https://orcid.org/0000-0002-7128-6208
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Figures
Abstract
Leishmania parasites cause a spectrum of diseases known as leishmaniases and must acquire nutrients like iron while surviving host defenses. Aquaglyceroporin 1 (AQP1) is a membrane channel that, in L. major, localizes to the flagellum and mediates antimony uptake and cell-volume regulation. Here, we show that in L. amazonensis AQP1 is instead targeted to glycosomes and that its expression is modulated by iron availability. A CRISPR-Cas9–mediated knockout of AQP1 in L. amazonensis revealed its multifunctional importance. AQP1-null promastigotes displayed a significant growth defect, particularly under iron-depleted conditions, and were impaired in regulating cell volume under osmotic stress. The mutant parasites contained approximately 50% less intracellular iron than wild-type cells and showed an increase in total superoxide dismutase activity, underscoring a role for AQP1 in iron homeostasis and oxidative stress management. AQP1 deletion also markedly reduced virulence in murine macrophages and in infected mice. Strikingly, loss of AQP1 increased resistance to trivalent antimony (SbIII), a first-line antileishmanial drug. AQP1-knockout promastigotes showed a 70% increase in SbIII IC50 and accumulated more Sb intracellularly than wild-type, suggesting an altered antimony handling. Altogether, L. amazonensis AQP1 is a glycosomal protein that links iron metabolism, osmoregulation, and antimony susceptibility. Its glycosomal targeting and multifaceted roles differ from those of AQP1 orthologs in other Leishmania species. These findings suggest the existence of additional antimony uptake mechanisms beyond AQP1, with implications for understanding drug resistance.
Author summary
Leishmaniases are neglected tropical diseases caused by parasites that survive and multiply inside vertebrates’ cells. These parasites rely on hosts’ nutrients like iron and must resist both host defenses and treatment with toxic drugs such as antimony. We studied a protein called Aquaglyceroporin 1 (AQP1) in Leishmania amazonensis, a species that causes skin lesions in South America. Unlike related species, where AQP1 is found on the parasite’s surface, we discovered that in L. amazonensis AQP1 is located in an internal organelle called glycosome. By deleting this protein from the parasite, we found that it plays a crucial role in iron balance, sensitivity to antimony drugs, and the parasite’s ability to cause disease. Unexpectedly, parasites without AQP1 were more resistant to antimony but still accumulated high levels of the drug, suggesting that Leishmania has other ways of taking up antimony. Our findings challenge the assumption that all Leishmania species use the same strategies to survive, and highlight the need to understand species-specific differences when designing treatments or analyzing parasite biology.
Citation: Boy RL, Zampieri RA, Aoki JI, Coelho AC, Floeter-Winter LM, Laranjeira-Silva MF (2026) Glycosomal Aquaglyceroporin 1 dual role in iron homeostasis and antimony susceptibility in Leishmania amazonensis. PLoS Negl Trop Dis 20(4): e0014141. https://doi.org/10.1371/journal.pntd.0014141
Editor: Syamal Roy, CSIR-Indian Institute of Chemical Biology, INDIA
Received: August 25, 2025; Accepted: March 11, 2026; Published: April 2, 2026
Copyright: © 2026 Boy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are fully available and included in the Supporting information.
Funding: Our research was supported by: Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (The São Paulo Research Foundation): Maria Fernanda Laranjeira- Silva (#2017/23933–3), Romario Lopes Boy (#2022/04551-0), and Lucile Maria Floeter-Winter (#2018/23512–0); Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Brazilian National Council for Scientific and Technological Development): Maria Fernanda Laranjeira-Silva (# 308191/2023-4), Adriano Cappellazzo Coelho (# 305598/2024-4); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (Brazilian Federal Agency for the Support and Evaluation of Graduate Education). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Leishmaniases are a group of neglected tropical diseases caused by kinetoplastid parasites of the genus Leishmania. They affect millions of people worldwide and remain a major public health problem, especially in tropical and subtropical regions where poverty and limited access to healthcare prevail. Clinical manifestations range from self-limiting cutaneous lesions to destructive mucocutaneous forms and fatal visceral disease if left untreated. Current estimates indicate approximately 12 million people are infected across 98 countries in Africa, Asia, the Americas, and Europe, with nearly one million new cases reported annually [1]. Additionally, around one billion people living in endemic areas are at risk of infection. This situation is further aggravated by deforestation and human expansion into forested areas, which facilitate sand fly vectors adapting to domestic environments and contribute to the urbanization of the disease [2].
Leishmania parasites have a heteroxenous life cycle, alternating between phlebotomine sand fly vectors and vertebrate hosts [3]. Within vertebrate hosts, the parasite proliferates as amastigotes inside macrophages and other cells. The survival of Leishmania in these cells depends on its ability to evade host immune defenses, especially within macrophages phagolysosomes where amastigotes confront adverse conditions such as low pH, oxidative stress, and restriction of essential nutrients like iron [4–6].
Iron is essential for Leishmania, supporting processes such as mitochondrial respiration and defense against reactive oxygen species. The parasite acquires iron as ferrous iron (Fe+2) and as heme [5]. Ferrous iron import is mediated by the Leishmania Iron Transporter 1 (LIT1), whereas heme uptake is mediated by heme transporters like Leishmania Heme Response-1 (LHR1) [7]. Maintaining iron homeostasis is crucial since excess iron can catalyze the formation of harmful reactive species, leading to cytotoxicity [8]. To prevent toxic iron accumulation, Leishmania rely on the iron exporter Leishmania
Iron Regulator 1 (LIR1), a plasma membrane protein shown to mediate iron efflux [9].
Trypanosomatids also possess unique peroxisome-related organelles called glycosomes that compartmentalize glycolysis and various other metabolic pathways, including purine salvage, pyrimidine biosynthesis, the pentose phosphate pathway, gluconeogenesis, fatty acid β-oxidation, ether-lipid biosynthesis, and oxidant defense [10,11]. Some of those pathways require iron-dependent enzymes, including the glycosomal superoxide dismutase isoform B (SODB) [12], underscoring the importance of coordinated iron availability for glycosomal function, although the mechanisms governing iron trafficking to this organelle remain poorly understood. The previous transcriptomic analysis of L. amazonensis under iron-deprived conditions identified several differential expressed genes encoding predicted transmembrane proteins with canonical peroxisomal targeting signals (PTS) [13]. One of these is the gene encoding a putative aquaglyceroporin-like protein. Aquaglyceroporins are members of the major intrinsic protein (MIP) superfamily, which form channels that facilitate the transport of water, glycerol and certain metalloids across biological membranes [14]. In L. major, an aquaglyceroporin 1 (AQP1) localized to the flagellum has been implicated in the uptake of trivalent antimony (SbIII) and in antimonial drug resistance [15,16].
Here, we report that L. amazonensis AQP1 contains a C-terminal peroxisomal targeting sequence type 1 (PTS1) and is targeted to glycosomes rather than the flagellum. Using a CRISPR/Cas9 knockout approach combined with phenotypic analyses, we demonstrate that AQP1 contributes to the regulation of intracellular iron content, osmotic balance, parasite growth, infectivity, and susceptibility to antimonials. These findings position AQP1 as a dual regulator of iron trafficking and metalloid transport, revealing a previously unrecognized link between glycosomal physiology, iron homeostasis, and drug response in Leishmania.
