A Plasmodium falciparum genetic cross reveals the contributions of pfcrt and plasmepsin II/III to piperaquine drug resistance.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.

A Phosphoinositide-Binding Protein Acts in the Trafficking Pathway of Hemoglobin in the Malaria Parasite Plasmodium falciparum.

Phosphoinositide lipids play key roles in a variety of processes in eukaryotic cells, but our understanding of their functions in the malaria parasite Plasmodium falciparum is still very much limited. To gain a deeper comprehension of the roles of phosphoinositides in this important pathogen, we attempted gene inactivation for 24 putative effectors of phosphoinositide metabolism. Our results reveal that 79% of the candidates are refractory to genetic deletion and are therefore potentially essential for parasite growth. Inactivation of the gene coding for a Plasmodium-specific putative phosphoinositide-binding protein, which we named PfPX1, results in a severe growth defect. We show that PfPX1 likely binds phosphatidylinositol-3-phosphate and that it localizes to the membrane of the digestive vacuole of the parasite and to vesicles filled with host cell cytosol and labeled with endocytic markers. Critically, we provide evidence that it is important in the trafficking pathway of hemoglobin from the host erythrocyte to the digestive vacuole. Finally, inactivation of PfPX1 renders parasites resistant to artemisinin, the frontline antimalarial drug. Globally, the minimal redundancy in the putative phosphoinositide proteins uncovered in our work supports that targeting this pathway has potential for antimalarial drug development. Moreover, our identification of a phosphoinositide-binding protein critical for the trafficking of hemoglobin provides key insight into this essential process. IMPORTANCE Malaria represents an enormous burden for a significant proportion of humanity, and the lack of vaccines and problems with drug resistance to all antimalarials demonstrate the need to develop new therapeutics. Inhibitors of phosphoinositide metabolism are currently being developed as antimalarials but our understanding of this biological pathway is incomplete. The malaria parasite lives inside human red blood cells where it imports hemoglobin to cover some of its nutritional needs. In this work, we have identified a phosphoinositide-binding protein that is important for the transport of hemoglobin in the parasite. Inactivation of this protein decreases the ability of the parasite to proliferate. Our results have therefore identified a potential new target for antimalarial development.

Assessment of Plasmodium falciparum Artemisinin Resistance Independent of kelch13 Polymorphisms and with Escalating Malaria in Bangladesh.

Emerging resistance to artemisinin drugs threatens the elimination of malaria. Resistance is widespread in South East Asia (SEA) and Myanmar. Neighboring Bangladesh, where 90% of infections occur in the Chittagong Hill Tracts (CHTs), lacks recent assessment. We undertook a prospective study in the sole district-level hospital in Bandarban, a CHT district with low population densities but 60% of reported malaria cases. Thirty patients presented with malaria in 2018. An increase to 68 patients in 2019 correlated with the district-level rise in malaria, rainfall, humidity, and temperature. Twenty-four patients (7 in 2018 and 17 in 2019) with uncomplicated Plasmodium falciparum monoinfection were assessed for clearing parasites after starting artemisinin combination therapy (ACT). The median (range) time to clear half of the initial parasites was 5.6 (1.5 to 9.6) h, with 20% of patients showing a median of 8 h. There was no correlation between parasite clearance and initial parasitemia, blood cell counts, or mutations of P. falciparum gene Pfkelch13 (the molecular marker of artemisinin resistance [AR]). The in vitro ring-stage survival assay (RSA) revealed one (of four) culture-adapted strains with a quantifiable resistance of 2.01% ± 0.1% (mean ± standard error of the mean [SEM]). Regression analyses of in vivo and in vitro measurements of the four CHT strains and WHO-validated K13 resistance mutations yielded good correlation (R2 = 0.7; ρ = 0.9, P < 0.005), strengthening evaluation of emerging AR with small sample sizes, a challenge in many low/moderate-prevalence sites. There is an urgent need to deploy multiple, complementary approaches to understand the evolutionary dynamics of the emergence of P. falciparum resistant to artemisinin derivatives in countries where malaria is endemic. IMPORTANCE Malaria elimination is a Millennium Development Goal. Artemisinins, fast-acting antimalarial drugs, have played a key role in malaria elimination. Emergence of artemisinin resistance threatens the global elimination of malaria. Over the last decade, advanced clinical and laboratory methods have documented its spread throughout South East Asia and Myanmar. Neighboring Bangladesh lies in the historical path of dissemination of antimalarial resistance to the rest of the world, yet it has not been evaluated by combinations of leading methods, particularly in the highland Chittagong Hill Tracts adjacent to Myanmar which contain >90% of malaria in Bangladesh. We show the first establishment of capacity to assess clinical artemisinin resistance directly in patients in the hilltops and laboratory adaptation of Bangladeshi parasite strains from a remote, sparsely populated malaria frontier that is responsive to climate. Our study also provides a generalized model for comprehensive monitoring of drug resistance for countries where malaria is endemic.

