Identifying antigens that elicit protective immunity is pivotal for developing effective vaccines and therapeutics against cutaneous leishmaniasis. Dihydrolipoyl dehydrogenase (DLD), a mitochondrial enzyme involved in oxidizing lipoamides to facilitate electron transfer for energy production and metabolism, plays a critical role in virulence of fungi and bacteria. However, its function in Leishmania virulence and pathogenesis remains unexplored. Using a CRISPR-Cas9-based approach, we generated DLD-deficient Leishmania (L.) major parasites and a complementary add-back strain by episomally reintroducing DLD gene into the knockout parasites. Loss of DLD significantly impaired parasite proliferation in axenic cultures and infected macrophages compared to wild-type (WT) and add-back control parasites. These defects were linked to reduced ROS production, impaired mitochondrial permeability, an enhanced oxygen consumption rate, and alterations in mitochondrial ultrastructure. In murine models, DLD-deficient parasites failed to cause observable lesions and exhibited significantly reduced parasite burdens compared to WT and add-back control strains. Notably, mice infected with DLD-deficient parasites displayed blunted immune responses compared to their WT controls. Importantly, vaccination with DLD-deficient parasites conferred robust protection against virulent L. major challenge, characterized by a strong IFN-γ-mediated immune response. These findings establish DLD as an essential metabolic enzyme for L. major intracellular survival and pathogenesis. Targeting DLD not only impairs parasite viability but also holds promise as a novel strategy for vaccine development to combat cutaneous leishmaniasis.

Leishmania donovani is a causative agent of the neglected tropical disease known as visceral leishmaniasis or Kala Azar. This disease is lethal when untreated, with more than 90 000 cases annually. Little is known about magnesium regulation in these parasites despite magnesium being the second most abundant intracellular cation and universally required for normal cell function. L. donovani contains two protein tyrosine phosphatase (PTP) proteins (PTP1 and PTP2) and a DUF21 protein (domain of unknown function 21), which are respectively homologous to mammalian PRL [phosphatases of regenerating liver; also known as protein tyrosine phosphatase type IVA (PTP4A)] and mammalian transmembrane protein CNNM (cyclin M family). In mammalian cells, the PRL and CNNM multiprotein complex has been shown to sense and modulate intracellular magnesium levels. Herein, we revealed that L. donovani PTP1 and DUF21 can also form a specific protein complex. Using CRISPR-Cas9 gene editing, four L. donovani knockouts, LdΔPTP1, LdΔPTP2, a double knockout termed LdΔPTP1/2, and LdΔDUF21 have been generated. Magnesium-dependent growth curves demonstrated that the LdΔPTP1/2 mutant could not survive in low magnesium and had a reduced level of survival in infected macrophages. In contrast, LdΔDUF21 is sensitive to high levels of magnesium and has an increased level of intracellular magnesium and an increased survival in macrophages compared to wild-type L. donovani. Taken together, these observations provide evidence that, similar to the PRL and CNNM proteins in mammalian cells, PTP and DUF21 homologs in L. donovani have the ability to complex and respond to environmental changes in magnesium.

Dogs are the main reservoir host of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis (ZVL). An effective vaccine against Canine Visceral Leishmaniasis (CVL) will help the control and elimination of ZVL. In this study, we evaluated in dogs the safety, immunogenicity, and efficacy of a live attenuated Leishmania major Centrin gene-deleted (LmCen−/−) as a vaccine. Two doses (106 or 107) of LmCen−/− vaccine were administered intradermally in a prime-boost regimen. Both vaccine doses induced equally high level of IgG anti-Leishmania and exhibited strong antigen-specific cellular responses with IFN-γ production by CD4 + T cells one-month post-immunization. A second cohort of dogs was vaccinated with 106 LmCen−/− parasites one month prior to their transfer to a CVL endemic focus in Northern Tunisia for exposure to sand fly bites during three successive transmission seasons. Dogs were exposed to bite from naturally infected sandflies for 3–5 months per year. Our results showed that only 1/11 vaccinated dogs became PCR positive for Leishmania and developed clinical signs of CVL. In contrast, 4/11 unvaccinated dogs were tested PCR positive for Leishmania and displayed oligosymptomatic CVL, demonstrating that immunization with LmCen−/− vaccine confers long-term protection with an efficacy of 82.5% against CVL in natural transmission settings.