Results
L. amazonensis AQP1 is a glycosomal protein and its expression is modulated during the parasite life cycle
Re-examination of a whole-genome transcriptome profile of L. amazonensis under iron deprivation indicated that gene LmxM.30.0020 is iron-responsive (fold-change = -1.2672; p = 0.043) [13]. This gene is annotated as Aquaglyceroporin 1 in L. mexicana and is conserved across the genus Leishmania, with potential orthologs also found in Crithidia fasciculata (TriTrypDB CFAC1_270006300.1), Endotrypanum monterogeii (TriTrypDB EMOLV88_310005100.1), Leptomonas pyrrhocoris (TriTrypDB rna_LpyrH10_32_1110), Leptomonas seymouri (TriTrypDB rna_Lsey_0007_0780–1), and Porcisia hertigi (TriTrypDB JKF63_03741_t1) [17]. The ortholog in L. amazonensis is annotated as a putative major intrinsic protein (MIP) of 314 amino acids with six predicted transmembrane domains (molecular mass ~34.778 kDa), which we hereafter refer to as L. amazonensis Aquaglyceroporin 1 (AQP1).
Proteins of the MIP superfamily form membrane channels that transport water, small neutral solutes, and in some cases metalloids. In L. major, AQP1 has been associated to drug resistance by mediating the transport of trivalent antimony (SbIII) [18]. Characterization of L. major AQP1 showed that the protein localizes to the promastigote flagellum and, in amastigotes, to both the flagellar pocket membrane and contractile vacuole. This previous study also demonstrated AQP1’s role in solute transport, volume regulation, and osmotaxis [15]. To extend these findings, we generated a three-dimensional model of L. amazonensis AQP1 with AlphaFold 3 [19]. The fully automated modeling, including homology search and multiple sequence alignment, returned per-residue confidence scores (pLDDT), with values below 70 interpreted cautiously. The predicted fold and subunit interface showed high confidence, with pTM and ipTM scores of 0.81, indicating a robust overall structure and inter-chain interaction (Fig 1a).
Quantitative RT-PCR analysis confirmed that aqp1 transcript levels are downregulated when promastigotes are cultured under iron-poor conditions. Specifically, after 18 hours in heme-depleted medium supplemented with the iron chelator deferoxamine (DFO), aqp1 mRNA levels were significantly lower than those in iron-replete control cells (Fig 1b). Besides, we observed a 4.65-fold increase in aqp1 expression in intracellular amastigotes compared to the transcript levels in promastigotes (Fig 1c).
Sequence analysis of L. amazonensis AQP1 revealed the presence of a canonical PTS1 motif (Pro-Asn-Phe, “PNF”) at the C-terminus of the protein (Fig 1d). This tripeptide is a well-characterized glycosomal targeting signal [11,20]. Notably, alignment of AQP1 orthologues from various trypanosomatids showed that the PTS1 motif is present only in L. amazonensis, L. mexicana, L. donovani, and L. infantum. This motif is absent from AQP1 in other Leishmania species (Fig 1d), including L. major AQP1, whose AQP1 localizes to the flagellum, consistent with the lack of PTS1 (Figarella et al., 2007). Pairwise alignment indicated ~74.8% amino acid identity between the L. amazonensis AQP1 and L. major AQP1 (S1 Fig). Moreover, a maximum-parsimony phylogenetic tree revealed that the species harboring the PTS1 motif in AQP1 form a monophyletic clade, comprising L. amazonensis, L. mexicana, L. donovani and L. infantum, distinct from other Leishmania species (Fig 1e). To verify the PTS1-based prediction, we generated a transgenic L. amazonensis line expressing AQP1 tagged with EGFP at the N-terminus. Confocal immunofluorescence microscopy revealed colocalization of EGFP–AQP1 with arginase, a glycosomal marker [21], confirming glycosomal targeting (Pearson correlation coefficient = 0.85) (Fig 2). In contrast, previous studies reported different AQP1 localizations in other species: cell surface/flagellum in L. major [15,22], and an undefined cytosolic signal in L. donovani [23]. Together, these data demonstrate that AQP1 is glycosomal in L. amazonensis. Moreover, the downregulation of aqp1 under iron deprivation and its upregulation in intracellular amastigotes support a role in intracellular iron trafficking, linking glycosomal physiology to iron homeostasis.
Confocal immunofluorescence images of L. amazonensis promastigotes expressing EGFP-AQP1. Colocalization between EGFP–AQP1 and the glycosomal marker arginase was quantified using the Pearson correlation coefficient (r). Green: EGFP-AQP1 fusion; Red: glycosomal marker (anti-arginase); Yellow: merged image; Blue: DNA (DAPI). Scale bar = 10 µm.
Loss of AQP1 impairs promastigote growth under heme-depleted conditions
To investigate AQP1’s role in L. amazonensis, we generated aqp1 knockout mutant lines using the LeishGEdit CRISPR/Cas9 system [24,25]. First, an L. amazonensis PH8 line ectopically expressing Cas9 nuclease and T7 RNA polymerase (C9/T7) was obtained and validated. Western blotting confirmed Cas9 expression in this line, and the C9/T7 parasites showed no growth defects or changes in antimony susceptibility or virulence compared to wild-type controls (S2 Fig). We then transfected C9/T7 promastigotes with a donor DNA construct containing a puromycin resistance gene (pac) flanked by 5′ and 3′ homology arms, along with two single-guide RNA templates targeting sequences immediately upstream and downstream of the aqp1 coding region (Fig 3a). Although aqp1 resides on the tetrasomic chromosome 30 [26], a single round of CRISPR editing was sufficient to knock out all alleles. We obtained two independent knockout clones (KO1 and KO2) in which the aqp1 open reading frame was replaced by the pac cassette, as confirmed by PCR (Fig 3b). For genetic complementation, we introduced an episome containing aqp1 ORF tagged with EGFP into the KO1 clone, generating add-back lines (AB1–AB4) that were also verified by PCR (Fig 3b). The KO1 and AB1 clones were selected for further phenotypic analyses. Before proceeding, we confirmed the complete absence of aqp1 transcripts in the knockout parasites and their restoration in the add-back line by qPCR (Fig 3c).
(a) Schematic of the CRISPR/Cas 9 strategy to delete aqp1 in L. amazonensis. Two sgRNAs (scissors) directed Cas9 to cut at 5′ and 3′ sites flanking the aqp1 ORF. A donor DNA containing a puromycin resistance marker (pac) with flanking 5′ and 3′ untranslated region (UTR) homology arms were delivered to replace the aqp1 ORF via homology-directed repair. Black bars indicate primer binding sites (F1–F4 and R1–R3) used for diagnostic PCR, producing amplicons PCR A–PCR E of the indicated sizes in base pairs (bp). (b) PCR confirmation of aqp1 deletion and complementation. Agarose gel images show diagnostic PCR products for the parental Cas9/T7 line (C9/T7), two aqp1 knockout clones (KO1, KO2), and four add-back clones (AB1–AB4). Water was used as a no-template control (NTC). DNA ladder sizes in bp. gapd = control PCR targeting the glyceraldehyde-3-phosphate dehydrogenase gene (indicating successful DNA amplification for each template). (c) aqp1 transcript levels in WT, aqp1 knockout (KO), and add-back (AB) promastigotes. Transcript levels were quantified by RT-qPCR and normalized to ubiquitin hydrolase (UbH) reference gene. Bars represent the mean ± SEM of three independent experiments. (d) Growth curves of WT, aqp1 knockout (KO), and add-back (AB) promastigotes in regular medium (solid lines) vs. heme-depleted medium (dashed lines). Parasite density (cells/mL) is shown as mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.005 (KO vs. WT). (e) Osmotic-stress responses of WT, KO, and AB promastigotes, measured as changes in optical density over 3 min following a hypo-osmotic shock. Data are mean ± SEM of three independent experiments. A non-linear sigmoidal fit (third-order polynomial) was applied, followed by one-way ANOVA and Tukey’s multiple comparisons test. **p = 0.0034 (WT vs. KO); *p = 0.0457 (KO vs. AB).
We next compared the in vitro growth of wild-type (WT), AQP1-knockout (KO), and add-back (AB) promastigotes under normal and iron-limited conditions. Loss of AQP1 caused a significant growth defect, most evident in heme-depleted medium (Fig 3d). As cultures progressed from logarithmic to stationary phase, KO parasites consistently reached lower densities than WT and AB, with the disparity becoming even more pronounced under heme deprivation.