Well-Tolerated Amphotericin B Derivatives That Effectively Treat Visceral Leishmaniasis.

Chemotherapy against the neglected tropical disease visceral leishmaniasis (VL) is suboptimal with only four licensed drugs. Amphotericin B (AmB), despite its toxicity, remained a second line drug for a long time. However, the demonstration that liposomal AmB is highly effective against VL propelled it, despite its cost, to a first line drug in many countries. While several ongoing efforts are aiming at finding cheaper and stable AmB-formulations, an alternative strategy is the development of less-toxic AmB derivatives. We show here that two less-toxic AmB derivatives with the carboxylate at position 16 of AmB derivatized to a methyl urea (AmB-MU) or amino urea (AmB-AU) are active in vitro against Leishmania donovani, both as free-living parasites as well as their intracellular form. Both less-toxic derivatives, similarly to AmB, target the ergosterol pathway of L. donovani. While the AmB-AU derivative showed female-specific liver toxicity in vivo, the AmB-MU derivative was well-tolerated and more effective than AmB against experimental VL. These studies are an important step for improving AmB-based therapy against a prevalent parasitic disease.

Total Synthesis and Antimalarial Activity of Dominicin, a Cyclic Octapeptide from a Marine Sponge.

Dominicin, a macrocyclic peptide isolated from the marine sponge Eurypon laughlini, has been synthesized for the first time by solid-phase peptide synthesis. The strategy uses oxime resin and takes advantage of the nucleophile susceptibility of the oxime ester bond. The synthesis relies on the preparation of a linear precursor followed by on-resin head-to-tail concomitant cyclization-cleavage. This is the first report of the use of a Boc/OtBu biorthogonal protection strategy on oxime resin to facilitate concomitant N-terminal and side-chain tert-butyl ether deprotection cyclization of unprotected peptides. Also, we report the first antimalarial investigation of dominicin. Interestingly, the natural macrocyclic peptide demonstrates effective low micromolar activity (1.8 µM) against the chloroquine-mefloquine-pyrimethamine-resistant Dd2 strain of Plasmodium falciparum.

New insights in the mode of action of anti-leishmanial drugs by using chemical mutagenesis screens coupled to next-generation sequencing.

Leishmania parasites are responsible for a range of clinical manifestations ranging from self-resolving cutaneous sores to life-threatening diseases. The management of leishmaniasis is complicated in part by the scarcity of treatment options but also by the emerging or established resistance to available drugs. A major driver of resistance in Leishmania is the amplification of resistance genes taking advantage of the highly repetitive genomic landscape of the parasite. The recent advent of whole genome gain of function screens gave new momentum to the study of such resistance mechanisms, leading to the identification of novel resistance factors and drug targets against approved drugs, which include antimony (SbIII), miltefosine (MIL), paromomycin (PMM), and amphotericin B. However, these screens do not pinpoint single nucleotide variations (SNVs), an important contributor of drug resistance. To fill the gap, our recent study describes the optimization of chemical mutagenesis coupled to next generation sequencing, an approach called Mut-seq, as a way to explore networks of drug resistance genes in organisms with a diploid to mosaic aneuploid genome like Leishmania. Our Mut-seq screen revealed associations between genes linked with lipid metabolism and resistance to MIL, and highlighted the role of a protein kinase in translation leading to resistance to PMM.