The ability to determine the essentiality of a gene in the protozoan parasite Leishmania is important to identify potential targets for intervention and understanding the parasite biology. CRISPR gene editing technology has significantly improved gene targeting efficiency in Leishmania. There are two commonly used CRISPR gene targeting methods in Leishmania; the stable expression of the gRNA and Cas9 using a plasmid containing a Leishmania ribosomal RNA gene promoter (rRNA-P stable protocol) and the T7 RNA polymerase based transient gRNA expression system in promastigotes stably expressing Cas9 (T7 transient protocol). There are distinct advantages with both systems. The T7 transient protocol is excellent for high throughput gene deletions and has been used to successfully delete hundreds of Leishmania genes to study mutant phenotypes and several research labs are now using this protocol to target all the genes in L. mexicana genome. The rRNA-P stable protocol stably expresses the plasmid derived gRNA and has been used to delete or disrupt single and multicopy Leishmania genes, perform single nucleotide changes and provide evidence for gene essentiality by directly observing null mutant promastigotes dying in culture. In this study, the rRNA-P stable protocol was used to target 22 Leishmania genes in which null mutants were not generated using the T7 transient protocol. Notably, the rRNA-P stable protocol was able to generate alive null mutants for 8 of the 22 genes. These results demonstrate the rRNA-P stable protocol could be used alone or in combination with the T7 transient protocol to investigate gene essentiality in Leishmania.

The protozoan parasite Leishmania donovani is a causative agent of the neglected tropical disease known as visceral leishmaniasis, which can be lethal when untreated. Studying Leishmania viru-lence factors is crucial in determining how the parasite causes disease and identifying new targets for treatment. One potential virulence factor is L. donovani's abundantly secreted protein: secreted acid phosphatase (SAcP). Whole-genome analysis revealed that the sacp gene was present in three copies in wild type L. donovani. Using CRISPR-Cas9 gene editing; we generated a sacp gene knockout termed LdΔSAcP, which demonstrated a loss of both the SAcP protein and an associated reduction in secreted acid phosphatase activity. Genome sequencing confirmed the precise dele-tion of the sacp gene in LdΔSAcP and identified several changes in the genome. LdΔSAcP demonstrated no significant changes in promastigote proliferation or its ability to infect and survive in macrophages compared to the wildtype strain. LdΔSAcP also demonstrated no change in murine liver infection; however, survival was impaired in the spleen. Taken together these results show that SAcP is not necessary for the survival of promastigotes in culture but may support long-term survival in the spleen. These observations also show that the use of CRISPR gene editing and WGS together are effective to investigate the function and phenotype of complex potential drug targets such as multicopy genes.

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen-/-) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen-/- parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen-/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (WT) parasites. We have also determined the safety and efficacy of LmexCen-/- in vivo using experimental murine models of L. mexicana. We demonstrate that LmexCen-/- parasites are safe and do not cause lesions in susceptible mouse models. Immunization with LmexCen-/- is also efficacious against challenge with WT L. mexicana parasites in genetically different BALB/c and C57BL/6 mouse models. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens at the infection site and draining lymph nodes compared to the control group. Overall, we demonstrate that LmexCen-/- parasites are safe and efficacious against New World cutaneous leishmaniasis in pre-clinical models.

Nonhomologous end joining (NHEJ) is the most efficient double-stranded DNA break (DSB) repair pathway in mammalian cells. In contrast, the protozoan parasite Leishmania has no functional NHEJ pathway but retains the core NHEJ factors of Ku70 and Ku80 proteins.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major ( LmCen −/− ) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani -infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen −/− parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen −/− immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen −/− infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen −/− immunized hamsters. This study demonstrates that the LmCen −/− parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.

