New drugs are needed to control leishmaniasis and efforts are currently on-going to counter the neglect of this disease. We discuss here the utility and the impact of associating drug resistance (DR) ...studies to drug discovery pipelines. We use as paradigm currently used drugs, antimonials and miltefosine, and complement our reflection by interviewing three experts in the field. We suggest DR studies to be involved at two different stages of drug development: (i) the efficiency of novel compounds should be confirmed on sets of strains including recent clinical isolates with DR; (ii) experimental DR should be generated to promising compounds at an early stage of their development, to further optimize them and monitor clinical trials.
Host-directed therapies (HDTs) constitute promising alternatives to traditional therapy that directly targets the pathogen but is often hampered by pathogen resistance. HDT could represent a new ...treatment strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. This protozoan develops exclusively within phagocytic cells, where infection relies on a complex molecular interplay potentially exploitable for drug targets. We previously identified naloxonazine, a compound specifically active against intracellular but not axenic Leishmania donovani. We evaluated here whether this compound could present a host cell-dependent mechanism of action. Microarray profiling of THP-1 macrophages treated with naloxonazine showed upregulation of vATPases, which was further linked to an increased volume of intracellular acidic vacuoles. Treatment of Leishmania-infected macrophages with the vATPase inhibitor concanamycin A abolished naloxonazine effects, functionally demonstrating that naloxonazine affects Leishmania amastigotes indirectly, through host cell vacuolar remodeling. These results validate amastigote-specific screening approaches as a powerful way to identify alternative host-encoded targets. Although the therapeutic value of naloxonazine itself is unproven, our results further demonstrate the importance of intracellular acidic compartments for host defense against Leishmania, highlighting the possibility of targeting this host cell compartment for anti-leishmanial therapy.
In the present review, we aim to provide a general introduction to different facets of the arms race between pathogens and their hosts/environment, emphasizing its evolutionary aspects. We focus on ...vector-borne parasitic protozoa, which have to adapt to both invertebrate and vertebrate hosts. Using Leishmania, Trypanosoma and Plasmodium as main models, we review successively (i) the adaptations and counter-adaptations of parasites and their invertebrate host, (ii) the adaptations and counter-adaptations of parasites and their vertebrate host and (iii) the impact of human interventions (chemotherapy, vaccination, vector control and environmental changes) on these adaptations. We conclude by discussing the practical impact this knowledge can have on translational research and public health.
Using a few pathogen models, the authors reviewed the different facets of the arms race between pathogens and hosts (vertebrate and invertebrate), with an emphasis on the evolutionary aspects of it. The authors discussed the impact of human interventions on these adaptations.
Graphical Abstract Figure.
Using a few pathogen models, the authors reviewed the different facets of the arms race between pathogens and hosts (vertebrate and invertebrate), with an emphasis on the evolutionary aspects of it. The authors discussed the impact of human interventions on these adaptations.
Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In ...several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples.
Leishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector-borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and ...over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment.
The protozoan parasite Trypanosoma brucei is lysed by apolipoprotein L-I, a component of human high-density lipoprotein (HDL) particles that are also characterized by the presence of ...haptoglobin-related protein. We report that this process is mediated by a parasite glycoprotein receptor, which binds the haptoglobin-hemoglobin complex with high affinity for the uptake and incorporation of heme into intracellular hemoproteins. In mice, this receptor was required for optimal parasite growth and the resistance of parasites to the oxidative burst by host macrophages. In humans, the trypanosome receptor also recognized the complex between hemoglobin and haptoglobin-related protein, which explains its ability to capture trypanolytic HDLs. Thus, in humans the presence of haptoglobin-related protein has diverted the function of the trypanosome haptoglobin-hemoglobin receptor to elicit innate host immunity against the parasite.
Leishmaniasis is a major health problem worldwide and tools available for their control are limited. Effective vaccines are still lacking, drugs are toxic and expensive, and parasites develop ...resistance to chemotherapy. In this context, new antimicrobials are urgently needed to control the disease in both human and animal. Here, we report the enzymatic and functional characterization of a
Leishmania
virulence factor,
Leishmania major
Protein disulfide isomerase (LmPDI) that could constitute a potential drug target. LmPDI possesses domain structure organization similar to other PDI family members (a, a′, b, b′ and c domains), and it displays the three enzymatic and functional activities specific of PDI family members: isomerase, reductase and chaperone. These results suggest that LmPDI plays a key role in assisting
Leishmania
protein folding via its capacity to catalyze formation, breakage, and rearrangement of disulfide bonds in nascent polypeptides. Moreover, Bacitracin, a reductase activity inhibitor, and Ribostamycin, a chaperone activity inhibitor, were tested in LmPDI enzymatic assays and versus
Leishmania
promastigote in vitro cultures and
Leishmania
amastigote multiplication inside infected THP-1-derived macrophages. Bacitracin inhibited both isomerase and reductase activities, while Ribostamycin had no effect on the chaperone activity. Interestingly, Bacitracin blocked in vitro promastigote growth as well as amastigote multiplication inside macrophages with EC
50
values of 39 μM. These results suggest that LmPDI may constitute an interesting target for the development of new anti-
Leishmania
drugs.
The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the ...assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.
High throughput sequencing techniques are poorly adapted for in vivo studies of parasites, which require prior in vitro culturing and purification. Trypanosomatids, a group of kinetoplastid ...protozoans, possess a distinctive feature in their transcriptional mechanism whereby a specific Spliced Leader (SL) sequence is added to the 5'end of each mRNA by trans-splicing. This allows to discriminate Trypansomatid RNA from mammalian RNA and forms the basis of our new multiplexed protocol for high-throughput, selective RNA-sequencing called SL-seq. We provided a proof-of-concept of SL-seq in Leishmania donovani, the main causative agent of visceral leishmaniasis in humans, and successfully applied the method to sequence Leishmania mRNA directly from infected macrophages and from highly diluted mixes with human RNA. mRNA profiles obtained with SL-seq corresponded largely to those obtained from conventional poly-A tail purification methods, indicating both enumerate the same mRNA pool. However, SL-seq offers additional advantages, including lower sequencing depth requirements, fast and simple library prep and high resolution splice site detection. SL-seq is therefore ideal for fast and massive parallel sequencing of parasite transcriptomes directly from host tissues. Since SLs are also present in Nematodes, Cnidaria and primitive chordates, this method could also have high potential for transcriptomics studies in other organisms.
In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells.
By screening a cDNA library of T. brucei ...with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time.
A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.
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