Abstract
Leishmaniasis is a neglected tropical disease caused by
Leishmania
protozoa transmitted by infected sand flies. Vaccination through leishmanization with live
Leishmania major
has been used ...successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited
L. major
strain (
LmCen
−/−
). Notably,
LmCen
−/−
is a genetically engineered
centrin
gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with
LmCen
−/−
have no visible lesions following challenge with
L. major
-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden.
LmCen
−/−
immunization results in protection and an immune response comparable to leishmanization.
LmCen
−/−
is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.
All cells are subject to structural damage that must be addressed for continued growth. A wide range of damage affects the genome, meaning multiple pathways have evolved to repair or bypass the ...resulting DNA lesions. Though many repair pathways are conserved, their presence or function can reflect the life style of individual organisms. To identify genome maintenance pathways in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens to identify genes important for survival following exposure to the alkylating agent methyl methanesulphonate. Amongst a cohort of broadly conserved and, therefore, early evolved repair pathways, we reveal multiple activities not so far examined functionally in T. brucei, including DNA polymerases, DNA helicases and chromatin factors. In addition, the screens reveal Trypanosoma- or kinetoplastid-specific repair-associated activities. We also provide focused analyses of repair-associated protein kinases and show that loss of at least nine, and potentially as many as 30 protein kinases, including a nuclear aurora kinase, sensitises T. brucei to alkylation damage. Our results demonstrate the potential for synthetic lethal genome-wide screening of gene function in T. brucei and provide an evolutionary perspective on the repair pathways that underpin effective responses to damage, with particular relevance for related kinetoplastid pathogens. By revealing that a large number of diverse T. brucei protein kinases act in the response to damage, we expand the range of eukaryotic signalling factors implicated in genome maintenance activities.
Epigenetic manipulation of host cells by intracellular pathogens has become increasingly evident. Lecoeur et al. show us how Leishmania amazonensis inhibits macrophage inflammasomes by modifying ...histone H3 activation marks on NF-κB-associated gene promoters that increase the expression of inhibitors and downmodulates activators of this pathway.
Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the ...infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking.
We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum.
Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies.
Leishmaniasis: the act of transmission Serafim, Tiago D.; Coutinho-Abreu, Iliano V.; Dey, Ranadhir ...
Trends in parasitology,
November 2021, 2021-11-00, 20211101, Volume:
37, Issue:
11
Journal Article
Peer reviewed
The contribution of vector transmission to pathogen establishment is largely underrated. For Leishmania, transmission by sand flies is critical to early survival involving an irreproducible myriad of ...parasite, vector, and host molecules acting in concert to promote infection at the bite site. Here, we review recent breakthroughs that provide consequential insights into how vector transmission of Leishmania unfolds. We focus on recent work pertaining to the effect of gut microbiota, sand fly immunity, and changes in metacyclogenesis upon multiple blood meals, on Leishmania development and transmission. We also explore how sand fly saliva, egested parasite molecules and vector gut microbiota, and bleeding have been implicated in modulating the early innate host response to Leishmania, affecting the phenotype of neutrophils and monocytes arriving at the bite site.
The feeding behavior of sand flies is intimately connected to vector competence and success of Leishmania transmission. In the sand fly, a postinfected blood meal prevents loss of parasites and promotes transmissible infections. More blood meals increase sand fly infectiousness through retroleptomonad parasites. In addition, prolonged host bleeding induces heme oxygenase-1 (HO-1) in macrophages, which dampens inflammation and promotes disease tolerance.Sand fly gut microbiota is critical to Leishmania development and promotes transmission through host inflammasome-mediated interleukin (IL)-1β that amplifies neutrophil recruitment.Sand fly yellow proteins in saliva are bona fide neutrophil chemoattractants that promote infection.Neutrophils are central to acute inflammation caused by bite-specific mediators, but dermal resident macrophages are gaining attention as participants that harbor Leishmania parasites early in infection.
Abstract
Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly ...characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (
P
< 0.0001) and increases parasite burden (
P
< 0.001) in mice upon co-injection with
Leishmania
parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.
