Increased copy number of the target gene squalene monooxygenase as the main resistance mechanism to terbinafine in Leishmania infantum.

We use here two genomic screens in an attempt to understand the mode of action and resistance mechanism of terbinafine, an antifungal contemplated as a potential drug against the parasite Leishmania. One screen consisted in in vitro drug evolution where 5 independent mutants were selected step-by-step for terbinafine resistance. Sequencing of the genome of the 5 mutants revealed no single nucleotide polymorphisms related to the resistance phenotype. However, the ERG1 gene was found amplified as part of a linear amplicon, and transfection of ERG1 fully recapitulated the terbinafine resistance phenotype of the mutants. The second screen, Cos-seq, consisted in selecting a gene overexpression library with terbinafine followed by the sequencing of the enriched cosmids. This screen identified two cosmids derived from loci on chromosomes 13 and 29 encoding the squalene monooxygenase (ERG1) and the C8 sterol isomerase (ERG2), respectively. Transfection of the ERG1-cosmid, but not the ERG2-cosmid, produced resistance to terbinafine. Our screens suggest that ERG1 is the main, if not only, target for terbinafine in Leishmania and amplification of its gene is the main resistance mechanism.

PfGBP2 is a novel G-quadruplex binding protein in Plasmodium falciparum.

Guanine-quadruplexes (G4s) are non-canonical DNA structures that can regulate key biological processes such as transcription, replication and telomere maintenance in several organisms including eukaryotes, prokaryotes and viruses. Recent reports have identified the presence of G4s within the AT-rich genome of Plasmodium falciparum, the protozoan parasite causing malaria. In Plasmodium, potential G4-forming sequences (G4FS) are enriched in the telomeric and sub-telomeric regions of the genome where they are associated with telomere maintenance and recombination events within virulence genes. However, there is a little understanding about the biological role of G4s and G4-binding proteins. Here, we provide the first snapshot of G4-interactome in P. falciparum using DNA pull-down assay followed by LC-MS/MS. Interestingly, we identified ~24 potential G4-binding proteins (G4-BP) that bind to a stable G4FS (AP2_G4). Furthermore, we characterised the role of G-strand binding protein 2 (PfGBP2), a putative telomere-binding protein in P. falciparum. We validated the interaction of PfGBP2 with G4 in vitro as well as in vivo. PfGBP2 is expressed throughout the intra-erythrocytic developmental cycle and is essential for the parasites in the presence of G4-stabilising ligand, pyridostatin. Gene knockout studies showed the role of PfGBP2 in the expression of var genes. Taken together, this study suggests that PfGBP2 is a bona fide G4-binding protein, which is likely to be involved in the regulation of G4-related functions in these malarial parasites. In addition, this study sheds light on this understudied G4 biology in P. falciparum.

Genome wide distribution of G-quadruplexes and their impact on gene expression in malaria parasites.

Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite's AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology.

A single amino acid substitution (H451Y) in Leishmania calcium-dependent kinase SCAMK confers high tolerance and resistance to antimony.

BACKGROUND: For almost a century, antimonials have remained the first-line drugs for the treatment of leishmaniasis. However, little is known about their mode of action and clinical resistance mechanisms. OBJECTIVES: We have previously shown that Leishmania nicotinamidase (PNC1) is an essential enzyme for parasite NAD+ homeostasis and virulence in vivo. Here, we found that parasites lacking the pnc1 gene (Δpnc1) are hypersusceptible to the active form of antimony (SbIII) and used these mutant parasites to better understand antimony's mode of action and the mechanisms leading to resistance. METHODS: SbIII-resistant WT and Δpnc1 parasites were selected in vitro by a stepwise selection method. NAD(H)/NADP(H) dosages and quantitative RT-PCR experiments were performed to explain the susceptibility differences observed between strains. WGS and a marker-free CRISPR/Cas9 base-editing approach were used to identify and validate the role of a new resistance mutation. RESULTS: NAD+-depleted Δpnc1 parasites were highly susceptible to SbIII and this phenotype could be rescued by NAD+ precursor or trypanothione precursor supplementation. Δpnc1 parasites could become resistant to SbIII by an unknown mechanism. WGS revealed a unique amino acid substitution (H451Y) in an EF-hand domain of an orphan calcium-dependent kinase, recently named SCAMK. When introduced into a WT reference strain by base editing, the H451Y mutation allowed Leishmania parasites to survive at extreme concentrations of SbIII, potentiating the rapid emergence of resistant parasites. CONCLUSIONS: These results establish that Leishmania SCAMK is a new central hub of antimony's mode of action and resistance development, and uncover the importance of drug tolerance mutations in the evolution of parasite drug resistance.

