We argue that the mode of reproduction of microorganisms in nature can only be decided by population genetic information. The evidence available indicates that many parasitic protozoa and unicellular fungi have clonal rather than sexual population structures, which has major consequences for medical research and practice. Plasmodium falciparum, the agent of malaria, is a special case: the scarce evidence available is contradictory, some suggesting that uniparental lineages may exist in nature. This is puzzling (because P. falciparum is known to have a sexual stage) and poses a challenge that can be readily settled by ascertaining the frequency distribution of genotypes in natural populations.Sexual reproduction is generally assumed to be a common mode of reproduction of eukaryotes. In the case of parasitic protozoa, the assumption of sexual reproduction relies largely on the presumption that these organisms are diploid, as well as on the occurrence of sexual recombination in the laboratory under appropriate circumstances (review in ref. 1), rather than on relevant evidence obtained from nature. Yet, whether or not sexual reproduction prevails in these organisms is of considerable medical and agronomic consequence as well as of scientific interest. These eukaryotic microorganisms include the agents of malaria, sleeping sickness, Chagas disease, and other parasitic diseases that affect more than 10% of the world population. The strategies for developing vaccines or curative drugs as well as for diagnosis and treatment are different for clonal and for sexual organisms.That sexual reproduction may occur in laboratory cultures or even occasionally in nature does not by itself settle the issue, since that simply manifests that the potentiality for sexual reproduction has not been lost. What remains to be determined is the prevailing mode of reproduction of these organisms in natural circumstances. The evidence to settle the matter exists for some of these organisms and could be obtained for others without massive investment of resources or new scientific or medical advances. We herein advance a sustained argument to show that population genetic evidence and population genetic theory is all that is needed to ascertain the extent to which, if at all, these (or any other) organisms reproduce sexually in nature. We have already reviewed the evidence for Trypanosoma cruzi, the agent of Chagas disease (2), and some other protozoa (3). Here we develop further the argument and present the results of a survey of the available evidence for parasitic protozoa and unicellular fungi. CLONALITY IN MICROBIAL EUKARIOTESThe two genetic consequences of sexual reproduction are segregation and recombination. Population genetic methods make it possible to ascertain whether or not the distribution of genotypes in natural populations is consistent with the occurrence of segregation and recombination. The kind of evidence that is needed is the frequency distribution ofgenotypes rather than the direct observation of sexual or clonal r...

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Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10−8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs.

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A study based on 28 microsatellite loci was performed on 32 isolates of Plasmodium falciparum from Pointe Noire (Republic of the Congo) and compared with a cosmopolitan sample of 21 isolates collected from different countries in Africa, Latin America, and Asia. The Pointe Noire population exhibited very high genetic diversity (A ‫ס‬ 7.8 ± 2.6, He ‫ס‬ 0.79 ± 0.11). Significant linkage disequilibria were observed in 28 of 378 pairs of microsatellite loci. This result could be explained by two non-exclusive hypotheses: 1) uniparental propagation (i.e., selfing), leading to nonpanmictic associations, and/or 2) a Wahlund effect (i.e., spatial population genetic heterogeneity). These observations are in agreement with data previously obtained from isozyme loci of the same isolates, but contrast with other population genetic analyses conducted in other hyperendemic zones.

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.

Temporins are anti-microbial peptides synthesized in the skin of frogs of the Ranidae family. The few studies to date that have examined their anti-viral properties have shown that they have potential as anti-viral therapies. In this work, we evaluated the anti-herpes simplex virus type 1 (HSV-1) activity of the temporin-SHa (SHa) and its synthetic analog [K3]SHa. Human cathelicidin LL-37 and temporin-Tb (Tb), previously demonstrated to have anti-HSV-1 properties, were used as positive controls. We observed that SHa and [K3]SHa significantly inhibit HSV-1 replication in human primary keratinocytes when used at micromolar concentrations. This anti-viral activity was equivalent to that of Tb, but lower than that of LL-37. Transcriptomic analyses revealed that SHa did not act through the modulation of the cell innate immune response, but rather, displayed virucidal properties by reducing infectious titer of HSV-1 in suspension. In contrast, pre-incubation of the virus with LL-37 suggests that this peptide does not act directly on the viral particle at non-cytotoxic concentrations tested. The anti-HSV-1 activity of LL-37 appears to be due to the potentiation of cellular anti-viral defenses through the induction of interferon stimulated gene expression in infected primary keratinocytes. This study demonstrated that SHa and [K3]SHa, in addition to their previously reported antibacterial and antiparasitic activities, are direct-acting anti-HSV-1 peptides. Importantly, this study extends the little studied anti-viral attributes of frog temporins and offers perspectives for the development of new anti-HSV-1 therapies.