Leishmania donovani parasites are the cause of visceral leishmaniasis and are transmitted by bites from phlebotomine sand flies. A prominent feature of vector-transmitted Leishmania is the ...persistence of neutrophils at bite sites, where they protect captured parasites, leading to enhanced disease. Here, we demonstrate that gut microbes from the sand fly are egested into host skin alongside Leishmania parasites. The egested microbes trigger the inflammasome, leading to a rapid production of interleukin-1β (IL-1β), which sustains neutrophil infiltration. Reducing midgut microbiota by pretreatment of Leishmania-infected sand flies with antibiotics or neutralizing the effect of IL-1β in bitten mice abrogates neutrophil recruitment. These early events are associated with impairment of parasite visceralization, indicating that both gut microbiota and IL-1β are important for the establishment of Leishmania infections. Considering that arthropods harbor a rich microbiota, its potential egestion after bites may be a shared mechanism that contributes to severity of vector-borne disease.
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•Gut microbes egested by Leishmania-infected sand flies prime the host inflammasome•Inflammasome-derived IL-1β sustains recruitment of neutrophils to bite sites•Giving antibiotics to sand flies before transmission abolishes neutrophil infiltration•Abolishing neutrophil infiltration at bite sites impairs Leishmania dissemination
Neutrophils recruited to sand fly bite sites shelter Leishmania, augmenting disease. Dey et al. demonstrate that egestion of sand fly gut microbes into host skin primes the inflammasome to produce IL-1β, which sustains neutrophil recruitment. Removing gut microbiota or blocking IL-1β before transmission abolishes neutrophil recruitment and impairs Leishmania dissemination.
Doehl et al. have combined empirical data with computer simulation to demonstrate that RAG-2 mice intravenously infected with Leishmania donovani form heterogeneous skin parasite patches that govern ...infectiousness to sand flies. This model provides a much-needed tool to explore the relevance of asymptomatic and symptomatic visceral leishmaniasis patients as infection reservoirs.
Leishmaniasis is a spectrum of diseases transmitted by sand fly vectors that deposit Leishmania spp. parasites in the host skin during blood feeding. Currently, available treatment options are ...limited, associated with high toxicity and emerging resistance. Even though a vaccine for human leishmaniasis is considered an achievable goal, to date we still do not have one available, a consequence (amongst other factors) of a lack of pre-clinical to clinical translatability. Pre-exposure to uninfected sand fly bites or immunization with defined sand fly salivary proteins was shown to negatively impact infection. Still, cross-protection reports are rare and dependent on the phylogenetic proximity of the sand fly species, meaning that the applicability of a sand fly saliva-based vaccine will be limited to a defined geography, one parasite species and one form of leishmaniasis. As a proof of principle of a future vector saliva-based pan-Leishmania vaccine, we engineered through a reverse vaccinology approach that maximizes translation to humans, a fusion protein consisting of immunogenic portions of PdSP15 and LJL143, sand fly salivary proteins demonstrated as potential vaccine candidates against cutaneous and visceral leishmaniasis, respectively. The in silico analysis was validated ex vivo, through T cell proliferation experiments, proving that the fusion protein (administered as a DNA vaccine) maintained the immunogenicity of both PdSP15 and LJL143. Additionally, while no significant effect was detected in the context of L. major transmission by P. duboscqi, this DNA vaccine was defined as partially protective, in the context of L. major transmission by L. longipalpis sand flies. Importantly, a high IFNγ response alone was not enough to confer protection, that mainly correlated with low T cell mediated Leishmania-specific IL-4 and IL-10 responses, and consequently with high pro/anti-inflammatory cytokine ratios. Overall our immunogenicity data suggests that to design a potentially safe vector-based pan-Leishmania vaccine, without geographic restrictions and against all forms of leishmaniasis is an achievable goal. This is why we propose our approach as a proof-of principle, perhaps not only applicable to the anti-Leishmania vector-based vaccines' field, but also to other branches of knowledge that require the design of multi-epitope T cell vaccines with a higher potential for translation.
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