Cos-Seq: A High-Throughput Gain-of-Function Screen for Drug Resistance Studies in Leishmania.

Leishmania is still a major cause of mortality and morbidity worldwide. Few efficient drugs are available, and resistance threatens actual treatments. In order to improve knowledge about the mode of action of current drugs and those in development, as well as to understand the mechanisms pertaining to their resistance, we recently described a sensitive and high-throughput method termed Cos-Seq. Here we provide a detailed protocol for every step of the procedure, from library construction to drug selection, cosmid extraction, and next-generation sequencing of extracted cosmids. A section on the bioinformatics of Cos-Seq is also included. Cos-Seq facilitates the identification of gain-of-function resistance mechanisms and drug targets and is a useful tool in resistance and drug development studies.

Protozoan Parasite Auxotrophies and Metabolic Dependencies.

Diseases caused by protozoan parasites have a major impact on world health. These early branching eukaryotes cause significant morbidity and mortality in humans and livestock. During evolution, protozoan parasites have evolved toward complex life cycles in multiple host organisms with different nutritional resources. The conservation of functional metabolic pathways required for these successive environments is therefore a prerequisite for parasitic lifestyle. Nevertheless, parasitism drives genome evolution toward gene loss and metabolic dependencies (including strict auxotrophy), especially for obligatory intracellular parasites. In this chapter, we will compare and contrast how protozoan parasites have perfected this metabolic adaptation by focusing on specific auxotrophic pathways and scavenging strategies used by clinically relevant apicomplexan and trypanosomatid parasites to access host's nutritional resources. We will further see how these metabolic dependencies have in turn been exploited for therapeutic purposes against these human pathogens.

Functional divergence of SIR2 orthologs between trypanosomatid parasites.

SIR2 proteins are NAD+-dependent deacetylases involved in epigenetic control of gene expression and metabolic regulation through post-translational modification of diverse target proteins. In pathogens, these enzymes are considered as attractive drug targets involved in key aspects of the infectious cycle. Leishmania infantum LiSIR2rp1 was among the first non-nuclear and essential SIR2 deacetylases described in eukaryotes. Here, we show that the two other LiSIR2rp2 and LiSIRrp3 paralogs are both located in mitochondria. Gene deletion experiments show that LiSIR2rp3 is not required for parasite survival. Surprisingly, multiple extrachromosomal amplicons bearing the LiSIR2rp2 gene are constitutively produced in wild type strains. Consequently, a knockout of this gene could not be obtained, even after episomal rescue experiments. We further provide genetic and biochemical evidence showing that SIR2rp2 protein directly affects parasite proliferation in relation to NAD+ bioavailability. Together, these results highlight unexpected genus-specific divergence of the SIR2 machinery among trypanosomatid parasites.

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania.

Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or "Cos-Seq"), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.

Drug resistance analysis by next generation sequencing in Leishmania.

The use of next generation sequencing has the power to expedite the identification of drug resistance determinants and biomarkers and was applied successfully to drug resistance studies in Leishmania. This allowed the identification of modulation in gene expression, gene dosage alterations, changes in chromosome copy numbers and single nucleotide polymorphisms that correlated with resistance in Leishmania strains derived from the laboratory and from the field. An impressive heterogeneity at the population level was also observed, individual clones within populations often differing in both genotypes and phenotypes, hence complicating the elucidation of resistance mechanisms. This review summarizes the most recent highlights that whole genome sequencing brought to our understanding of Leishmania drug resistance and likely new directions.