Because AQP1 has been implicated in osmoregulation in L. major [22], we assessed whether loss of AQP1 affected the ability of L. amazonensis promastigotes to cope with osmotic stress. Cell volume changes in response to a hypo-osmotic shock were measured for WT, KO, and AB lines (Fig 3e). KO cells exhibited a markedly smaller volume increase upon osmotic challenge compared to WT, indicating an impaired capacity for osmotic regulation in the absence of AQP1. The AB line showed partial restoration of the WT phenotype, which is a common outcome for episomal add-backs in Leishmania [9,27,28]. These results reveal a previously unrecognized role for AQP1 in promoting promastigote replication, especially under iron-poor conditions, and reinforce its involvement in maintaining osmotic balance.
Glycosomal AQP1 links antimony susceptibility to iron and redox homeostasis in L. amazonensis
Leishmania susceptibility to antimonials has been linked to AQP1 expression, which would be involved in antimony uptake [29–31]. We therefore tested whether AQP1 deletion affects SbIII susceptibility in L. amazonensis. MTT assays revealed that KO promastigotes were significantly more resistant to SbIII than the WT (Fig 4a). The IC50 for SbIII was 315 µM in the KO, 187 µM in the WT, and 121 µM in the AQP1 add-back line. Notably, the L. amazonensis WT strain was ~ 7-fold more resistant than a L. major WT strain (IC50 27 µM), consistent with species-specific differences in drug susceptibility [16]. We next measured antimony uptake by inductively coupled plasma mass spectrometry (ICP-MS) after exposing promastigotes to SbIII for 15 minutes (Fig 4b). Surprisingly, despite its higher resistance, the AQP1 KO line accumulated more SbIII than WT parasites. This finding suggests that the presence of AQP1 may actually limit net Sb uptake, promote efflux reducing the intracellular accumulation of antimony, or detoxify Sb in L. amazonensis.
(a) MTT viability assays of promastigotes from L. major wild type (Lm WT), L. amazonensis wild type (La WT), aqp1 knockout (KO), and add-back (AB) lines exposed to increasing SbIII concentrations (0–1000 µM). Percent survival is shown for each strain. IC50 values are listed in the inset table. Data are presented as mean ± SEM from three independent experiments, each performed in triplicate. **** p < 0.0001 (WT vs. KO; KO vs. AB). (b) Total antimony content in WT, KO, and AB promastigotes following 15 min exposure to potassium antimonyl tartrate trihydrate (SbIII), measured by ICP-MS. Values were normalized to total cellular protein. Bars represent the mean ± SEM of three independent experiments, each performed in duplicate. **** p < 0.0001. (c) Total iron content in WT, KO, and AB promastigotes at the end-log growth phase, measured by ICP-MS and normalized to total protein. Bars represent the mean ± SEM of three independent experiments, each performed in duplicate. *** p < 0.001. (d) Total superoxide dismutase (SOD) activity in WT, KO, and AB promastigote lysates, expressed as units (U) per mg of protein. Bars represent the mean ± SEM of three independent experiments. ** p < 0.01. (e) Intracellular reactive oxygen species (ROS) levels in WT, KO, and AB promastigotes. Untreated parasites served as the negative control. ROS levels were quantified using H2DCFDA. Bars represent the mean ± SEM of four independent experiments. * p < 0.05 (WT vs. KO; KO vs. AB).
Given the iron-responsive expression of aqp1 and the growth defect of the KO under iron-poor conditions, we examined whether AQP1 influences iron homeostasis. We quantified total cellular iron in WT, KO, and AB promastigotes by ICP-MS (Fig 4c). The AQP1 KO contained roughly 50% less iron than WT cells, whereas iron levels in the AB line were restored to near-WT values. These results indicate that AQP1 is important for maintaining normal iron content in L. amazonensis. Given that trypanosomatids SOD enzymes utilize iron as a cofactor (unlike most eukaryotic SODs, which use manganese) and that the essential SODB isoform is glycosomal in Leishmania, we also examined SOD gene and protein expression and activity in the AQP1 mutant lines. Immunoblotting and qPCR detected no differences in SODA or SODB protein and transcript levels between WT, KO, and AB parasites (S3 Fig). However, total SOD enzymatic activity was significantly elevated in the AQP1 KO compared to WT (Fig 4d). Consistent with this, we observed increased reactive oxygen species (ROS) levels in the KO (Fig 4e), indicating that loss of AQP1 disrupts redox homeostasis and triggers a compensatory increase in antioxidant activity, rather than changes in SOD expression.
Together, these data highlight a novel link between antimony resistance and iron metabolism in L. amazonensis. Glycosomal AQP1 appears to be a key factor connecting these pathways, influencing both metal ion homeostasis and the oxidative stress response.
AQP1 is essential for intracellular proliferation and in vivo pathogenicity
Infection of murine bone marrow–derived macrophages demonstrated that AQP1 is critical for the intracellular replication of L. amazonensis amastigotes (Fig 5a). At 4 hours post-infection, similar numbers of WT, KO, and AB parasites were observed inside macrophages, indicating that AQP1 deletion does not impair initial host cell entry. However, by 48 hours post-infection, the number of KO amastigotes per macrophage had significantly decreased, whereas WT and AB parasites continued to replicate, indicating that KO parasites fail to proliferate intracellularly.
(a) Quantification of intracellular amastigotes in murine bone marrow–derived macrophages (BMM) infected with WT, KO, or AB metacyclic parasites (MOI = 5). Bars indicate the mean number of amastigotes per macrophage (± SEM) at 4, 24, 48, 72, and 96-hours post-infection from three independent experiments. *** p<0.001; ****p<0.0001. No statistically significant differences were observed at 4h. (b) Lesion development in C57BL/6 mice inoculated with 106 metacyclic promastigotes of WT, KO, and AB lines. Points represent mean footpad thickness (± SEM, n = 5 mice per group) over time. * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001 for (WT vs. KO; AB vs. KO). (c) Parasite load in footpad lesions at 10 weeks post-infection for WT (when lesions reached the maximum allowed) and 12 weeks for KO and AB (when lesions became apparent). Bars represent the mean ± SEM for 5 mice per group. ** p = 0.0079 (WT vs. KO; KO vs. AB).
We next examined the importance of AQP1 for L. amazonensis virulence in vivo. C57BL/6 mice were inoculated in the footpad with metacyclic promastigotes of WT, KO, or AB lines, and lesion development was monitored weekly. All mice infected with WT parasites developed progressive lesions by 3–4 weeks post-infection. In contrast, mice infected with AQP1 KO parasites showed only minimal pathology, with footpad swelling only becoming apparent around 11–12 weeks post-infection (Fig 5b). Mice infected with AB parasites developed lesions of intermediate severity, with footpad thickness approximately two-fold greater than in the KO group. Parasite load measurements at the end of the experiment confirmed that KO-infected footpads harbored significantly fewer parasites than those infected with WT or AB lines (Fig 5c).
These findings, together with the macrophage infection data, demonstrate that AQP1 is a pivotal virulence factor for L. amazonensis. Loss of AQP1 severely compromises the parasite’s ability to replicate inside host cells and cause disease in the mammalian host.
Discussion
Our study reveals that AQP1 in L. amazonensis is a glycosomal protein with multiple roles in parasite physiology (Fig 6), highlighting how even highly conserved proteins can have distinct localizations and functions across Leishmania species. In L. major, AQP1 localizes to the flagellum and flagellar pocket membranes, where it participates in osmoregulation and antimony uptake, and its distribution can be further modulated by phosphorylation [15,22,32]. In contrast, we found that L. amazonensis AQP1 carries a C-terminal PTS1 motif—absent in L. major and several other species—that directs it to glycosomes. To our knowledge, this is the first report of an aquaglyceroporin targeted to the glycosomal compartment in trypanosomatids [33].
