Lipophosphoglycan Polymorphisms Do Not Affect \u3cem\u3eLeishmania amazonensis\u3c/em\u3e Development in the Permissive Vectors \u3cem\u3eLutzomyia migonei\u3c/em\u3e and \u3cem\u3eLutzomyia longipalpis\u3c/em\u3e

Background: Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmaniapromastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species. Results: Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 β-glucose or 1 to 3 β-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi. Conclusions: LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector

The Phlebotomus papatasi systemic transcriptional response to trypanosomatid-contaminated blood does not differ from the non-infected blood meal

Background: Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that affects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control require a better understanding of the key step for transmission, namely the establishment of infection inside the fly. Methods: The aim of this work was to identify sand fly systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand flies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, L. donovani and Herpetomonas muscarum, the latter being a parasite not transmitted to humans. Results: Of the trypanosomatids studied, only L. major was able to successfully establish an infection in the host P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not specific to success or failure of a specific infection and they did not differ from each other. The transcriptional signatures were also indistinguishable after a non-contaminated blood meal. Conclusions: The results imply that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect

Visualisation of Leishmania donovani Fluorescent Hybrids during Early Stage Development in the Sand Fly Vector

hybrids were produced by co-infecting sand flies with two strains carrying different drug resistance markers. However, the location and timing of hybridisation events in sand flies has not been described. strains carrying hygromycin or neomycin resistance genes and red or green fluorescent markers. Fed females were dissected at different times post bloodmeal (PBM) and examined by fluorescent microscopy or fluorescent activated cell sorting (FACS) followed by confocal microscopy. In mixed infections strains LEM3804 and Gebre-1 reached the cardia and stomodeal valves more rapidly than strains LEM4265 and LV9. Hybrids unequivocally expressing both red and green fluorescence were seen in single flies of both vectors tested, co-infected with LEM4265 and Gebre-1. The hybrids were present as short (procyclic) promastigotes 2 days PBM in the semi-digested blood in the endoperitrophic space. Recovery of a clearly co-expressing hybrid was also achieved by FACS. However, hybrids could not sustain growth in vitro. has profound epidemiological significance, because it facilitates the emergence and spread of new phenotypic traits

A clinical study to optimise a sand fly biting protocol for use in a controlled human infection model of cutaneous leishmaniasis (the FLYBITE study)

Background: Leishmaniasis is a globally important yet neglected parasitic disease transmitted by phlebotomine sand flies. With new candidate vaccines in or near the clinic, a controlled human challenge model (CHIM) using natural sand fly challenge would provide a method for early evaluation of prophylactic efficacy. Methods : We evaluated the biting frequency and adverse effects resulting from exposure of human volunteers to bites of either Phlebotomus papatasi or P. duboscqi, two natural vectors of Leishmania major. 12 healthy participants were recruited (mean age 40.2 ± 11.8 years) with no history of significant travel to regions where L. major-transmitting sand flies are prevalent. Participants were assigned to either vector by 1:1 allocation and exposed to five female sand flies for 30 minutes in a custom biting chamber. Bite frequency was recorded to confirm a bloodmeal was taken. Participant responses and safety outcomes were monitored using a visual analogue scale (VAS), clinical examination, and blood biochemistry. Focus groups were subsequently conducted to explore participant acceptability. Results: All participants had at least one successful sand fly bite with none reporting any serious adverse events, with median VAS scores of 0-1/10 out to day 21 post-sand fly bite. Corresponding assessment of sand flies confirmed that for each participant at least 1/5 sand flies had successfully taken a bloodmeal (overall mean 3.67±1.03 bites per participant). There was no significant difference between P. papatasi and P. duboscqi in the number of bites resulting from 5 sand flies applied to human participants (3.3±0.81 vs 3.00±1.27 bites per participant; p=0.56) .  In the two focus groups (n=5 per group), themes relating to positive participant-reported experiences of being bitten and the overall study, were identified. Conclusions: These results validate a protocol for achieving successful sand fly bites in humans that is safe, well-tolerated and acceptable for participants. Clinicaltrials.gov registration: NCT03999970 (27/06/2019)

