BACKGROUND: The present study investigates implications of a sub-chromosomal deletion in Leishmania infantum strains, the causative agent of American Visceral Leishmaniasis (AVL). Primarily found in New World strains, the deletion leads to the absence of the ecto-3'-nucleotidase/nuclease enzyme, impacting parasite virulence, pathogenicity, and drug susceptibility. The factors favoring prevalence and the widespread geographic distribution of these deleted mutant parasites (DEL) in the NW (NW) are discussed under the generated data. METHODS: We conducted phenotypic assessments of the sub-chromosomal deletion through in vitro assays with axenic parasites and experimental infections in both in vitro and in vivo models of vertebrate and invertebrate hosts using geographically diverse mutant field isolates. RESULTS: Despite reduced pathogenicity, the DEL strains efficiently infect vertebrate hosts and exhibit relevant differences, including enhanced metacyclogenesis and colonization rates in sand flies, potentially facilitating transmission. This combination may represent a more effective way to maintain and disperse the transmission cycle of DEL strains. CONCLUSIONS: Phenotypic assessments reveal altered parasite fitness, with potential enhanced transmissibility at the population level. Reduced susceptibility of DEL strains to miltefosine, a key drug in VL treatment, further complicates control efforts. The study underscores the importance of typing parasite genomes for surveillance and control, advocating for the sub-chromosomal deletion as a molecular marker in AVL management.

• MeSH
• Gene Deletion * MeSH
• Leishmania infantum * genetics   pathogenicity   MeSH
• Leishmaniasis, Visceral * parasitology   transmission   epidemiology   MeSH
• Humans MeSH
• Mice MeSH
• Psychodidae parasitology   MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Several new species of Leishmania have recently emerged in Europe, probably as the result of global changes and increased human migration from endemic areas. In this study, we tested whether two sand fly species, the Western Mediterranean Phlebotomus perniciosus and the Eastern Mediterranean P. tobbi, are competent vectors of L. donovani, L. major and L. martiniquensis. METHODOLOGY/PRINCIPAL FINDINGS: Sand flies were infected through the chick skin membrane using Leishmania species and strains of various geographical origins. Leishmania infections were evaluated by light microscopy and qPCR, and the representation of morphological forms was assessed from Giemsa-stained gut smears. Neither P. perniciosus nor P. tobbi supported the development of L. martiniquensis, but L. major and L. donovani in both species survived defecation of blood meal remnants, colonized the stomodeal valve and produced metacyclic stages. The results with L donovani have shown that infection rates in sand flies can be strain-specific; therefore, to determine vector competence or refractoriness, it is optimal to test at least two strains of Leishmania. CONCLUSIONS, SIGNIFICANCE: Both sand fly species tested are potential vectors of L. donovani and L. major in Mediterranean area. However, further studies will be needed to identify European vectors of L. martiniquensis and to test the ability of other European sand fly species to transmit L. major, L. donovani, L. tropica and L. infantum.

• MeSH
• Insect Vectors * parasitology   physiology   MeSH
• Chickens parasitology   MeSH
• Leishmania * physiology   classification   genetics   MeSH
• Leishmaniasis transmission   parasitology   MeSH
• Humans MeSH
• Phlebotomus * parasitology   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe MeSH

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread. One of the key life cycle stages is the haptomonad form, which attaches to insect tissues through its flagellum. This adhesion, conserved across kinetoplastid parasites, is implicated in having an important function within their life cycles and hence in disease transmission. Here, we discover the kinetoplastid-insect adhesion proteins (KIAPs), which localise in the attached Leishmania flagellum. Deletion of these KIAPs impairs cell adhesion in vitro and prevents Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. Additionally, loss of parasite adhesion in the sand fly results in reduced physiological changes to the fly, with no observable damage of the stomodeal valve and reduced midgut swelling. These results provide important insights into a comprehensive understanding of the Leishmania life cycle, which will be critical for developing transmission-blocking strategies.

• MeSH
• Cell Adhesion MeSH
• Flagella * metabolism   MeSH
• Insect Vectors parasitology   MeSH
• Insect Proteins metabolism   genetics   MeSH
• Host-Parasite Interactions MeSH
• Leishmania * physiology   genetics   metabolism   MeSH
• Leishmaniasis parasitology   transmission   MeSH
• Protozoan Proteins metabolism   genetics   MeSH
• Psychodidae * parasitology   MeSH
• Life Cycle Stages MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Insect Proteins MeSH
• Protozoan Proteins MeSH