Schematic overview of the major phenotypes observed in the L. amazonensis aqp1 knockout (La AQP1 KO). This study identifies AQP1 as a glycosomal protein and links its role in iron and redox homeostasis to the parasite’s antimony response. Loss of AQP1 is associated with impaired promastigote growth under low-iron conditions, decreased intracellular iron content, altered osmoregulatory capacity, increased SbIII resistance despite higher intracellular antimony accumulation, increased SOD activity and ROS levels, and reduced virulence. Abbreviations: AQP1, aquaglyceroporin 1; G, glycosome; H₂O₂, hydrogen peroxide; LIR1, Leishmania iron regulator 1; LIT1, Leishmania iron transporter 1; LMIT1, Leishmania mitochondrial iron transporter 1; M, mitochondrion; N, nucleus; O₂, oxygen; O₂⁻, superoxide anion; ROS, reactive oxygen species; SODA, superoxide dismutase A; SODB, superoxide dismutase B; SOD, superoxide dismutase isoforms.
These findings underscore the risk of extrapolating protein function across species. Large-scale localization efforts often rely on a single reference species and implicitly assume conservation across the genus or even the Trypanosomatidae family [34]. Our data argue that such assumptions can obscure biologically meaningful adaptations. The acquisition of a PTS1 motif by AQP1 in species from the L. mexicana and L. donovani complexes suggests lineage-specific rewiring of protein localization with important physiological consequences.
Glycosomes compartmentalize essential metabolic processes, including glycolysis, β-oxidation, and detoxification pathways, some of which rely on iron-dependent enzymes [35]. Yet the mechanisms governing iron trafficking into glycosomes remain poorly understood [5,33]. We found that aqp1 expression is iron responsive and downregulated under iron‑depleted conditions, and that AQP1-null parasites retain approximately half the intracellular iron content of wild-type cells. Together, these observations indicate a role for AQP1 in maintaining iron homeostasis. One plausible model is that AQP1 facilitates the movement of small solutes required for glycosomal metabolism or osmotic balance. In this context, it is notable that Leishmania encodes a glycosomal superoxide dismutase (SODB) that uses iron as a cofactor The growth defect of AQP1-null parasites under iron-poor conditions further supports the importance of AQP1 during iron limitation, potentially by facilitating iron recycling or redistribution when availability is restricted. Overall, these findings position glycosomal AQP1 as a previously unrecognized contributor to iron and redox homeostasis in L. amazonensis.
Another major finding is that L. amazonensis AQP1 modulates antimonial susceptibility in an unexpected manner. In several Leishmania species, AQP1 has been proposed as the main route for uptake of trivalent antimony (SbIII), and reduced AQP1 expression correlates with drug resistance [15,18,32,37]. Our data from L. amazonensis challenge this model. Although deletion of AQP1 increased resistance to SbIII, AQP1-null parasites paradoxically accumulated higher intracellular antimony levels than wild-type cells (Fig 6). This indicates that SbIII can enter L. amazonensis through alternative routes when AQP1 is absent, consistent with previous reports of low-affinity or non-saturable Sb uptake pathways [38].
Beyond uptake, the increased Sb content in AQP1-null parasites suggests that wild-type AQP1 may normally limit net Sb accumulation. Given its glycosomal localization, AQP1 could facilitate compartmentalization of Sb or Sb-related metabolites, thereby reducing cytosolic toxicity. In the absence of AQP1, Sb may remain in the cytosol and be neutralized through other mechanisms [31]. Importantly, the AQP1 knockout exhibits alterations in iron metabolism and redox balance, including reduced iron levels, elevated ROS, and increased SOD activity (Fig 6). Because antimonials are known to exacerbate oxidative stress, either directly or via thiol depletion [39], a parasite with diminished iron-driven Fenton chemistry and reinforced antioxidant defenses would be better equipped to tolerate Sb-induced damage. Thus, changes in metal homeostasis and redox state provide a coherent explanation for the increased Sb tolerance observed in AQP1-null parasites.
These results call for a refinement of prevailing models of antimonial action and resistance, which have largely emphasized drug transport and thiol metabolism. Our findings suggest that iron availability and redox balance are additional, underappreciated determinants of drug sensitivity. It will also be important to identify the alternative Sb uptake route in AQP1-deficient L. amazonensis, whether through another aquaglyceroporin, a different type of channel, or simply passive diffusion, and to determine if similar pathways operate in other Leishmania species or in clinical scenarios.
Finally, our data establish as a virulence factor in L. amazonensis. AQP1 expression is upregulated in intracellular amastigotes, consistent with a role in adapting to the hostile phagolysosomal environment, where parasites face iron limitation, osmotic stress, and oxidative challenges [5]. In the absence of AQP1, amastigotes fail to proliferate in macrophages and produce markedly attenuated lesions in mice (Fig 6).
In summary, this study identifies L. amazonensis AQP1 as a multifunctional glycosomal protein linking iron homeostasis, redox balance, virulence, and antimonial response. Rather than acting solely as a drug transport channel, AQP1 emerges as a central regulator of metabolic adaptation in this species. Our findings also highlight that Leishmania species are not biologically interchangeable, more comparative work across species will be essential to capture the diversity of strategies for nutrient handling and drug response. The lineage-specific glycosomal targeting of AQP1 points to an expanded role for glycosomes in stress adaptation and survival in the host environment. Finally, the coupling of metal ion homeostasis to antimonial susceptibility suggests actionable therapeutic directions, modulating parasite iron availability or redox balance may offer routes to improve treatment efficacy. Overall, these results challenge assumptions derived from other species and emphasize that species-specific biology must be dissected to design more effective, targeted interventions against leishmaniasis.
Materials and methods
Ethics statement
All animal experimental procedures were approved by the Animal Care and Use Committee at the Institute of Bioscience of the University of São Paulo (CEUA 342/2019) and were conducted in accordance with the recommendations and the policies for the Care and Use of Laboratory Animals of São Paulo State (Lei Estadual 11.977, de 25/08/2005) and the Brazilian government (Lei Federal 11.794, de 08/10/2008).
Animals
Female C57BL/6 mice (6–8 weeks old) (five animals per group) were obtained from the Animal Centre of the Medical School of the University of São Paulo and maintained at the Animal Centre of the Department of Physiology at the Institute of Bioscience of the University of São Paulo, receiving food and water ad libitum.
Leishmania cultivation
Promastigote forms of the L. amazonensis IFLA/BR/67/PH8 and L. major MHOM/IL/81/Friedlin strains were cultured in vitro at 26°C in M199: medium 199 (Gibco, Invitrogen) pH 7.2 supplemented with 10% heat inactivated fetal bovine serum (FBS), 40 mM HEPES, 0.1 mM adenine, 0.0001% biotin, 5 μg/ml hemin (25 mg/ml in 50% triethanolamine), 5 mM L-Glutamine and 5% penicillin-streptomycin. Heme-depleted medium was prepared similarly, omitting hemin addition and replacing regular FBS by heme-depleted FBS. Heme-depleted FBS was prepared as previously described [13] by treating heat inactivated FBS with 10 mM ascorbic acid for 16 h at room temperature, followed by verification of heme depletion by measuring the optical absorbance at 405 nm, 3 rounds of dialysis in cold phosphate-buffered saline (PBS) and filter-sterilization.
Leishmania immunofluorescence microscopy
Promastigotes were fixed with 4% paraformaldehyde and attached to poly L-lysine coated slides. The fixed cells were quenched with 50 mM NH4Cl for 1 h and permeabilized with PBS containing 0.1% Triton X-100 for 15 min, prior to blocking with TBS Blocking Buffer (LI-COR Bioscience) for 1 h at room temperature. For arginase detection, a rabbit anti-arginase was used as primary antibody [21] (1:500 in blocking buffer) for 1 h, followed by an anti-rabbit AlexaFluor 546 (Life Technologies) (1:500) as secondary antibody. All samples were incubated with 1 μg/mL DAPI (4’,6-diamidino- 2-phenylindole) for nuclear staining. Slides were mounted with ProLong Gold Antifade Mountant (Invitrogen), and images were acquired using a confocal microscope (Zeiss LSM 780 NLO) at the Core Facility for Research Support (Centro de Facilidades para Pesquisa, CEFAP) from University of São Paulo.
