Biology subjects

Matlashewski, G.

Publications and source records attributed to Matlashewski, G..

9 recordsLinked to original sources

bioRxiv2022

Leishmania major centrin knock-out parasites alter the Kynurenine- aryl hydrocarbon receptor signaling to produce a pro-inflammatory response

Leishmaniasis is a parasitic disease that is prevalent in approximately 88 countries, and yet no licensed human vaccine exists against it. Towards control of leishmaniasis, we have developed Leishmania major centrin gene deletion mutant strains (LmCen-/-) as a live attenuated vaccine, which induces a strong Th1 response to provide IFN-{gamma}-mediated protection to the host. However, the immune mechanisms of such protection remain to be understood. Metabolomic reprogramming of the host cells following Leishmania-infection has been shown to play a critical role in pathogenicity and shaping the immune response following infection. Here, we applied untargeted mass spectrometric analysis to study the metabolic changes induced by infection with LmCen-/- and compared those with virulent L. major parasite infection to identify the immune mechanism of protection. Our data shows that immunization with LmCen-/- parasites, in contrast to virulent L. major infection, alters tryptophan metabolism to down-regulate kynurenine-AhR signaling and promote a pro-inflammatory response.

systems biology↗

bioRxiv2022

Leishmania mexicana Centrin Knock out Parasites Promote M1-polarizing Metabolic Changes

Leishmaniasis is a tropical disease present in more than 90 countries. Presently, there is no approved vaccine for human use. We have previously developed live attenuated L. mexicana Cen-/- (LmexCen-/-) as a vaccine candidate that showed excellent efficacy that was characterized by reduced activation of Th2 responses and enhanced Th1 responses, contrary to wild type L. mexicana (LmexWT) infection. Towards understanding the interplay between immune mechanisms of protection and metabolic reprogramming, we applied untargeted mass spectrometric analysis to LmexCen-/- and compared them with LmexWT infection. Data showed that enriched pentose phosphate pathway (PPP) in ears immunized with LmexCen-/- parasites, compared to naive and LmexWT-infected ears. This pathway is known to promote an M1 phenotype in macrophages, suggesting a switch to a pro-inflammatory phenotype following LmexCen-/- inoculation. Accordingly, inhibition of the PPP in macrophages cultured with LmexCen-/- parasites led to diminished production of nitric oxide, IL-12, and IL-1{beta}, hallmarks of classical activation. Overall, our study revealed novel immune regulatory mechanisms that may be critical for the induction of protective immunity.

microbiology↗

bioRxiv2022

Centrin-deficient Leishmania mexicana confers protection against Old World visceral leishmaniasis

Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen-/-) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen-/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We show that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver and lack of mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the Th2 response as indicated by a significant reduction in the anti-inflammatory cytokines such as IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-{gamma} resulting in higher IFN-{gamma}/IL-10 and IFN-{gamma}/IL4 ratios compared to non-immunized animals. This contrasts with our studies with L. major centrin deletion mutants that showed a dominant Th1 response compared to L. major wild-type infection suggesting the divergent mechanisms of protection in the two mutant parasites. LmexCen-/- immunization resulted in long-lasting protection against L. donovani infection. Further, since the efficacy of LmCen-/- has not been determined against Leishmania strains prevalent in the Americas, LmexCen-/- may be a viable alternative. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against old world visceral leishmaniasis.

immunology↗

bioRxiv2021

Skin resident memory cells generated by the L. major centrin gene deleted parasites mediate protective immune response analogous to leishmanization

Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene deleted parasite strain (LmCen-/-) that induced protection against a homologous and heterologous challenges. The protection is mediated by IFN{gamma} secreting CD4+ T effector cells and multifunctional T cells, which is analogous to leishmanization. Previously, skin tissue resident memory T cells (TRM cells) were shown to be crucial for host protection in a leishmanization model. In this study, we evaluated generation and function of skin TRM cells following immunization with LmCen-/- parasites and compared those with leishmanization. In the absence of recoverable LmCen-/- parasites, the skin of immunized mice showed functional TRM cells comparable to leishmanized mice. The generation of the skin TRM cells was supported by the induction of cytokines and chemokines essential for their production and survival. Following challenge infection with wild type L. major, TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice which was similar to leishmanization. Further, upon challenge, CD4+ TRM cells induced higher levels of IFN{gamma} and Granzyme B in the immunized and leishmanized mice than non-immunized mice. Taken together, our studies demonstrate that a genetically modified live attenuated Leishmania vaccine generates functional CD4+ TRM cells that mediate protection and can be a safer alternative to leishmanization.

immunology↗

bioRxiv2020

Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

Visceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen-/-). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen-/- against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen-/- did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen-/--immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen-/- immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-{gamma} and TNF-, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen-/- which showed equal protection as laboratory-grade LmCen-/- parasites in hamsters. Importantly, GLP-grade LmCen-/- parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

immunology↗

bioRxiv2020

Characterization of a new Leishmania major isolate for use in a controlled human infection model

Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIM) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical isolate of Leishmania major. Two fresh isolates of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one (L. major_MRC-02) to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.

microbiology↗

bioRxiv2020

Evidence that interspecies Leishmania hybrids contribute to changes in disease pathology