Transmission potential of antimony-resistant leishmania field isolates.

We studied the development of antimony-resistant Leishmania infantum in natural vectors Lutzomyia longipalpis and Phlebotomus perniciosus to ascertain the risk of parasite transmission by sand flies. All three resistant strains produced fully mature late-stage infections in sand flies; moreover, the resistant phenotype was maintained after the passage through the vector. These results highlight the risk of circulation of resistant Leishmania strains and question the use of human drugs for treatment of dogs as Leishmania reservoirs.

Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide.

Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.

Leishmania infantum nicotinamidase is required for late-stage development in its natural sand fly vector, Phlebotomus perniciosus.

Leishmania infantum nicotinamidase, encoded by the Lipnc1 gene, converts nicotinamide into nicotinicacid to ensure Nicotinamide­Adenine­Dinucleotide (NAD+) biosynthesis. We were curious to explore the role of this enzyme during L. infantum development in its natural sand fly vector, Phlebotomus perniciosus (Diptera, Phlebotominae), using null mutants with a deleted Lipnc1 gene. The null mutants developed as well as the wild type L. infantum at the early time points post their ingestion within the bloodmeal. In contrast, once the blood meal digestion was completed, the null mutants were unable to develop further and establish late-stage infections. Data highlight the importance of the nicotinamide degradation pathway for Leishmania development in sand flies. They indicate that the endogenous nicotinamidase is essential for Leishmania development in the sand fly after the blood meal has been digested and the remnants defecated.

Leishmania antimony resistance: what we know what we can learn from the field.

Leishmania is the causative agent of various forms of leishmaniasis, a significant cause of morbidity and mortality. The clinical manifestations of the disease range from self-healing cutaneous and mucocutaneous skin ulcers to a fatal visceral form named visceral leishmaniasis or kala-azar. In the absence of any effective vaccine, the only means to treat and control leishmaniasis is affordable medication. The treatment choice is essentially directed by economic considerations; therefore, for a large majority of countries, chemotherapy relies only on the use of cheaper antimonial compounds. The emergence of antimonial therapy failure in India linked to proven parasite resistance has stressed questions about selective factors as well as transmission risk of drug resistance. Unfortunately, in most parts of the world, the frequency of parasite antimony resistance linked to treatment failure is unknown because of a lack of information on Leishmania antimony susceptibility. This information is crucial for addressing the risk of selection and transmission of drug-resistant parasites, particularly in areas where antimony is the only chemotherapeutic alternative. However, the poor knowledge about factors that favor selection of resistant parasites, the multiplicity of the agents that can play a role in the in vivo antileishmanial activity of antimony, and the lack of a standard protocol to diagnose and survey parasite resistance all contribute to insufficient monitoring of antimony resistance. In this review, we discuss on the factors potentially involved in the selection of antimony resistance in the field and discuss on the methods available for its diagnosis.

In vitro activity of nicotinamide/antileishmanial drug combinations.

To improve the management of leishmaniasis, new drugs and/or alternative therapeutic strategies are required. Combination therapy of antileishmanial drugs is currently considered as one of the most rational approaches to lower treatment failure rate and limit drug resistance spreading. Nicotinamide (NAm), also known as vitamin B3 that is already is used in human therapy, exerts in vitro antileishmanial activity. Drug combination studies, performed on L. infantum axenic amastigotes, revealed that NAm significantly improves the antileishmanial activity of trivalent antimony in a synergistic manner while it shows additive activity with amphotericin B and slightly antagonizes pentamidine activity. NAm also significantly increases the toxicity of pentavalent antimony against the intracellular forms of L. infantum, L. amazonensis and L. braziliensis. The potential of NAm to be used as adjuvant during leishmaniasis chemotherapy is further discussed.