Skin parasite landscape determines host infectiousness in visceral leishmaniasis

Increasing evidence suggests that the infectiousness of patients for the sand fly vector of visceral leishmaniasis is linked to parasites found in the skin. Using a murine model that supports extensive skin infection with Leishmania donovani, spatial analyses at macro-(quantitative PCR) and micro-(confocal microscopy) scales indicate that parasite distribution is markedly skewed. Mathematical models accounting for this heterogeneity demonstrate that while a patchy distribution reduces the expected number of sand flies acquiring parasites, it increases the infection load for sand flies feeding on a patch, increasing their potential for onward transmission. Models representing patchiness at both macro- and micro-scales provide the best fit with experimental sand fly feeding data, pointing to the importance of the skin parasite landscape as a predictor of host infectiousness. Our analysis highlights the skin as a critical site to consider when assessing treatment efficacy, transmission competence and the impact of visceral leishmaniasis elimination campaigns.Parasitemia has been considered the main determinant of visceral leishmaniasis transmission. By combining imaging, qPCR and experimental xenodiagnoses with mathematical models, Doehl et al. argue that the patchy landscape of parasites in the skin is necessary to explain infectiousness

Infection Parameters in the Sand Fly Vector That Predict Transmission of Leishmania major

To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic “cutoff” value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite

Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism

Leishmania-Specific Surface Antigens Show Sub-Genus Sequence Variation and Immune Recognition

Single-celled Leishmania parasites, transmitted by sand flies, infect humans and other mammals in many tropical and sub-tropical regions, giving rise to a spectrum of diseases called the leishmaniases. Species of parasite within the Leishmania genus can be divided into two groups (referred to as sub-genera) that are separated by up to 100 million years of evolution yet are highly related at the genome level. Our research is focused on identifying gene differences between these sub-genera that may identify proteins that impact on the transmission and pathogenicity of different Leishmania species. Here we report the presence of a highly-variant genomic locus (OHL) that was previously described as absent in parasites of the L. (Viannia) subgenus (on the basis of lack of key genes) but is present and well-characterised (as the LmcDNA16 locus) in all members of the alternative subgenus, L. (Leishmania). We demonstrate that the proteins encoded within the LmcDNA16 and OHL loci are similar in their structure and surface localisation in mammalian-infective amastigotes, despite significant differences in their DNA sequences. Most importantly, we demonstrate that the OHL locus proteins, like the HASP proteins from the LmcDNA16 locus, contain highly variable amino acid repeats that are antigenic in man and may therefore contribute to future vaccine development

Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group

In eastern Mediterranean, leishmaniasis represents a major public health problem with considerable impact on morbidity and potential to spread. Cutaneous leishmaniasis (CL) caused by L. major or L. tropica accounts for most cases in this region although visceral leishmaniasis (VL) caused by L. infantum is also common. New foci of human CL caused by L. donovani complex strains were recently described in Cyprus and Turkey. Herein we analyzed Turkish strains from human CL foci in Çukurova region (north of Cyprus) and a human VL case in Kuşadasi. These were compared to Cypriot strains that were previously typed by Multilocus Enzyme Electrophoresis (MLEE) as L. donovani MON-37. Nevertheless, they were found genetically distinct from MON-37 strains of other regions and therefore their origin remained enigmatic. A population study was performed by Multilocus Microsatellite Typing (MLMT) and the profile of the Turkish strains was compared to previously analyzed L. donovani complex strains. Our results revealed close genetic relationship between Turkish and Cypriot strains, which form a genetically distinct L. infantum monophyletic group, suggesting that Cypriot strains may originate from Turkey. Our analysis indicates that the epidemiology of leishmaniasis in this region is more complicated than originally thought