Pearson correlation coefficients were calculated using the Coloc 2 plugin in ImageJ/Fiji [40,41], based on pixel-by-pixel intensity correlations between the red arginase signal (red channel) and the GFP–AQP1 signal (green channel) within manually defined regions of interest (ROIs), following automatic thresholding using the Costes method.
aqp1 mRNA expression
Total RNA was obtained using PureLink RNA Mini Kit (Invitrogen) following manufacturers’ instructions. cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For a final reaction volume of 20 μL, 2 μg of total RNA mixed with 0.5 μg oligo(dT)12–18, 0.8 μL of dNTPs 25 mM, 2 μL of Buffer 10X, and 50 U of MultiScribe Reverse Transcriptase (Applied Biosystems) was incubated at 25oC for 10 min, 37oC for 120 min, 85oC for 5 min, and storage at -20oC. A negative control containing all reaction components except the enzyme was included and analyzed by real-time PCR to exclude the possibility of DNA contamination in the RNA samples.
For real-time PCR, 1/20 of the reverse transcription product was used as a template. The reactions were performed in a StepOnePlus Real-Time PCR System (Applied Biosystems) with 200 nM of each gene-specific pair of primers and LuminoCt SYBR Green qPCR ReadyMix (Sigma Aldrich). The specific primers used were AQP1-RT-F and AQP1-RT-R for L. amazonensis aqp1, GAPD-RT-F and GAPD-RT-R for glyceraldehyde-3-phosphate dehydrogenase (gapd), UbH-RT-F and UbH-RT-F for ubiquitin hydrolase (UbH) (S1 Table). The PCR reaction consisted of an initial denaturation step of 95oC for 20 s followed by 40 cycles of 3 s at 95oC and 30 s at 60oC. The target gene expression levels were quantified according to a standard curve prepared from a ten-fold serial dilution of a quantified and linearized plasmid containing the DNA segment to be amplified.
Generation of L. amazonensis AQP1 overexpressor, knockout and complemented cell lines
To generate L. amazonensis parasites expressing AQP1 tagged with EGFP, the gene open reading frame (ORF) was amplified using the primers AQP1-GFP-N-F and AQP1-GFP-N-R for N-terminal fusion (S1 Table). The amplicon was purified and cloned into the BamHI site of the Leishmania expression vector pXG-GFP2+ [42]. The construction was transfected by electroporation [43]. Clones were selected in semi-solid M199 containing G418 (20 μg/mL).
For gene deletion, we used the LeishGEdit toolkit based on the CRISPR/Cas9 strategy provided by Prof. Eva Gluenz group [25]. Briefly, we transfected L. amazonensis with the plasmid pT007_Cas9_T7 to generate our parental line expressing Cas9 nuclease and T7 RNA polymerase. The online primer design tool www.LeishGEdit.net was used to design primers for amplification of the 5′ and 3′ sgRNA templates and for amplification of donor DNA from pTPuro_v1 plasmid (S1 Table) (Fig 3a). Following transfection, parasites were plated onto semisolid M199 (1% agar) complemented with puromycin (20 μg/mL) for selection and isolation of knockout clones. For genetic complementation, the aqp1 ORF fused to EGFP at the N-terminus, was previously cloned into the episomal expression vector pXG-GFP2+ and reintroduced into selected knockout lines by electroporation. Transfectants were selected and cloned in semisolid M199 containing G418 (20 μg/mL).
To confirm replacement of the aqp1 ORF by the puromycin resistance cassette and aqp1 episomal complementation, genomic DNA from the selected clones was extracted using the salting out deproteination extraction (SODE) method [44,45]. The resulting DNA samples were used as template in PCR amplification analyses.
Cell volume measurements
Cell volume changes in response to hypo-osmotic stress were evaluated as previously described [16,46], with minor modifications. Briefly, log-phase promastigotes were collected, washed with PBS, and resuspended in a 150 mOsm buffer to a final concentration of 109 promastigotes/mL. Aliquots of 100 μL were transferred to the wells of a microtiter plate. Hypo-osmotic shock was induced by mixing equal volumes of the isotonic cell suspension and deionized water. Absorbance at 550 nm was measured every 15 sec over a 3 min period in a microplate reader (Spectramax 340, Molecular Devices). A decrease in absorbance reflects an increase in cell volume. For isosmotic controls, cell suspensions were diluted with isosmotic buffer without addition of water.
In vitro antimony susceptibility assay
The susceptibility of the Leishmania lines of interest to trivalent antimony (SbIII) (Sigma-Aldrich) was determined using the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay (Sigma-Aldrich), as previously described [47,48]. 2 × 106 log-phase promastigotes were incubated for 24 h at 25°C in 96-well plates with 200 μL of M199 containing increasing concentrations of potassium antimonyl tartrate trihydrate (SbIII) (Sigma-Aldrich): 0, 7.81, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 μM. Then, 30 μL of the MTT solution (5 mg/mL) was added and plates were incubated for 3 h at 25°C. The reaction was stopped by addition of 50 μL of 20% Sodium Dodecyl Sulfate (SDS). Absorbance was measured at 595 nm using a microplate reader (Spectramax 250, Molecular Devices). The half-maximal inhibitory concentration (IC50) was determined using a nonlinear sigmoidal regression model of the concentration-response curves in GraphPad Prism 8.
Elemental antimony and iron quantification
Inductively coupled plasma mass spectrometry (ICP-MS) was performed as previously described [49]. Briefly, the concentrations of Sb isotope (121Sb) and Fe isotope (56Fe) were quantified using internal standards and normalized by total protein content of digested promastigote cells. For Sb uptake quantification, 108 L. amazonensis promastigotes were incubated with 10 μM of potassium antimonyl tartrate trihydrate (SbIII) (Sigma-Aldrich) for 15 min and washed three times with PBS. All samples were digested for 1 h in 100 μL of HNO3 70%. Lysates were diluted 10-fold into 1% nitric acid for the analysis.
Western blot
Approximately 107 promastigotes were lysed with 100 μL of lysis buffer (1% Triton, 150 mM NaCl, 50 mM Tris-HCl pH 7.6) supplemented with a protease-inhibitor cocktail (Roche). Lysates were clarified at 14,000 × g for 15 min at 4 °C. Laemmli sample buffer was added to the supernatant and samples were boiled for 5 min. Equal protein amounts (20 μg per lane) were resolved by SDS-PAGE and analyzed by western blot using anti-SODA (1:500) [50], anti-SODB (1:500) [50], anti-FLAG M2 (1:4,000, for Cas9 detection; F1804-MG, Sigma-Aldrich), anti-alpha Tubulin (1:5,000, loading control; 62204, Thermo Fisher) and anti-arginase (1:500, loading control) [21] antibodies diluted in TBS Blocking Buffer (LI-COR Bioscience). After primary incubation, membranes were washed three times in TBST (TBS, 0.1% Tween-20) and incubated for 1 h at room temperature, protected from light, with IRDye 800CW goat anti-Rabbit or IRDye 680RD anti-Mouse (LICORbio; 1:10,000 in TBS Blocking Buffer). Blots were washed again in TBST and scanned on an Odyssey CLx imager (LICORbio). Band intensities (near-infrared fluorescence) were quantified using Image Studio Software 6.1 (LICORbio).