BackgroundLeishmaniasis is a widespread neglected tropical disease present in over 90 countries with diverse pathologies associated with different species of Leishmania parasites transmitted by infected sand flies. Leishmania donovani causes visceral leishmaniasis, a highly virulent fatal infection of the visceral organs. Leishmania major and Leishmania tropica cause less virulent cutaneous leishmaniasis where the infection remains in the skin at the site of the sandfly bite. A major molecular epidemiological question is why some variants of L. donovani in Sri Lanka cause cutaneous disease rather than the typical visceral disease. MethodsWhole genome sequencing data for 684 L. donovani samples was used to perform sequence alignments and worldwide phylogenetic analyses to determine the source of the atypical L. donovani strains from Sri Lanka. L. donovani genome sequences originating from Sri Lanka were further analyzed for evidence of hybridization with other Leishmania species by determining the density of heterozygous alleles. Polymorphisms from potential Leishmania hybrids were used to reconstruct the parental genetic sequences to identify the potential parental species and quantify their genetic contribution through sequence comparison of the reconstructed parental sequences with all Old World Leishmania genomes. FindingsHere we show that L. donovani in Sri Lanka contains genes with widespread gene polymorphisms derived from African L. major and L. tropica genomes that were likely obtained as a result of diploid genome hybridization and recombination resulting in progeny with mosaic genomes. Furthermore, evidence is presented that multiple L. donovani hybrid parasites originating from visceral leishmaniasis endemic Africa have entered Sri Lanka yet visceral leishmaniasis remains non-existent raising the possibility that environmental factors favour the establishment of atypical L. donovani strains in Sri Lanka. InterpretationThe discovery of L. major and L. tropica genome sequences in L. donovani provides a compelling rationale how some L. donovani strains in Sri Lanka may be able to cause cutaneous rather than visceral leishmaniasis. The identification of L. donovani hybrid parasites in cutaneous leishmaniasis lesions provides a unique opportunity to investigate environmental and parasite genetic factors controlling disease epidemiology and pathogenesis. FundingCanadian Institutes of Health Research and Fonds de recherche du Quebec - Sante Research in contextO_ST_ABSEvidence before this studyC_ST_ABSDifferent Leishmania species parasites cause either benign cutaneous leishmaniasis or fatal visceral leishmaniasis. It is unknown why some variants of Leishmania donovani that typically causes visceral leishmaniasis in Asia and Africa can cause cutaneous leishmaniasis in specific geographic locations including Sri Lanka. Leishmania has a diploid genome and hybrid parasites have been identified in nature and generated experimentally. In the context of this study, hybrids are considered to be progeny derived from a single outcross event between two diverse parents. Uncertainty remains whether interspecies hybrids with visceral and cutaneous leishmaniasis causing species in nature are associated with different disease outcomes. Added value of this studyEvidence for genetic hybridization between visceral and cutaneous disease causing Leishmania species is described from Sri Lanka where cutaneous leishmaniasis is highly endemic yet there is no ongoing visceral leishmaniasis transmission. This provides a potential explanation how L. donovani can become attenuated for visceral disease and could help to identify geographic environmental factors associated with selection for parasite attenuation. Implications of all the available evidenceHybrid Leishmania parasites may be one source of atypical cutaneous leishmaniasis. Epidemiological studies are needed to determine why diverse L. donovani hybrid parasites have become ubiquitous in specific geographic locations where the incidence of cutaneous leishmaniasis is increasing. This has implications for understanding the genetic control of disease pathogenesis and for the prevention of cutaneous or visceral leishmaniasis locally and in neighboring countries.

microbiology↗

bioRxiv2020

A Second Generation Leishmanization Vaccine with a Markerless Attenuated Leishmania major Strain using CRISPR gene editing

Leishmaniasis is a debilitating and often fatal 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 unacceptable skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is the first genetically engineered gene deleted Leishmania strain that is antibiotic resistant marker free and does not have any off-target mutations. Mice immunized with LmCen-/- had virtually no visible lesions following challenge with L. major-infected sand flies while non-immunized animals developed large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization showed protection and an immune response comparable to leishmanization. LmCen-/- is safe since it was unable to cause disease even 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.

microbiology↗

bioRxiv2019

The hunger games: sensing host arginine is essential for Leishmania parasite virulence

Arginine homeostasis in lysosomes is critical for growth and metabolism of mammalian cells. They employ a specific sensor (SLC38A9) that monitors intra-lysosome arginine sufficiency and subsequently up-regulates cellular mTORC1 activity. Lysosomes of macrophages (phagolysosomes) are the niche where the parasitic protozoan Leishmania resides and causes important human disease. Several years ago, we discovered that upon arginine starvation, cultured Leishmania parasites promptly activate a MAPK2-mediated Arginine Deprivation Response (ADR) pathway, resulting in up-regulation of the Leishmania arginine transporter (AAP3), as well as a small group of other transporters. Significantly, ADR is also activated during macrophage infection, implying that the intracellular parasite actively depletes arginine within the host phagolysosome, likely to prevent mTORC1 activation and enhance intracellular development. We hypothesize that ADR-mediated up-regulation of AAP3 activity is necessary to withstand the resultant arginine starvation. Both copies of the AAP3 genes are located (in tandem) on a tetrasomic chromosome (chr31), but only one (AAP3.2) is responsive to arginine deprivation. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport (mediated by AAP3.1), but lack a functional copy of AAP3.2 and are therefore not responsive to arginine starvation. While these mutants grow normally in culture as promastigotes, they were impaired in their ability to develop inside THP1 macrophages grown under physiological concentrations of arginine (0.1 mM). However, flooding the macrophage growth medium with arginine (1.5 mM) restored parasite infectivity and intracellular growth to that of wild type. The results indicate that inside the host macrophage, Leishmania must overcome the arginine \"Hunger Games\" by up-regulating transport of arginine via the ADR. Furthermore, the AAP3.2 mutants were ~70-80% less virulent in Balb/C mice, showing, for the first time, that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.

cell biology↗