The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies. Leishmania are protozoan parasites, transmitted between mammals by the bite of phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is motile and used for anchoring the parasites to prevent clearance with the digested blood meal remnants. To dissect flagellar functions and their importance in life cycle progression, we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant swimming speeds showed that about half behaved differently compared to the wild type: a few swam faster, many slower and some were completely paralysed. We also found a group of uncoordinated swimmers. To test whether flagellar motility is required for parasite migration from the fly midgut to the foregut from where they reach the next host, we infected sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking these tags up to nine days after infection showed that paralysed and uncoordinated Leishmania were rapidly lost from flies. These data indicate that directional swimming is important for successful colonisation of sand flies.

Leishmaniases are parasitic diseases present worldwide that are transmitted to the vertebrate host by the bite of an infected sand fly during a blood feeding. Phlebotomine sand flies inoculate into the mammalian host Leishmania parasites embedded in promastigote secretory gel (PSG) with saliva, which is composed of a diverse group of molecules with pharmacological and immunomodulatory properties. In this review, we focus on 3 main aspects of sand fly salivary molecules: (1) structure and composition of salivary glands, including the properties of salivary molecules related to hemostasis and blood feeding, (2) immunomodulatory properties of salivary molecules and the diverse impacts of these molecules on leishmaniasis, ranging from disease exacerbation to vaccine development, and (3) use of salivary molecules for field applications, including monitoring host exposure to sand flies and the risk of Leishmania transmission. Studies showed interesting differences between salivary proteins of Phlebotomus and Lutzomyia species, however, no data were ever published on salivary proteins of Sergentomyia species. In the last 15 years, numerous studies have characterized sand fly salivary proteins and, in parallel, have addressed the impact of such molecules on the biology of the host–sand fly–parasite interaction. The results obtained shall pave the way for the development of field-application tools that could contribute to the management of leishmaniasis in endemic areas.

The co-infection cases involving dixenous Leishmania spp. (mostly of the L. donovani complex) and presumably monoxenous trypanosomatids in immunocompromised mammalian hosts including humans are well documented. The main opportunistic parasite has been identified as Leptomonas seymouri of the sub-family Leishmaniinae. The molecular mechanisms allowing a parasite of insects to withstand elevated temperature and substantially different conditions of vertebrate tissues are not understood. Here we demonstrate that L. seymouri is well adapted for the environment of the warm-blooded host. We sequenced the genome and compared the whole transcriptome profiles of this species cultivated at low and high temperatures (mimicking the vector and the vertebrate host, respectively) and identified genes and pathways differentially expressed under these experimental conditions. Moreover, Leptomonas seymouri was found to persist for several days in two species of Phlebotomus spp. implicated in Leishmania donovani transmission. Despite of all these adaptations, L. seymouri remains a predominantly monoxenous species not capable of infecting vertebrate cells under normal conditions. In this work we performed a comprehensive evaluation of the infective potential of Leptomonas seymouri, repeatedly isolated from kala-azar patients infected by Leishmania donovani in India and neighboring countries, and have tested the capacity of this monoxenous trypanosomatid to utilize the sand fly vectors permissive for Leishmania donovani. We concluded that despite several genetic adaptations it has developed, Leptomonas seymouri remains a predominantly monoxenous species not able to infect mammalian macrophages either alone or in co-infection with Leishmania. Under certain circumstances it is able to infect mammals, but probably only when the host is immunocompromised by infection with another pathogen, such as Leishmania donovani or HIV.

Phlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs. Sera from dogs naturally exposed to P. perniciosus bites over two consecutive transmission seasons in a site endemic for canine leishmaniasis (CanL) were tested at different time points by ELISA for the antibodies recognizing whole saliva, single salivary 43 kDa yellow-related recombinant protein (rSP03B), and a combination of two salivary recombinant proteins, 43 kDa yellow-related protein and 35.5 kDa apyrase (rSP01). Dogs were also tested for Leishmania infantum positivity by serology, culture, and PCR and the infection status was evaluated prospectively. We found a significant association between active CanL infection and the amount of anti-P. perniciosus saliva antibodies. Importantly, we detected a high correlation between IgG antibodies recognizing rSP03B protein and the whole salivary antigen. The kinetics of antibody response showed for both a whole saliva and rSP03B a similar pattern that was clearly related to the seasonal abundance of P. perniciosus. These results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas. Canine leishmaniasis (CanL) is a widespread zoonosis caused by protozoan parasite Leishmania infantum. CanL is endemic in more than 70 countries, including regions of southwestern Europe, where the main vector is Phlebotomus perniciosus. Saliva inoculated into the host during blood feeding provoke species-specific antibody response. They could be used as a serological marker of exposure to sand flies and, in leishmaniasis endemic areas, also as an indication of Leishmania infection risk. However, in case of large-scale serological studies, it is difficult to obtain appropriate amounts of crude salivary antigen from laboratory-reared sand flies. Therefore, we studied if specific salivary recombinant proteins could be suitable to replace whole saliva as an antigen for serological assays. Antibodies recognizing recombinant salivary protein in naturally bitten dogs were highly correlated with antibody response against whole saliva. Seasonal exposure of dogs to sand flies led to antibody response fluctuations related to the period of activity and abundance of vectors. In a context of high CanL endemicity, we found a significant association between progressive CanL infections and antibodies against sand fly saliva. Thus, salivary recombinant proteins could be used as suitable tool for investigations host exposure to sand flies in endemic settings.