Superoxide dismutase (SOD) activity
Superoxide dismutase (SOD) activity was quantified with the riboflavin–nitroblue tetrazolium (NBT) photoreduction method as described previously [51,52]. Briefly, 108 promastigotes were washed three times in PBS (KCl 2.7 mM, KH2PO4 1.5 mM, NaHPO4 8 mM and NaCl 136.9 mM, pH 7.0) and lysed in 50 mM Tris-HCl pH 7.5 containing 2.5% [v/v] protease inhibitor cocktail (Sigma) by ten cycles of rapid freezing in liquid nitrogen followed by thawing in a 42°C water bath. The lysate was clarified by centrifugation (12,000 × g, 10 min, 4°C) and the protein concentration measured by the Bradford assay [53]. 10 µg of total protein was added to the reaction buffer (50 mM Tris-HCl pH 7.5, 13 mM L-methionine, 75 µM NBT, 0.1 mM EDTA) to a final volume of 200 µL. The reaction was initiated with addition of 4 µM of riboflavin and the mixtures were exposed to white-fluorescent light for 30 minutes at room temperature. Tubes kept in the dark served as blanks, and a riboflavin-containing reaction without lysate represented 100% NBT reduction. Absorbance was measured at 560 nm, and one unit (U) of SOD activity was defined as the amount of enzyme that inhibits NBT-diformazan formation by 50% under these conditions.
Intracellular ROS quantification
Intracellular reactive oxygen species (ROS) levels were quantified using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Thermo Scientific), as previously described [54,55]. Briefly, stationary-phase Leishmania promastigotes (2 × 10⁷ cells/mL) were incubated with 100 µM H₂O₂ (positive control) or PBS (negative control and experimental conditions), followed by the addition of H₂DCFDA to a final concentration of 10 µM. After incubation for 1 h at 25°C, cells were washed with PBS, and 200 µL of each cell suspension were transferred to black 96-well plates in technical triplicates. Fluorescence was measured at excitation and emission wavelengths of 495 nm and 527 nm, respectively, and values were corrected by subtracting cell autofluorescence.
In vitro mouse macrophage infections
Mouse bone marrow derived macrophages (BMM) were prepared from C57BL/6 mice as previously described [56]. A total of 106 BMMs per well were plated on glass coverslips in 6-well plates and incubated for 24 h at 37°C 5% CO2 in BMM media: RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 5% FBS, and 20% L929 cell-conditioned medium, as a source of macrophage differentiation factors. Infective metacyclic forms were purified from stationary phase promastigote cultures (second to third day after entering stationary phase) using the 3A.1 monoclonal antibody as described [57]. Purified metacyclics were added at a ratio of 5 parasites per macrophage for 4 h at 34°C. The cells were washed 3 times with PBS and fixed or incubated in BMM media to complete 24, 48, 72 and 96 h of infection. Coverslips were fixed in 4% paraformaldehyde and incubated with 1 μg/mL DAPI for 1 h, after permeabilization with 0.1% Triton X-100 for 10 min. The number of intracellular parasites was determined by counting the total macrophages and the total intracellular parasites per microscopic field (Leica DMI8 inverted fluorescence microscope, Department of Zoology, Institute of Biosciences, University of São Paulo). At least 200 host cells, in triplicate, were analyzed for each time point.
In vivo infection and parasite tissue load determination
Six-week-old female C57BL/6 or BALB/c mice were inoculated as previously described [58] with 106 purified metacyclics from L. amazonensis WT, C9/T7, AQP1 KO and add-back (AB) in the left hind footpad in a volume of 0.01 mL. Lesion progression was monitored once a week by measuring the difference in thickness between the left and right hind footpads with a caliper (Mitutoyo Corp., Japan). The parasite load in the infected tissue was determined after 8–12 weeks in the infected tissue collected from footpad lesions of sacrificed mice by limiting dilution [59].
Phylogenetic analysis
Putative AQP1 orthologues from trypanosomatid parasites were retrieved from TriTrypDB using the Leishmania mexicana AQP1 sequence (LmxM.30.0020) as a query [17]. The database was also searched for putative AQP1 homologues in Homo sapiens. Amino acid sequences were aligned in MEGA 12 with MUSCLE (default parameters), and phylogenetic relationships were inferred by Maximum Likelihood (ML) under the Jones–Taylor–Thornton substitution model [60,61]. The topology with the highest log-likelihood (–5,648.66) was chosen for presentation. Branch support was evaluated by 1,000 bootstrap replicates; percentages of replicate trees in which the associated taxa clustered are indicated at the corresponding nodes [62]. The final data set comprised 13 sequences and 342 unambiguously aligned positions.
Supporting information
S1 Fig. Sequence identity of trypanosomatid aquaglyceroporins.
(a) Multiple-sequence alignment of aquaglyceroporins from trypanosomatids. The Pro-Asn-Phe (PNF) peroxisomal‐targeting motif is boxed in red. Dots (.) denote residues identical to the reference (top) sequence; dashes (–) indicate gaps. (b) Pairwise percent-identity matrix for the same proteins, calculated as (1 − p-distance) × 100. Protein IDs: L. amazonensis LAMAPH8_000653100; L. mexicana LmxM.30.0020; L. donovani LdBPK_310030; L. infantum LINF_310005100; L. major LmjF.31.0020; L. tarentolae LtaP31.0020; L. braziliensis LbrM.00.0079; Trypanosoma brucei Tb927.6.1520, Tb927.10.14160, Tb927.10.14170; Leptomonas pyrrhocoris LpyrH10_32_1110; Lept. seymouri Lsey_0007_0780; H. sapiens NP_001161.1.
https://doi.org/10.1371/journal.pntd.0014141.s001
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S2 Fig. Validation of the Cas9/T7-expressing L. amazonensis parental line.
(a) Western blot for Cas9 in total lysates of wild-type (WT) and Cas9/T7 (C9/T7) L. amazonensis promastigotes. α-tubulin is shown as loading control. (b) Growth curves of WT and C9/T7 promastigotes in regular medium (solid lines) vs. heme-depleted medium (dashed lines). Parasite density (cells/mL) is shown as mean ± SEM of 3 independent experiments. (c) MTT viability assays for promastigotes of WT and C9/T7 exposed to increasing SbIII concentrations (0–1000 µM). Percent survival is plotted for each strain. Data represent the mean ± SEM from 3 independent experiments, each performed in triplicate. (d) Lesion development in BALB/c mice inoculated with 106 metacyclic promastigotes of WT and C9/T7 lines. Points represent the mean footpad thickness ± SEM (n = 5 mice per group) over time. (e) Parasite load in footpad lesions when lesions reached the maximum allowed: 7 weeks post-infection for WT and 9 weeks for C9/T7. Bars show the mean ± SEM for 5 mice per group.
https://doi.org/10.1371/journal.pntd.0014141.s002
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S3 Fig. Superoxide dismutase expression in AQP1 mutant lines.
(a) Western blots for SOD A (SODA) and SOD B (SODB) in total lysates of WT, KO, and AB promastigotes. α-tubulin is shown as loading control. (b) Densitometry of SOD bands normalized to α-tubulin. Data represent the mean of two independent experiments ± SEM. (c) Transcript levels of SODA, SOB1, and SODB2 in end-log phase promastigotes were quantified by RT-qPCR and normalized to the ubiquitin hydrolase (UbH) reference gene. Data represent the mean ± SEM of three independent experiments.
https://doi.org/10.1371/journal.pntd.0014141.s003
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S1 Table. Oligonucleotides used in this study.
https://doi.org/10.1371/journal.pntd.0014141.s004
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S1 Data. Source data for Fig 1.
Amino acid sequences and numerical values underlying all graphs in Fig 1.
https://doi.org/10.1371/journal.pntd.0014141.s005
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S2 Data. Source data for Fig 2.
Raw confocal microscopy image data. For reference, S2 Data.czi can be opened with ZEISS ZEN lite (free software from ZEISS) or with Fiji/ImageJ using the Bio-Formats importer (open access). Please note that standard ImageJ may require the Bio-Formats plugin, while Fiji typically includes it.
https://doi.org/10.1371/journal.pntd.0014141.s006
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S3 Data. Source data for Fig 3.