Leishmania major is responsible for zoonotic cutaneous leishmaniasis. Therapy is mainly based on the use of antimony-based drugs; however, treatment failures and illness relapses were reported. Although studies were developed to understand mechanisms of drug resistance, the interactions of resistant parasites with their reservoir hosts and vectors remain poorly understood. Here we compared the development of two L. major MON-25 trivalent antimony-resistant lines, selected by a stepwise in vitro Sb(III)-drug pressure, to their wild-type parent line in the natural vector Phlebotomus papatasi. The intensity of infection, parasite location and morphological forms were compared by microscopy. Parasite growth curves and IC50 values have been determined before and after the passage in Ph. papatasi. qPCR was used to assess the amplification rates of some antimony-resistance gene markers. In the digestive tract of sand flies, Sb(III)-resistant lines developed similar infection rates as the wild-type lines during the early-stage infections, but significant differences were observed during the late-stage of the infections. Thus, on day 7 p. i., resistant lines showed lower representation of heavy infections with colonization of the stomodeal valve and lower percentage of metacyclic promastigote forms in comparison to wild-type strains. Observed differences between both resistant lines suggest that the level of Sb(III)-resistance negatively correlates with the quality of the development in the vector. Nevertheless, both resistant lines developed mature infections with the presence of infective metacyclic forms in almost half of infected sandflies. The passage of parasites through the sand fly guts does not significantly influence their capacity to multiply in vitro. The IC50 values and molecular analysis of antimony-resistance genes showed that the resistant phenotype of Sb(III)-resistant parasites is maintained after passage through the sand fly. Sb(III)-resistant lines of L. major MON-25 were able to produce mature infections in Ph. papatasi suggesting a possible circulation in the field using this vector.

• Keywords
• Antimony resistance, Fitness, Leishmania major, Phlebotomus papatasi, experimental infection, Virulence,
• MeSH
• Gene Amplification MeSH
• Antimony * pharmacology   MeSH
• Genetic Fitness MeSH
• Leishmania major * drug effects   genetics   pathogenicity   MeSH
• Drug Resistance * genetics   MeSH
• Phlebotomus * parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antimony * MeSH

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.

• MeSH
• Cell Division MeSH
• Cell Cycle MeSH
• Leishmania mexicana * genetics   MeSH
• Leishmania * MeSH
• Parasites * MeSH
• Psychodidae * parasitology   MeSH
• Mammals MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Sand fly transmitted Leishmania species are responsible for severe, wide ranging, visceral and cutaneous leishmaniases. Genetic exchange can occur among natural Leishmania populations and hybrids can now be produced experimentally, with limitations. Feeding Phlebotomus orientalis or Phlebotomus argentipes on two strains of Leishmania donovani yielded hybrid progeny, selected using double drug resistance and fluorescence markers. Fluorescence activated cell sorting of cultured clones derived from these hybrids indicated diploid progeny. Multilocus sequence typing of the clones showed hybridisation and nuclear heterozygosity, although with inheritance of single haplotypes in a kinetoplastid target. Comparative genomics showed diversity of clonal progeny between single chromosomes, and extraordinary heterozygosity across all 36 chromosomes. Diversity between progeny was seen for the HASPB antigen, which has been noted previously as having implications for design of a therapeutic vaccine. Genomic diversity seen among Leishmania strains and hybrid progeny is of great importance in understanding the epidemiology and control of leishmaniasis. As an outcome of this study we strongly recommend that wider biological archives of different Leishmania species from endemic regions should be established and made available for comparative genomics. However, in parallel, performance of genetic crosses and genomic comparisons should give fundamental insight into the specificity, diversity and limitations of candidate diagnostics, vaccines and drugs, for targeted control of leishmaniasis.

• MeSH
• Genomics MeSH
• Crosses, Genetic MeSH
• Leishmania donovani * genetics   MeSH
• Leishmaniasis, Cutaneous * MeSH
• Leishmaniasis, Visceral * diagnosis   prevention & control   epidemiology   MeSH
• Phlebotomus * genetics   MeSH
• Psychodidae * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

INTRODUCTION: Sand flies (Diptera: Phlebotominae) belonging to the Lutzomyia genus transmit Leishmania infantum parasites. To understand the complex interaction between the vector and the parasite, we have been investigating the sand fly immune responses during the Leishmania infection. Our previous studies showed that genes involved in the IMD, Toll, and Jak-STAT immunity pathways are regulated upon Leishmania and bacterial challenges. Nevertheless, the parasite can thrive in the vectors' gut, indicating the existence of mechanisms capable of modulating the vector defenses, as was already seen in mammalian Leishmania infections. METHODS RESULTS AND DISCUSSION: In this study, we investigated the expression of Lutzomyia longipalpis genes involved in regulating the Toll pathway under parasitic infection. Leishmania infantum infection upregulated the expression of two L. longipalpis genes coding for the putative repressors cactus and protein tyrosine phosphatase SHP. These findings suggest that the parasite can modulate the vectors' immune response. In mammalian infections, the Leishmania surface glycoprotein GP63 is one of the inducers of host immune depression, and one of the known effectors is SHP. In L. longipalpis we found a similar effect: a genetically modified strain of Leishmania amazonensis over-expressing the metalloprotease GP63 induced a higher expression of the sand fly SHP indicating that the L. longipalpis SHP and parasite GP63 increased expressions are connected. Immuno-stained microscopy of L. longipalpis LL5 embryonic cells cultured with Leishmania strains or parasite conditioned medium showed cells internalization of parasite GP63. A similar internalization of GP63 was observed in the sand fly gut tissue after feeding on parasites, parasite exosomes, or parasite conditioned medium, indicating that GP63 can travel through cells in vitro or in vivo. When the sand fly SHP gene was silenced by RNAi and females infected by L. infantum, parasite loads decreased in the early phase of infection as expected, although no significant differences were seen in late infections of the stomodeal valve. CONCLUSIONS: Our findings show the possible role of a pathway repressor involved in regulating the L. longipalpis immune response during Leishmania infections inside the insect. In addition, they point out a conserved immunosuppressive effect of GP63 between mammals and sand flies in the early stage of parasite infection.