Human visceral leishmaniasis caused by Leishmania donovani is considered an anthroponosis; however, Leishmania-infected animals have been increasingly reported in L. donovani foci, and the role of these animals as reservoirs for human L. donovani infection remains unclear. We conducted a study of domestic animals (goats, sheep, cows, dogs, and donkeys) in three L. donovani foci in northwestern Ethiopia. Domestic animals were screened for Leishmania DNA and for anti-L. donovani IgG. Serum anti-sand fly saliva antibodies were used as a marker of exposure to the vector sand fly, Phlebotomus orientalis. Of 546 animals tested, 32 (5.9 %) were positive for Leishmania DNA, with positive animals identified among all species studied. Sequencing indicated that the animals were infected with parasites of the L. donovani complex but could not distinguish between L. infantum and L. donovani. A total of 18.9 % of the animals were seropositive for anti-L. donovani IgG, and 23.1 % of the animals were seropositive for anti-P. orientalis saliva IgG, with the highest seroprevalence observed in dogs and sheep. A positive correlation was found between anti-P. orientalis saliva and anti-L. donovani IgGs in cows, goats, and sheep. The detection of L. donovani complex DNA in the blood of domestic animals, the reported seroprevalence to the L. donovani antigen, and the widespread exposure to sand fly saliva among domestic animals indicate that they are frequently exposed to Leishmania infection and are likely to participate in the epidemiology of Leishmania infection, either as potential blood sources for sand flies or possibly as parasite hosts.

Leishmania parasites are transmitted to vertebrate hosts by female phlebotomine sand flies as they bloodfeed by lacerating the upper capillaries of the dermis with their barbed mouthparts. In the sand fly midgut secreted proteophosphoglycans from Leishmania form a biological plug known as the promastigote secretory gel (PSG), which blocks the gut and facilitates the regurgitation of infective parasites. The interaction between the wound created by the sand fly bite and PSG is not known. Here we nanoinjected a sand fly egested dose of PSG into BALB/c mouse skin that lead to the differential expression of 7,907 transcripts. These transcripts were transiently up-regulated during the first 6 hours post-wound and enriched for pathways involved in inflammation, cell proliferation, fibrosis, epithelial cell differentiation and wound remodelling. We found that PSG significantly accelerated wound healing in vitro and in mice; which was associated with an early up-regulation of transcripts involved in inflammation (IL-1β, IL-6, IL-10, TNFα) and inflammatory cell recruitment (CCL2, CCL3, CCL4, CXCL2), followed 6 days later by enhanced expression of transcripts associated with epithelial cell proliferation, fibroplasia and fibrosis (FGFR2, EGF, EGFR, IGF1). Dermal expression of IGF1 was enhanced following an infected sand fly bite and was acutely responsive to the deposition of PSG but not the inoculation of parasites or sand fly saliva. Antibody blockade of IGF1 ablated the gel’s ability to promote wound closure in mouse ears and significantly reduced the virulence of Leishmania mexicana infection delivered by an individual sand fly bite. Dermal macrophages recruited to air-pouches on the backs of mice revealed that IGF1 was pivotal to the PSG’s ability to promote macrophage alternative activation and Leishmania infection. Our data demonstrate that through the regurgitation of PSG Leishmania exploit the wound healing response of the host to the vector bite by promoting the action of IGF1 to drive the alternative activation of macrophages. Female phlebotomine sand flies efficiently transmit Leishmania parasites, yet the underlying mechanisms remain poorly understood. During transmission, promastigote secretory gel (PSG) regurgitated from the blocked sand fly gut promote Leishmania infection and exacerbates disease. Here we investigate mouse ear-skin response to PSG and find that a significant proportion of up-regulated transcripts are involved in wound healing. Following a wound to skin preconditioned with PSG, key transcripts associated with inflammation and cell recruitment were enhanced by 4 hours and those associated with epithelial cell differentiation, proliferation and fibrosis were enhanced in the late phase of wound healing; including insulin growth factor-1 (IGF1) and its receptor. The presence of PSG significantly accelerated wound closure in vitro and in vivo, and this was directed by the presence of IGF1. IGF1 was essential to both the wound healing and disease-exacerbating properties of PSG, arising from its ability to alternatively activate macrophages. IGF1-blockade efficiently reduced the virulence of Leishmania infection from sand fly bite transmission indicating that it is essential to the function of PSG during natural infection. Our findings open the dual possibility of exploiting PSG, or defined components thereof, as a novel wound healing therapy and provide new targets for anti-leishmanial therapeutic design.