Coupling chemical mutagenesis to next generation sequencing for the identification of drug resistance mutations in Leishmania.

Current genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging. Here, we use chemical mutagenesis, drug selection and next generation sequencing to characterize miltefosine and paromomycin resistant clones of the parasite Leishmania. We highlight several genes involved in drug resistance by sequencing the genomes of 41 resistant clones and by concentrating on recurrent SNVs. We associate genes linked to lipid metabolism or to ribosome/translation functions with miltefosine or paromomycin resistance, respectively. We prove by allelic replacement and CRISPR-Cas9 gene-editing that the essential protein kinase CDPK1 is crucial for paromomycin resistance. We have linked CDPK1 in translation by functional interactome analysis, and provide evidence that CDPK1 contributes to antimonial resistance in the parasite. This screen is powerful in exploring networks of drug resistance in an organism with diploid to mosaic aneuploid genome, hence widening the scope of its applicability.

A pan-apicomplexan phosphoinositide-binding protein acts in malarial microneme exocytosis.

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is an essential step in the development of the disease. Consequently, the molecular players involved in host cell invasion represent important targets for inhibitor design and vaccine development. The process of merozoite invasion is a succession of steps underlined by the sequential secretion of the organelles of the apical complex. However, little is known with regard to how their contents are exocytosed. Here, we identify a phosphoinositide-binding protein conserved in apicomplexan parasites and show that it is important for the attachment and subsequent invasion of the erythrocyte by the merozoite. Critically, removing the protein from its site of action by knock sideways preferentially prevents the secretion of certain types of micronemes. Our results therefore provide evidence for a role of phosphoinositide lipids in the malaria invasion process and provide further insight into the secretion of microneme organelle populations, which is potentially applicable to diverse apicomplexan parasites.

A Review of FOXI3 Regulation of Development and Possible Roles in Cancer Progression and Metastasis.

Development and cancer share a variety of functional traits such as EMT, cell migration, angiogenesis, and tissue remodeling. In addition, many cellular signaling pathways are noted to coordinate developmental processes and facilitate aspects of tumor progression. The Forkhead box superfamily of transcription factors consists of a highly conserved DNA binding domain, which binds to specific DNA sequences and play significant roles during adult tissue homoeostasis and embryogenesis including development, differentiation, metabolism, proliferation, apoptosis, migration, and invasion. Interestingly, various studies have implicated the role of key Fox family members such as FOXP, FOXO, and FOXA during cancer initiation and metastases. FOXI3, a member of the Forkhead family affects embryogenesis, development, and bone remodeling; however, no studies have reported a role in cancer. In this review, we summarize the role of FOXI3 in embryogenesis and bone development and discuss its potential involvement in cancer progression with a focus on the bone metastasis. Moreover, we hypothesize possible mechanisms underlying the role of FOXI3 in the development of solid tumor bone metastasis.

Novel classification for global gene signature model for predicting severity of systemic sclerosis.