Numerical values underlying all graphs in Fig 3.
https://doi.org/10.1371/journal.pntd.0014141.s007
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S4 Data. Source data for Fig 4.
Numerical values underlying all graphs shown in Fig 4.
https://doi.org/10.1371/journal.pntd.0014141.s008
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S5 Data. Source data for Fig 5.
Numerical values underlying all graphs shown in Fig 5.
https://doi.org/10.1371/journal.pntd.0014141.s009
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S6 Data. Source data for S2 Fig.
Numerical values underlying S2 Fig.
https://doi.org/10.1371/journal.pntd.0014141.s010
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S7 Data. Source data for S3 Fig.
Numerical data underlying S3 Fig.
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Acknowledgments
We thank Nassib Saab Daniel for his assistance in preparing the diagrammatic scheme. We thank Eva Gluenz (University of Bern) and Stephen Beverley (Washington University) for generous gift of plasmids. We are grateful to Bruno Lemos Batista and Camila Neves Lange (Federal University of ABC) for assistance with ICP-MS analyses; to Mario Costa Cruz for confocal microscopy support at the Core Facility of the Centro de Facilidades para Pesquisa (CEFAP), University of São Paulo; and to Federico David Brown Almeida for providing access to the fluorescence microscope in his laboratory at the University of São Paulo.
References
- 1.
WHO. Leishmaniasis fact sheet Geneva: World Health Organization; 2023 [cited 2023 28 Jul]. Available from: https://www.who.int/news-room/fact-sheets/detail/leishmaniasis
- 2. Hong A, Zampieri RA, Shaw JJ, Floeter-Winter LM, Laranjeira-Silva MF. One Health Approach to Leishmaniases: Understanding the Disease Dynamics through Diagnostic Tools. Pathogens. 2020;9(10):809. pmid:33019713
- 3. Serafim TD, Coutinho-Abreu IV, Dey R, Kissinger R, Valenzuela JG, Oliveira F, et al. Leishmaniasis: the act of transmission. Trends Parasitol. 2021;37(11):976–87. pmid:34389215
- 4. Tripathi P, Singh V, Naik S. Immune response to leishmania: paradox rather than paradigm. FEMS Immunol Med Microbiol. 2007;51(2):229–42. pmid:17714488
- 5. Laranjeira-Silva MF, Hamza I, Pérez-Victoria JM. Iron and Heme Metabolism at the Leishmania-Host Interface. Trends Parasitol. 2020;36(3):279–89. pmid:32005611
- 6. Goto Y, Ito T, Ghosh S, Mukherjee B. Access and utilization of host-derived iron by Leishmania parasites. J Biochem. 2023;175(1):17–24. pmid:37830941
- 7. Huynh C, Yuan X, Miguel DC, Renberg RL, Protchenko O, Philpott CC, et al. Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1. PLoS Pathog. 2012;8(7):e1002795. pmid:22807677
- 8. Mach J, Sutak R. Iron in parasitic protists - from uptake to storage and where we can interfere. Metallomics. 2020;12(9):1335–47. pmid:32627796
- 9. Laranjeira-Silva MF, Wang W, Samuel TK, Maeda FY, Michailowsky V, Hamza I, et al. A MFS-like plasma membrane transporter required for Leishmania virulence protects the parasites from iron toxicity. PLoS Pathog. 2018;14(6):e1007140. pmid:29906288
- 10.
Lentini G, Dumoulin PC, Carter NS. Intracellular parasites: Kinetoplastids. In: Bradshaw RA, Hart GW, Stahl PD, editors. Encyclopedia of Cell Biology. Second ed. Oxford: Academic Press. 2023. p. 371–85.
- 11. Andrade-Alviárez D, Bonive-Boscan AD, Cáceres AJ, Quiñones W, Gualdrón-López M, Ginger ML, et al. Delineating transitions during the evolution of specialised peroxisomes: Glycosome formation in kinetoplastid and diplonemid protists. Front Cell Dev Biol. 2022;10:979269. pmid:36172271
- 12. Plewes KA, Barr SD, Gedamu L. Iron superoxide dismutases targeted to the glycosomes of Leishmania chagasi are important for survival. Infect Immun. 2003;71(10):5910–20. pmid:14500512
- 13. Mittra B, Cortez M, Haydock A, Ramasamy G, Myler PJ, Andrews NW. Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels. J Exp Med. 2013;210(2):401–16. pmid:23382545
- 14.
Verma RK, Gupta AB, Sankararamakrishnan R. Major intrinsic protein superfamily: Channels with unique structural features and diverse selectivity filters. In: Shukla AK, editor. Methods in Enzymology. Academic Press. 2015. p. 485–520.
- 15. Figarella K, Uzcategui NL, Zhou Y, LeFurgey A, Ouellette M, Bhattacharjee H, et al. Biochemical characterization of Leishmania major aquaglyceroporin LmAQP1: possible role in volume regulation and osmotaxis. Mol Microbiol. 2007;65(4):1006–17. pmid:17640270
- 16. Mandal G, Mandal S, Sharma M, Charret KS, Papadopoulou B, Bhattacharjee H, et al. Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1. PLoS Negl Trop Dis. 2015;9(2):e0003500. pmid:25714343
- 17. Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010;38(Database issue):D457-62. pmid:19843604
- 18. Marquis N, Gourbal B, Rosen BP, Mukhopadhyay R, Ouellette M. Modulation in aquaglyceroporin AQP1 gene transcript levels in drug-resistant Leishmania. Mol Microbiol. 2005;57(6):1690–9. pmid:16135234
- 19. Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630(8016):493–500. pmid:38718835
- 20. Acosta H, Burchmore R, Naula C, Gualdrón-López M, Quintero-Troconis E, Cáceres AJ, et al. Proteomic analysis of glycosomes from Trypanosoma cruzi epimastigotes. Mol Biochem Parasitol. 2019;229:62–74. pmid:30831156
- 21. da Silva ER, da Silva MFL, Fischer H, Mortara RA, Mayer MG, Framesqui K, et al. Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme. Mol Biochem Parasitol. 2008;159(2):104–11. pmid:18400316
- 22. Sharma M, Mandal G, Mandal S, Bhattacharjee H, Mukhopadhyay R. Functional role of lysine 12 in Leishmania major AQP1. Mol Biochem Parasitol. 2015;201(2):139–45. pmid:26259900
- 23. Biyani N, Mandal S, Seth C, Saint M, Natarajan K, Ghosh I, et al. Characterization of Leishmania donovani aquaporins shows presence of subcellular aquaporins similar to tonoplast intrinsic proteins of plants. PLoS One. 2011;6(9):e24820. pmid:21969862
- 24. Beneke T, Madden R, Makin L, Valli J, Sunter J, Gluenz E. A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. R Soc Open Sci. 2017;4(5):170095. pmid:28573017
- 25. Beneke T, Gluenz E. LeishGEdit: A Method for Rapid Gene Knockout and Tagging Using CRISPR-Cas9. Methods Mol Biol. 2019;1971:189–210. pmid:30980304
- 26. Goes WM, Brasil CRF, Reis-Cunha JL, Coqueiro-Dos-Santos A, Grazielle-Silva V, de Souza Reis J, et al. Complete assembly, annotation of virulence genes and CRISPR editing of the genome of Leishmania amazonensis PH8 strain. Genomics. 2023;115(5):110661. pmid:37263313
- 27. Tran KD, Rodriguez-Contreras D, Vieira DP, Yates PA, David L, Beatty W, et al. KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes. J Biol Chem. 2013;288(31):22721–33. pmid:23766511