Leishmaniasis is a widespread neglected tropical disease transmitted by infected sand flies resulting in either benign cutaneous infection or fatal visceral disease. Leishmania donovani is the principal species responsible for visceral leishmaniasis, yet an atypical L . donovani has become attenuated in several countries including Sri Lanka and causes cutaneous leishmaniasis. Previous studies have identified 91 genes altered in the atypical cutaneous L . donovani compared to typical visceral disease associated L . donovani including mutations in the RagC and Raptor genes that are part of the eukaryotic conserved TOR pathway and its upstream sensing pathway. In the present study, we investigate whether the RagC R231C mutation present in atypical cutaneous L . donovani introduced into the virulent L . donovani 1S2D chromosome by CRISPR gene editing could affect virulence for survival in visceral organs. Through bioinformatic analysis, we further investigated the presence of sensing pathway components upstream of TOR in L . donovani including RagC complexing proteins, RagA and Raptor. L . donovani 1S2D edited to express mutant RagC R231C were viable in promastigote but had reduced visceral parasitemia in infected BALB/c mice. The RagC R231C mutant retained the ability to interact with RagA and gene knockout experiments revealed that although the RagA gene was essential, the RagC gene was not essential under promastigote culture conditions but was essential for survival in the liver of experimentally infected mice. These results provide evidence that the TOR associated sensing pathway plays a prominent role in L . donovani visceral disease and the RagC R231C mutation contributed to the atypical pathology of cutaneous L . donovani in Sri Lanka.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were similar to 70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis. IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

CRISPR-Cas9 is an RNA guided endonuclease derived from the bacterium Streptococcus pyogenes. Due to its simplicity, versatility, and high efficiency, it has been widely used for genome editing in a variety of organisms including the protozoan parasite Leishmania, the causative agent of human leishmaniasis. Compared to the traditional homologous recombination gene targeting method, CRISPR-Cas9 has been shown to be a more efficient method to delete or disrupt Leishmania genes, generate point mutations, and add tags to endogenous genes. Notably, the stable CRISPR expression systems were shown to delete multicopy family Leishmania genes and genes present in multiploid chromosomes, identify essential Leishmania genes, and create specific chromosome translocations. In this chapter, we describe detailed procedures on using the stable CRISPR expression system for genome editing in Leishmania. These procedures include CRISPR targeting site selection, gRNA design, cloning single and double gRNA coding sequences into the Leishmania CRISPR vector pLdCN, oligonucleotide donor and drug resistance selection donor design, Leishmania cell transfection, screening, and isolation of CRISPR-edited mutants. As the principles of gene editing are generally similar, many of these procedures could also apply to the transient Leishmania CRISPR systems described by other labs.

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.

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-γ and TNF-α, 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.

Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania .

Background: Visceral leishmaniasis (VL), caused by Leishmania donovani complex parasites, is a neglected parasitic disease that is generally fatal if untreated. Despite decades of research to develop a sensitive VL diagnostic test, definitive diagnosis of VL still mainly relies on the visualization of the parasite in aspirates from the spleen, liver or bone marrow, an invasive and dangerous process with variable sensitivity. A sensitive assay that can detect Leishmania antigen from blood samples will help confirm cause, cure or recurrence of VL. Methods: In this study, rabbit polyclonal antibodies were raised against eight recombinant Leishmania proteins that are highly abundant in Leishmania. The antibodies were purified and labeled with biotin for developing a prototype sandwich enzyme-linked immunosorbent assay (ELISA). Results: The ELISA for the Leishmania 40S ribosomal protein S12 detected target antigen with the highest sensitivity and specificity and could detect 1 pg of purified protein or as few as 60 L. donovani parasites. The 40S ribosomal protein S12 sandwich ELISA could detect the target antigen from Peripheral Blood Mononuclear Cell (PBMC) samples in 68% of VL patients and post-kala-azar dermal leishmaniasis (PKDL) patients, providing an estimation of parasitemia ranging from 15 to 80 amastigotes per ml of blood. Conclusion: These results indicate that the 40S ribosomal protein S12 sandwich ELISA warrants further tests with more clinical samples of VL patients and other parasitic diseases. It is hopeful that this ELISA could become a useful tool for confirming VL diagnosis, monitoring treatment progress, disease recurrence and possibly detecting asymptomatic Leishmania infections with a high parasite load.