Progression of systemic scleroderma (SSc), a chronic connective tissue disease that causes a fibrotic phenotype, is highly heterogeneous amongst patients and difficult to accurately diagnose. To meet this clinical need, we developed a novel three-layer classification model, which analyses gene expression profiles from SSc skin biopsies to diagnose SSc severity. Two SSc skin biopsy microarray datasets were obtained from Gene Expression Omnibus. The skin scores obtained from the original papers were used to further categorize the data into subgroups of low (<18) and high (≥18) severity. Data was pre-processed for normalization, background correction, centering and scaling. A two-layered cross-validation scheme was employed to objectively evaluate the performance of classification models of unobserved data. Three classification models were used: support vector machine, random forest, and naive Bayes in combination with feature selection methods to improve performance accuracy. For both input datasets, random forest classifier combined with correlation-based feature selection (CFS) method and naive Bayes combined with CFS or support vector machine based recursive feature elimination method yielded the best results. Additionally, we performed a principal component analysis to show that low and high severity groups are readily separable by gene expression signatures. Ultimately, we found that our novel classification prediction model produced global gene signatures that significantly correlated with skin scores. This study represents the first report comparing the performance of various classification prediction models for gene signatures from SSc patients, using current clinical diagnostic factors. In summary, our three-classification model system is a powerful tool for elucidating gene signatures from SSc skin biopsies and can also be used to develop a prognostic gene signature for SSc and other fibrotic disorders.

Plasmepsin II-III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum.

Multidrug resistant Plasmodium falciparum in Southeast Asia endangers regional malaria elimination and threatens to spread to other malaria endemic areas. Understanding mechanisms of piperaquine (PPQ) resistance is crucial for tracking its emergence and spread, and to develop effective strategies for overcoming it. Here we analyze a mechanism of PPQ resistance in Cambodian parasites. Isolates exhibit a bimodal dose-response curve when exposed to PPQ, with the area under the curve quantifying their survival in vitro. Increased copy number for plasmepsin II and plasmepsin III appears to explain enhanced survival when exposed to PPQ in most, but not all cases. A panel of isogenic subclones reinforces the importance of plasmepsin II-III copy number to enhanced PPQ survival. We conjecture that factors producing increased parasite survival under PPQ exposure in vitro may drive clinical PPQ failures in the field.

Inactivation of Plasmepsins 2 and 3 Sensitizes Plasmodium falciparum to the Antimalarial Drug Piperaquine.

Dihydroartemisinin-piperaquine (DHA-PPQ), the current frontline artemisinin combination therapy used to treat Plasmodium falciparum malaria in multiple Southeast Asian countries, is now increasingly failing in Cambodia, where artemisinin resistance is nearly fixed, which suggests that PPQ resistance has emerged and is spreading rapidly in the Greater Mekong Subregion. Recent reports have shown that amplification of the genes encoding plasmepsins 2 and 3 is a molecular marker of PPQ resistance; however, whether these enzymes play a role in the mechanism of resistance is currently unknown. We show here that inactivating the genes encoding plasmepsin 2 or 3 individually in P. falciparum reference strain 3D7 results in hypersusceptibility to PPQ. Interestingly, no significant differences in the susceptibility to other antimalarials were observed, which suggests specific roles of plasmepsins 2 and 3 in PPQ susceptibility. The piperaquine hyper-sensitivity of the plasmepsin-2-and-3-inactivated lines provides direct evidence that these enzymes modulate parasite susceptibility to PPQ in the context of a single copy of PfMDR1 and independent of Kelch13 mutations conferring artemisinin resistance.

A map of the subcellular distribution of phosphoinositides in the erythrocytic cycle of the malaria parasite Plasmodium falciparum.

Despite representing a small percentage of the cellular lipids of eukaryotic cells, phosphoinositides (PIPs) are critical in various processes such as intracellular trafficking and signal transduction. Central to their various functions is the differential distribution of PIP species to specific membrane compartments through the actions of kinases, phosphatases and lipases. Despite their importance in the malaria parasite lifecycle, the subcellular distribution of most PIP species in this organism is still unknown. We here localise several species of PIPs throughout the erythrocytic cycle of Plasmodium falciparum. We show that PI3P is mostly found at the apicoplast and the membrane of the food vacuole, that PI4P associates with the Golgi apparatus and the plasma membrane and that PI(4,5)P2, in addition to being detected at the plasma membrane, labels some cavity-like spherical structures. Finally, we show that the elusive PI5P localises to the plasma membrane, the nucleus and potentially to the transitional endoplasmic reticulum (ER). Our map of the subcellular distribution of PIP species in P. falciparum will be a useful tool to shed light on the dynamics of these lipids in this deadly parasite.

Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia.

BACKGROUND: Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. METHODS: Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA0-3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. RESULTS: Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA0-3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. CONCLUSIONS: This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance.

African Americans with pancreatic ductal adenocarcinoma exhibit gender differences in Kaiso expression.

Kaiso, a bi-modal transcription factor, regulates gene expression, and is elevated in breast, prostate, and colon cancers. Depletion of Kaiso in other cancer types leads to a reduction in markers for the epithelial-mesenchymal transition (EMT) (Jones et al., 2014), however its clinical implications in pancreatic ductal adenocarcinoma (PDCA) have not been widely explored. PDCA is rarely detected at an early stage but is characterized by rapid progression and invasiveness. We now report the significance of the subcellular localization of Kaiso in PDCAs from African Americans. Kaiso expression is higher in the cytoplasm of invasive and metastatic pancreatic cancers. In males, cytoplasmic expression of Kaiso correlates with cancer grade and lymph node positivity. In male and female patients, cytoplasmic Kaiso expression correlates with invasiveness. Also, nuclear expression of Kaiso increases with increased invasiveness and lymph node positivity. Further, analysis of the largest PDCA dataset available on ONCOMINE shows that as Kaiso increases, there is an overall increase in Zeb1, which is the inverse for E-cadherin. Hence, these findings suggest a role for Kaiso in the progression of PDCAs, involving the EMT markers, E-cadherin and Zeb1.

Altered drug susceptibility during host adaptation of a Plasmodium falciparum strain in a non-human primate model.

Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model.

Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease.

Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11(-/-) parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania.

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment.

Telomeric gene deletion and intrachromosomal amplification in antimony-resistant Leishmania.

Antimonials are still the mainstay of treatment against leishmaniasis but drug resistance is increasing. We carried out short read next-generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony-resistant mutants. Copy number variations were consistently detected with both NGS and CGH. A major attribute of antimony resistance was a novel terminal deletion of variable length (67 kb to 204 kb) of the polyploid chromosome 31 in the three mutants. Terminal deletions in two mutants occurred at the level of inverted repeated sequences. The AQP1 gene coding for an aquaglyceroporin was part of the deleted region and its transfection into resistant mutants reverted resistance to SbIII. We also highlighted an intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded for ascorbate-dependent peroxidase (APX) and glucose-6-phosphate dehydrogenase (G6PDH). Overexpression of these genes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS). Generation of a G6PDH null mutant in one revertant exhibited SbIII sensitivity and a decreased protection of ROS. Our genomic analyses and functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania.

Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.

In most organisms, the primary function of homologous recombination (HR) is to allow genome protection by the faithful repair of DNA double-strand breaks. The vital step of HR is the search for sequence homology, mediated by the RAD51 recombinase, which is stimulated further by proteins mediators such as the tumor suppressor BRCA2. The biochemical interplay between RAD51 and BRCA2 is unknown in Leishmania or Trypanosoma. Here we show that the Leishmania infantum BRCA2 protein possesses several critical features important for the regulation of DNA recombination at the genetic and biochemical level. A BRCA2 null mutant, generated by gene disruption, displayed genomic instability and gene-targeting defects. Furthermore, cytological studies show that LiRAD51 can no longer localize to the nucleus in this mutant. The Leishmania RAD51 and BRCA2 interact together and the purified proteins bind single-strand DNA. Remarkably, LiBRCA2 is a recombination mediator that stimulates the invasion of a resected DNA double-strand break in an undamaged template by LiRAD51 to form a D-loop structure. Collectively, our data show that LiBRCA2 and LiRAD51 promote HR at the genetic and biochemical level in L. infantum, the causative agent of visceral leishmaniasis.