- 28. Onwah SS, Mou Z, Gupta G, Obi P, Ikeogu N, Jia P, et al. Leishmania major Dihydrolipoyl dehydrogenase (DLD) is a key metabolic enzyme that drives parasite proliferation, pathology and host immune response. PLoS Pathog. 2025;21(3):e1012978. pmid:40096189
- 29. Maharjan M, Singh S, Chatterjee M, Madhubala R. Role of aquaglyceroporin (AQP1) gene and drug uptake in antimony-resistant clinical isolates of Leishmania donovani. Am J Trop Med Hyg. 2008;79(1):69–75. pmid:18606765
- 30. Monte-Neto R, Laffitte M-CN, Leprohon P, Reis P, Frézard F, Ouellette M. Intrachromosomal amplification, locus deletion and point mutation in the aquaglyceroporin AQP1 gene in antimony resistant Leishmania (Viannia) guyanensis. PLoS Negl Trop Dis. 2015;9(2):e0003476. pmid:25679388
- 31. Frézard F, Monte-Neto R, Reis PG. Antimony transport mechanisms in resistant leishmania parasites. Biophys Rev. 2014;6(1):119–32. pmid:28509965
- 32. Plourde M, Ubeda J-M, Mandal G, Monte-Neto RL do, Mukhopadhyay R, Ouellette M. Generation of an aquaglyceroporin AQP1 null mutant in Leishmania major. Mol Biochem Parasitol. 2015;201(2):108–11. pmid:26222914
- 33. Jeacock L, Baker N, Wiedemar N, Mäser P, Horn D. Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity. PLoS Pathog. 2017;13(3):e1006307. pmid:28358927
- 34. LeishGEM Team. LeishGEM: genome-wide deletion mutant fitness and protein localisations in Leishmania. Trends Parasitol. 2024;40(8):675–8. pmid:39030136
- 35. Michels PAM, Bringaud F, Herman M, Hannaert V. Metabolic functions of glycosomes in trypanosomatids. Biochim Biophys Acta. 2006;1763(12):1463–77. pmid:17023066
- 36. Taylor MC, Kelly JM. Iron metabolism in trypanosomatids, and its crucial role in infection. Parasitology. 2010;137(6):899–917. pmid:20152063
- 37. Gourbal B, Sonuc N, Bhattacharjee H, Legare D, Sundar S, Ouellette M, et al. Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin. J Biol Chem. 2004;279(30):31010–7. pmid:15138256
- 38. Dos Reis PG, do Monte-Neto RL, Melo MN, Frézard F. Biophysical and Pharmacological Characterization of Energy-Dependent Efflux of Sb in Laboratory-Selected Resistant Strains of Leishmania (Viannia) Subgenus. Front Cell Dev Biol. 2017;5:24. pmid:28393067
- 39. Mandal G, Wyllie S, Singh N, Sundar S, Fairlamb AH, Chatterjee M. Increased levels of thiols protect antimony unresponsive Leishmania donovani field isolates against reactive oxygen species generated by trivalent antimony. Parasitology. 2007;134(Pt 12):1679–87. pmid:17612420
- 40. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. pmid:22743772
- 41. Dunn KW, Kamocka MM, McDonald JH. A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol. 2011;300(4):C723-42. pmid:21209361
- 42. Ha DS, Schwarz JK, Turco SJ, Beverley SM. Use of the green fluorescent protein as a marker in transfected Leishmania. Mol Biochem Parasitol. 1996;77(1):57–64. pmid:8784772
- 43. Robinson KA, Beverley SM. Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania. Mol Biochem Parasitol. 2003;128(2):217–28. pmid:12742588
- 44. Rotureau B, Gego A, Carme B. Trypanosomatid protozoa: a simplified DNA isolation procedure. Exp Parasitol. 2005;111(3):207–9. pmid:16139269
- 45. Andrade Zampieri R, Ide Aoki J, Müller KE, Jon Shaw J, Maria Floeter-Winter L. Comparison of Sampling Procedures for the Molecular Diagnosis of Leishmaniases. Am J Trop Med Hyg. 2023;108(3):548–54. pmid:36689945
- 46. Rohloff P, Rodrigues CO, Docampo R. Regulatory volume decrease in Trypanosoma cruzi involves amino acid efflux and changes in intracellular calcium. Mol Biochem Parasitol. 2003;126(2):219–30. pmid:12615321
- 47. Ferreira BA, Santos G de A, Coser EM, Sousa JM, Gama MEA, Júnior LLB, et al. In Vitro Drug Susceptibility of a Leishmania (Leishmania) infantum Isolate from a Visceral Leishmaniasis Pediatric Patient after Multiple Relapses. Trop Med Infect Dis. 2023;8(7):354. pmid:37505650
- 48. Zauli-Nascimento RC, Miguel DC, Yokoyama-Yasunaka JKU, Pereira LIA, Pelli de Oliveira MA, Ribeiro-Dias F, et al. In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B. Trop Med Int Health. 2010;15(1):68–76. pmid:19874570
- 49. Ma S, Lee S-G, Kim EB, Park TJ, Seluanov A, Gorbunova V, et al. Organization of the Mammalian Ionome According to Organ Origin, Lineage Specialization, and Longevity. Cell Rep. 2015;13(7):1319–26. pmid:26549444
- 50. Mittra B, Laranjeira-Silva MF, Miguel DC, Perrone Bezerra de Menezes J, Andrews NW. The iron-dependent mitochondrial superoxide dismutase SODA promotes Leishmania virulence. J Biol Chem. 2017;292(29):12324–38. pmid:28550086
- 51. Veronica J, Chandrasekaran S, Dayakar A, Devender M, Prajapati VK, Sundar S, et al. Iron superoxide dismutase contributes to miltefosine resistance in Leishmania donovani. The FEBS Journal. 2019;286(17):3488–503.
- 52. Durak I, Yurtarslanl Z, Canbolat O, Akyol O. A methodological approach to superoxide dismutase (SOD) activity assay based on inhibition of nitroblue tetrazolium (NBT) reduction. Clin Chim Acta. 1993;214(1):103–4. pmid:8453769
- 53. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. pmid:942051
- 54. Oparka M, Walczak J, Malinska D, van Oppen LMPE, Szczepanowska J, Koopman WJH, et al. Quantifying ROS levels using CM-H 2 DCFDA and HyPer. Methods. 2016;109:3–11.
- 55. Keshav P, Goyal DK, Kaur S. Promastigotes of Leishmania donovani exhibited sensitivity towards the high altitudinal plant Cicer microphyllum. Curr Res Parasitol Vector Borne Dis. 2021;1:100040. pmid:35284854
- 56. Sarkar A, Khan YA, Laranjeira-Silva MF, Andrews NW, Mittra B. Quantification of intracellular growth inside macrophages is a fast and reliable method for assessing the virulence of Leishmania parasites. J Vis Exp. 2018;2018(133). pmid:29608175
- 57. Pinto-da-Silva LH, Fampa P, Soares DC, Oliveira SMP, Souto-Padron T, Saraiva EM. The 3A1-La monoclonal antibody reveals key features of Leishmania (L) amazonensis metacyclic promastigotes and inhibits procyclics attachment to the sand fly midgut. Int J Parasitol. 2005;35(7):757–64. pmid:15885694
- 58. Aoki JI, Hong A, Zampieri RA, Floeter-Winter LM, Laranjeira-Silva MF. In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice. J Vis Exp. 2020;2020(156). pmid:32150165
- 59. Titus RG, Marchand M, Boon T, Louis JA. A limiting dilution assay for quantifying Leishmania major in tissues of infected mice. Parasite Immunol. 1985;7(5):545–55. pmid:3877902
- 60. Kumar S, Stecher G, Suleski M, Sanderford M, Sharma S, Tamura K. MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Molecular Biology and Evolution. 2024;41(12):msae263.
- 61. Jones DT, Taylor WR, Thornton JM. The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992;8(3):275–82. pmid:1633570
- 62. Felsenstein J. CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP. Evolution. 1985;39(4):783–91. pmid:28561359