, Li DUSP^+/-, Li WT and Li WT + pIR1_SAT-DUSP.

Relative MAPK1, MAPK3 and MAPK10 transcript level in DUSP heterozygous knockout (A) promastigote and (B) axenic amastigote-like forms, and in DUSP overexpressing (C) promastigote and (D) axenic amastigote-like forms. Relative transcript levels were determined by RT-qPCR. Relative quantification was performed using the 2^⁻ΔΔCt method, with the DNA polymerase gene used as an internal reference for normalization. The T test was used to compare control parasite Li Cas9 and Li DUSP^+/- or Li WT and Li WT + pIR1_SAT-DUSP parasites. Data represents the mean ± standard deviation of three independent experiments performed in triplicates. T test was used to compare control parasite Li Cas9 and Li DUSP^+/- or Li WT and Li WT + pIR1_SAT-DUSP parasites. *Represents significant differences in relation to the control parasite (* p < 0.05, ** p < 0.01 and **** p < 0.0001).

https://doi.org/10.1371/journal.pntd.0014330.g006

Together, their evaluation provides an integrated view of how DUSP modulation may influence the overall dynamics of MAPK signaling. Transcript expression levels were quantified by RT-qPCR in both promastigote and axenic amastigote-like stages of the DUSP heterozygous knockout and DUSP-overexpressing L. infantum lines, along with their respective control lines. Because MAPK activation primarily occurs via post-translational phosphorylation, transcriptional profiling was used as an indirect approach to infer regulatory adjustments in MAPK signaling, potentially reflecting compensatory responses to altered DUSP expression.

In heterozygous knockout Li DUSP + / − promastigotes, transcript levels of MAPK1 and MAPK3 were reduced by 1.7- and 1.4-fold, respectively, whereas MAPK10 expression remained comparable to that of the Li Cas9 control (Fig 6A). In contrast, in the axenic amastigote-like stage, all three MAPKs exhibited marked transcriptional downregulation, with approximately 4.0-, 3.1-, and 2.3-fold reductions in MAPK1, MAPK3, and MAPK10 transcript levels, respectively (Fig 6B). These results indicate a broad perturbation of MAPK signaling homeostasis in the DUSP heterozygous knockout line. Collectively, these findings suggest that partial depletion of DUSP disrupts the regulatory balance of kinase signaling, potentially triggering compensatory feedback mechanisms that adjust MAPK gene expression in response to altered phosphorylation dynamics.

Conversely, DUSP-overexpressing promastigotes exhibited significant upregulation of MAPK1, MAPK3, and MAPK10 transcripts, with increases of 2.9, 1.7, and 2.1-fold, respectively (Fig 6C). This pattern suggests a compensatory transcriptional response to DUSP overexpression aimed at restoring signaling equilibrium. In contrast, no significant changes in MAPK transcript levels were detected in the axenic amastigote-like stage (Fig 6D), indicating that stage-specific regulatory mechanisms may limit the transcriptional compensation observed in promastigotes.