Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum.

Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.

Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism.

The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of RAD50 was possible in a MRE11 null mutant that we had previously generated, providing good evidence that RAD50 may be dispensable in the absence of MRE11. Inactivation of the MRE11 and RAD50 genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the MRE11-/-RAD50-/- parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability.

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.

Deep-sequencing revealing mutation dynamics in the miltefosine transporter gene in Leishmania infantum selected for miltefosine resistance.

Miltefosine is the first oral drug used in chemotherapy against leishmaniasis. In vitro studies found that resistance to miltefosine in Leishmania is often associated with the acquisition of point mutations in the miltefosine transporter, leading to a decrease in drug uptake. In this study, the dynamics of mutations upon miltefosine selection was studied by deep-sequencing of the miltefosine transporter gene. Deep-sequencing data revealed that no mutation was detected in the miltefosine transporter at sub-inhibitory concentrations of miltefosine. We show that the prevalence of mutated alleles was increasing when the drug pressure heightened, that more mutations were observed in highly resistant mutants, and that most mutations remained when parasites were cultured for a few passages in the absence of miltefosine.

Plasticity of the Leishmania genome leading to gene copy number variations and drug resistance.

Leishmania has a plastic genome, and drug pressure can select for gene copy number variation (CNV). CNVs can apply either to whole chromosomes, leading to aneuploidy, or to specific genomic regions. For the latter, the amplification of chromosomal regions occurs at the level of homologous direct or inverted repeated sequences leading to extrachromosomal circular or linear amplified DNAs. This ability of Leishmania to respond to drug pressure by CNVs has led to the development of genomic screens such as Cos-Seq, which has the potential of expediting the discovery of drug targets for novel promising drug candidates.

Intrachromosomal amplification, locus deletion and point mutation in the aquaglyceroporin AQP1 gene in antimony resistant Leishmania (Viannia) guyanensis.

BACKGROUND: Antimony resistance complicates the treatment of infections caused by the parasite Leishmania. METHODOLOGY/PRINCIPAL FINDINGS: Using next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR) mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1). Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion. CONCLUSIONS/SIGNIFICANCE: This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.

DNA repair pathways in trypanosomatids: from DNA repair to drug resistance.

All living organisms are continuously faced with endogenous or exogenous stress conditions affecting genome stability. DNA repair pathways act as a defense mechanism, which is essential to maintain DNA integrity. There is much to learn about the regulation and functions of these mechanisms, not only in human cells but also equally in divergent organisms. In trypanosomatids, DNA repair pathways protect the genome against mutations but also act as an adaptive mechanism to promote drug resistance. In this review, we scrutinize the molecular mechanisms and DNA repair pathways which are conserved in trypanosomatids. The recent advances made by the genome consortiums reveal the complete genomic sequences of several pathogens. Therefore, using bioinformatics and genomic sequences, we analyze the conservation of DNA repair proteins and their key protein motifs in trypanosomatids. We thus present a comprehensive view of DNA repair processes in trypanosomatids at the crossroads of DNA repair and drug resistance.