• Keywords
• SHP-2, immunity, protein-tyrosine phosphatase, sand fly, signaling pathway, vector-parasite interaction,
• MeSH
• Immunosuppression Therapy MeSH
• Culture Media, Conditioned MeSH
• Leishmania infantum * MeSH
• Leishmaniasis * MeSH
• Phlebotomus * MeSH
• Psychodidae * MeSH
• Mammals MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Culture Media, Conditioned MeSH

Introduction: Production of different antimicrobial peptides (AMPs) is one of the insect's prominent defense strategies, regulated mainly by Toll and immune deficiency (IMD) humoral pathways. Here we focused mainly on two AMPs of Phlebotomus papatasi, vector of Leishmania major parasites, their association with the relish transcription factor and the effective participation on Leishmania infection. Methods and results: We further characterized the role of previously described gut-specific P. papatasi defensin (PpDef1) and identified the second defensin (PpDef2) expressed in various sand fly tissues. Using the RNAi-mediated gene silencing, we report that the silencing of PpDef1 gene or simultaneous silencing of both defensin genes (PpDef1 and PpDef2) resulted in increased parasite levels in the sand fly (detectable by PCR) and higher sand fly mortality. In addition, we knocked down relish, the sole transcription factor of the IMD pathway, to evaluate the association of the IMD pathway with AMPs expression in P. papatasi. We demonstrated that the relish gene knockdown reduced the expression of PpDef2 and attacin, another AMP abundantly expressed in the sand fly body. Conclusions: Altogether, our experiments show the importance of defensins in the sand fly response toward L. major and the role of the IMD pathway in regulating AMPs in P. papatasi.

• Keywords
• antimicrobial peptides, defensin, innate immunity, knockdown, leishmania, relish, sand fly,
• Publication type
• Journal Article MeSH

Parasites of the genus Porcisia, together with the genus Endotrypanum, form a sister clade to the species-rich and medically important genus Leishmania. Both Porcisia species, P. hertigi and P. deanei, are dixenous parasites of Neotropical porcupines. Almost 50 years after their first discovery, knowledge of their life cycle remains poor and their insect vectors are unknown. Because competent vectors of their closest phylogenetic relatives, genera Endotrypanum and Leishmania, are phlebotomine sand flies (Diptera: Psychodidae) and/or biting midges (Diptera: Ceratopogonidae), we examined here the potential of both sand flies and biting midges to transmit Porcisia parasites. The insects (Lutzomyia longipalpis, L. migonei and Culicoides sonorensis) were exposed to parasites through the chicken skin membrane and dissected at various time intervals post bloodmeal. Potentially infected females were also allowed to feed on the ears of anaesthetized BALB/c mice and the presence of parasite DNA was subsequently confirmed in the mice by PCR. Porcisia hertigi did not survive defecation in L. longipalpis or L. migonei, suggesting that these sand fly species are unlikely to serve as natural vectors of this parasite. Similarly, P. hertigi infections were lost in Culicoides midges. In contrast, mature P. deanei infections developed in 51-61% of L. longipalpis females, 7.3% of L. migonei females and 7.7% of Culicoides sonorensis females. In all three vector species, P. deanei colonized predominantly Malpighian tubules and produced metacyclic infective forms. Transmission of P. daenei to BALB/c mice was demonstrated via the prediuresis of L. longipalpis females. This mode of transmission, as well the colonization of Malpighian tubules as the dominant tissue of the vector, is unique among trypanosomatids. In conclusion, we demonstrated the vector competence of L. longipalpis for P. deanei but not for P. hertigi, and further studies are needed to evaluate competence of other Neotropical vectors for these neglected parasites.

• Keywords
• Culicoides, Lutzomyia, Malpighian tubules, Porcisia deanei, Porcisia hertigi, contaminative transmission, prediuresis,
• MeSH
• Phylogeny MeSH
• Insect Vectors MeSH
• Leishmania * MeSH
• Mice MeSH
• Psychodidae * MeSH
• Life Cycle Stages MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

• MeSH
• Leishmania donovani * genetics   MeSH
• Leishmaniasis, Visceral * parasitology   MeSH
• Humans MeSH
• Genomic Instability MeSH
• Proteomics MeSH
• Gene Expression Regulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH