The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

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.

Periampullary tumors in a patient with pancreatic divisum and neurofibromatosis type 1: a case report.

INTRODUCTION: We present a case of a male patient with neurofibromatosis type 1 diagnosed with pancreatic divisum and several gastrointestinal tumors. A 55-year-old man was admitted to the hospital with recurrent chronic pancreatitis, indicating a large mass in the ampulla. In addition, genetic testing revealed two unique germline mutations in the neurofibromin (NF1) gene, and their potential interaction in promoting cancer was further investigated. CONCLUSION: The first similar case was reported in 2020. The current case was distinct from other cases since an additional two NF1 mutations were found in the patient. In conjunction with prior case reports, our findings imply that genetic testing in patients diagnosed with neurofibromatosis type 1 could be helpful in the development of effective treatments.

Comprehensively Understanding Isomorphism and Photoluminescent Nature of Two-Dimensional Coordination Polymers of Cd(II) and Mn(II) with 1,1'-Ethynebenzene-3,3',5,5'-tetracarboxylic Ligand.

Four isomorphic two-dimensional (2D) homo- and heterometallic coordination polymers (CPs), [(Cd xMn1- x)3(HEBTC)2(DMSO)6] with x = 1 (1), 1/3 (2), 0.5 (3), and 2/3 (4) were prepared by conventional one-pot self-assembly approach, using Cd2+ or mixtures of Cd2+ and Mn2+ with 1,1'-ethynebenzene-3,3',5,5'-tetracarboxylic (H4EBTC) under solvothermal conditions. The crystal structures of four isomorphic CPs are composed of center-symmetric trinuclear metal clusters building units linked by HEBTC3- ligands, extending into (3, 6)-connected topological 2D nets. Four CPs are isomorphic to the Mn-CP, [Mn3(HEBTC)2(DMSO)6], we recently reported. The solid-state photoluminescence of 1-4 shows dual emissions at ambient condition, where the emission bands centered at ca. 390 and 562 nm in 1 are assigned to the fluorescence and phosphorescence within HEBTC3- ligand, respectively; however, the emission bands centered at around 397 and 470 nm in 2-4 are attributed to fluorescence, corresponding to electron transition within HEBTC3- ligand and MLCT transition between HEBTC3- ligand and Mn2+ ion. In addition, the origin of isomorphism between 1 and Mn-CP is also discussed.

Development of a sandwich ELISA to detect Leishmania 40S ribosomal protein S12 antigen from blood samples of visceral leishmaniasis patients.

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.

In(III)-TMSBr-Catalyzed Cascade Reaction of Diarylalkynes with Acrylates for the Synthesis of Aryldihydronaphthalene Derivatives.

A combined Lewis acid system comprising of two or more Lewis acids occasionally exhibits augmented catalytic activity in organic transformations which are otherwise unrealizable by either of the components exclusively. On the other hand, the efficient construction of multiple new C-C bonds and polycyclic structures in minimal steps remains a subject of great interest in both academia and industry. Herein we report an efficient method to assemble aryldihydronaphthalene derivatives via a cascade reaction of diarylalkynes with acrylates under the catalysis of a combined Lewis acid derived from In(III) salt and TMSBr.

Genetic analysis of Leishmania donovani tropism using a naturally attenuated cutaneous strain.

A central question in Leishmania research is why most species cause cutaneous infections but others cause fatal visceral disease. Interestingly, L. donovani causes both visceral and cutaneous leishmaniasis in Sri Lanka. L. donovani clinical isolates were therefore obtained from cutaneous leishmaniasis (CL-SL) and visceral leishmaniasis (VL-SL) patients from Sri Lanka. The CL-SL isolate was severely attenuated compared to the VL-SL isolate for survival in visceral organs in BALB/c mice. Genomic and transcriptomic analysis argue that gene deletions or pseudogenes specific to CL-SL are not responsible for the difference in disease tropism and that single nucleotide polymorphisms (SNPs) and/or gene copy number variations play a major role in altered pathology. This is illustrated through the observations within showing that a decreased copy number of the A2 gene family and a mutation in the ras-like RagC GTPase enzyme in the mTOR pathway contribute to the attenuation of the CL-SL strain in visceral infection. Overall, this research provides a unique perspective on genetic differences associated with diverse pathologies caused by Leishmania infection.

Adaptation of Leishmania donovani to cutaneous and visceral environments: in vivo selection and proteomic analysis.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa. Two main forms are found in the Old World, self-limited cutaneous leishmaniasis and potentially fatal visceral leishmaniasis, with parasite dissemination to liver, bone marrow, and spleen. The Leishmania donovani species complex is the causative agent of visceral leishmaniasis worldwide, but atypical L. donovani strains can cause cutaneous leishmaniasis. We hypothesized that L. donovani can adapt to survive in response to restrictions imposed by the host environment. To assess this, we performed in vivo selection in BALB/c mice with a cutaneous L. donovani clinical isolate to select for parasites with increased capacity to survive in visceral organs. We then performed whole cell proteomic analysis and compared this visceral-selected strain to the original cutaneous clinical isolate and to a visceral leishmaniasis clinical isolate. Overall, there were no major shifts in proteomic profiles; however, translation, biosynthetic processes, antioxidant protection, and signaling were elevated in visceral strains. Conversely, transport and trafficking were elevated in the cutaneous strain. Overall, these results provide new insight into the adaptability of Leishmania parasites to the host environment and on the factors that mediate their ability to survive in different organs.

Determinants for the development of visceral leishmaniasis disease.

Leishmaniasis is a vector-borne neglected tropical disease associated with a spectrum of clinical manifestations, ranging from self-healing cutaneous lesions to fatal visceral infections. Among the most important questions in Leishmania research is why some species like L. donovani infect visceral organs, whereas other species like L. major remain in the skin. The determinants of visceral leishmaniasis are still poorly understood, although genomic, immunologic, and animal models are beginning to provide important insight into this disease. In this review, we discuss the vector, host, and pathogen factors that mediate the development of visceral leishmaniasis. We examine the progression of the parasite from the initial site of sand fly bite to the visceral organs and its ability to survive there. The identification of visceral disease determinants is required to understand disease evolution, to understand visceral organ survival mechanisms, and potentially to develop better interventions for this largely neglected disease.

Role of cytosolic glyceraldehyde-3-phosphate dehydrogenase in visceral organ infection by Leishmania donovani.

The initial 7 steps of the glycolytic pathway from glucose to 3-phosphoglycerate are localized in the glycosomes in Leishmania, including step 6, catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In L. donovani and L. mexicana, there exists a second GAPDH enzyme present in the cytosol that is absent in L. braziliensis and that has become a pseudogene in L. major. To investigate the role of the cytosolic GAPDH (cGAPDH), an L. donovani cGAPDH-null mutant was generated, and conversely, the functional L. donovani cGAPDH was introduced into L. major and the resulting engineered parasites were characterized. The L. donovani cGAPDH-null mutant was able to proliferate at the same rate as the wild-type parasite in glucose-deficient medium. However, in the presence of glucose, the L. donovani cGAPDH-null mutant consumed less glucose and proliferated more slowly than the wild-type parasite and displayed reduced infectivity in visceral organs of experimentally infected mice. This demonstrates that cGAPDH is functional in L. donovani and is required for survival in visceral organs. Restoration of cGAPDH activity in L. major, in contrast, had an adverse effect on L. major proliferation in glucose-containing medium, providing a possible explanation of why it has evolved into a pseudogene in L. major. This study indicates that there is a difference in glucose metabolism between L. donovani and L. major, and this may represent an important factor in the ability of L. donovani to cause visceral disease.

[Cerebral microbleeds detected on T2-weighted gradient echo magnetic resonance and its clinical significance].

OBJECTIVE: To explore the diagnostic value of T2-weighted gradient-echo magnetic resonance imaging (MRI) in cerebral microbleeds (CMBs) and its clinical significance. METHODS: The distribution of CMBs and follow-up observations were performed by routine T1WI, T2WI and T2-weighted gradient echo sequence in 634 patients clinically suspected for stroke. RESULTS: In 149 patients, a total of 1140 CMBs occurred predominantly in cortex-subcortical area (n = 471, 41.31%), basal ganglia (n = 289, 25.35%), thalamus (n = 199, 17.45%), brain stem (n = 90, 7.89%) and cerebellum (n = 91, 7.98%). Among them, 137 patients had various degrees of ischemic brain changes, displayed iso-intensity or hypo-intensity on T1WI, hyper-intensity on T2WI and FLAIR in basal ganglia, white matter around sided ventricle and brain stem. There were 23 patients with cerebral infarction and 5 with CMBs after hemorrhagic brain stroke. And 12 of them had new hemorrhagic stroke in 2-6 months. CONCLUSION: T2-weighted gradient echo MRI has obvious advantages in the detection of CMBs. The presence of CMBs suggests a risk of cerebral hemorrhage.

Three novel isomeric zinc metal-organic frameworks from a tetracarboxylate linker.

Three porous supramolecular isomers (IZE-1, IZE-2, and IZE-3) with the same framework component [Zn(2)(EBTC)(H(2)O)(2)] (EBTC = 1,1'-ethynebenzene-3,3',5,5'-tetracarboxylate) were successfully constructed by finely tuning the reaction condition. Although both IZE-1 and IZE-2 are constructed from the linear EBTC subunits and one kind of regular [Zn(2)(CO(2))(4)] paddlewheels, their frameworks exhibit two different (3,4)-c net of fof (sqc1575) and sqc1572, respectively, resulting in cavities with different size and shape. However, as for isomer IZE-3, the EBTC ligands are bent and one-half of the [Zn(2)(CO(2))(4)] paddlewheels are distorted, leading to a novel (3,4,4)-c hyx net with point symbol (6.7(2))(4)(6(2).8(2).10(2))(7(2).8(2).11(2)) and vertex symbol (6.7.7)(4)(7(2).7(2).8.8.12.12)(6.6.8.8.10(2).10(2)). Quantum chemical calculations by DFT indicate that the three isomers have very close thermodynamic stabilities, which may explain that subtle condition change leads to variation of the frameworks. Further theoretical semiempirical investigation on the interactions between solvent molecules and compounds shows different hydrogen binding patterns in good agreement with the experimental observations. Furthermore, they exhibit good solid-state luminescence properties with long lifetime.

Cotton gene expression profiles in resistant Gossypium hirsutum cv. Zhongzhimian KV1 responding to Verticillium dahliae strain V991 infection.

Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease that is caused by the soil-borne fungus pathogen Verticillium dahliae (V. dahliae). To study the molecular mechanism in wilt tolerance, suppression subtractive hybridization (SSH) and dot blot techniques were used to identify the specifically expressed genes in a superior wilt-resistant cotton cultivar (G. hirsutum cv. Zhongzhimian KV1) after inoculation with pathogen. cDNAs from the root tissues of Zhongzhimian KV1 inoculated with V. dahliae strain V991 or water mock were used to construct the libraries that contain 4800 clones. Based on the results from dot blot analysis, 147 clones were clearly induced by V. dahliae and selected from the SSH libraries for sequencing. A total of 92 up-regulated and 7 down-regulated non-redundant expressed sequences tags (ESTs) were identified as disease responsive genes and classified into 9 functional groups. Two important clues regarding wilt-resistant G. hirsutum were obtained from this study. One was Bet v 1 family; the other was UbI gene family that may play an important role in the defense reaction against Verticillium wilt. The result from real-time quantitative reverse transcription polymerase chain reaction showed that these genes were activated quickly and transiently after inoculation with V. dahliae.

catena-Poly[[[triaqua-cobalt(II)]-µ-10-methyl-phenothia-zine-3,7-dicarboxyl-ato] monohydrate].

The polymeric title compound, {[Co(C(15)H(9)NO(4)S)(H(2)O)(3)]·H(2)O}(n), consists of chains along [001] made up from Co(2+) ions bridged by 10-methyl-phenothia-zine-3,7-dicarboxyl-ate anions. The Co(2+) ion, coordinated by three O atoms from two different carboxyl-ate groups and three water mol-ecules, displays a distorted octa-hedral environment. In the crystal, π-π inter-chain inter-actions, with centroid-centroid distances of 3.656 (2) and 3.669 (2) Šbetween the benzene rings of the ligands, assemble the chains into sheets parallel to (100). O-H⋯O hydrogen-bonding inter-actions between the coordinating water mol-ecules and carboxyl-ate O atoms link the sheets into a three-dimensional network.

Reconstitution of Mycobacterium marinum Nonhomologous DNA End Joining Pathway in Leishmania.

In mammalian cells, DNA double-strand breaks (DSBs) are mainly repaired by nonhomologous end joining (NHEJ) pathway. Ku (a heterodimer formed by Ku70 and Ku80 proteins) and DNA ligase IV are the core NHEJ factors. Ku could also be involved in other cellular processes, including telomere length regulation, DNA replication, transcription, and translation control. Leishmania, an early branching eukaryote and the causative agent of leishmaniasis, has no functional NHEJ pathway due to its lack of DNA ligase IV and other NHEJ factors but retains Ku70 and Ku80 proteins. In this study, we generated Leishmania donovani Ku70 disruption mutants and Ku70 and Ku80 double gene (Ku70/80) disruption mutants. We found that Leishmania Ku is still involved in DSB repair, possibly through its binding to DNA ends to block and slowdown 5' end resections and Ku-Ku or other protein interactions. Depending on location of a DSB between the direct repeat genomic sequences, Leishmania Ku could have an inhibiting effect, no effect or a promoting effect on the DSB repair mediated by single strand annealing (SSA), the most frequently used DSB repair pathway in Leishmania. Ku70/80 proteins are also required for the healthy proliferation of Leishmania cells. Interestingly, unlike in Trypanosoma brucei and L. mexicana, Ku70/80 proteins are dispensable for maintaining the normal lengths of telomeres in L. donovani. We also show it is possible to reconstitute the two components (Ku and Ligase D) NHEJ pathway derived from Mycobacterium marinum in Leishmania. This improved DSB repair fidelity and efficiency in Leishmania and sets up an example that the bacterial NHEJ pathway can be successfully reconstructed in an NHEJ-deficient eukaryotic parasite. IMPORTANCE 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. In this study, we found that Leishmania Ku heterodimers are still participating in DSB repair possibly through blocking 5' end resections and Ku-Ku protein interactions. Depending on the DSB location, Ku could have an inhibiting or promoting effect on DSB repair mediated by the single-strand annealing repair pathway. Ku is also required for the normal growth of the parasite but surprisingly dispensable for maintaining the telomere lengths. Further, we show it is possible to introduce Mycobacterium marinum NHEJ pathway into Leishmania. Understanding DSB repair mechanisms of Leishmania may improve the CRISPR gene targeting specificity and efficiency and help identify new drug targets for this important human parasite.

Investigating the Leishmania donovani sacp Gene and Its Role in Macrophage Infection and Survival in Mice.

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.

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 showed 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 as well as reduced mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the disease promoting cytokines IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-γ resulting in higher IFN-γ/IL-10 and IFN-γ/IL4 ratios compared to non-immunized animals. LmexCen-/- immunization also resulted in long-lasting protection against L. donovani infection. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against the Old World visceral leishmaniasis.

Centrin-deficient Leishmania mexicana confers protection against New World cutaneous leishmaniasis.

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.

Screening Leishmania donovani-specific genes required for visceral infection.

Comparison of the Leishmania infantum genome with Leishmania braziliensis and Leishmania major genomes has identified 25 L. infantum species-specific genes that are absent or pseudogenes in L. major and L. braziliensis. To determine whether these L. infantum species-specific genes are involved in visceral Leishmania infection, we cloned the orthologues of 14 L. infantum species-specific genes from the genetically closely related Leishmania donovani and introduced them into L. major. Two of these L. donovani species-specific genes were found to significantly increase L. major survival in visceral organs in BALB/c mice. One (orthologue of LinJ28_V3.0340; Ld2834) of these two genes was further investigated. The L. donovani Ld2834 null mutants displayed dramatically reduced virulence in BALB/c mice and were unable to survive in axenic amastigote culture conditions arguing that Ld2834 plays a crucial role in enabling L. donovani survive at the increased temperature typically associated with visceral organs. Ld2834 encodes a 50 kDa protein that is localized in the cytoplasma and has no significant sequence similarity with other known genes. This study validates the importance of comparative genomics for understanding Leishmania species pathology and argues that Leishmania species-specific genes play important roles in tissue tropism and virulence.

Expression of a Leishmaniadonovani nucleotide sugar transporter in Leishmaniamajor enhances survival in visceral organs.

Leishmania donovani and Leishmaniainfantum infections cause fatal visceral leishmaniasis, and Leishmaniamajor causes self healing cutaneous lesions. It is poorly understood what genetic differences between these Leishmania species are responsible for the different pathologies of infection. To investigate whether L.donovani species-specific genes are involved in visceral Leishmania infection, we have examined a L.donovani species-specific gene Ld1590 (ortholog of LinJ15_V3.0900) that is a pseudogene in L.major. We have previously shown that transgenic expression of L.donovani Ld1590 in L.major significantly increased the liver and spleen parasite burdens in infected BALB/c mice. In this study we report that Ld1590 potentially encodes a nucleotide sugar transporter (NST) which localizes in the L.donovani Golgi apparatus. Surprisingly, although transgenic expression of the Ld1590 NST increased L.major survival in visceral organs, deletion of Ld1590 NST in L.donovani had no significant effect on L.donovani survival in mice. These observations suggest that loss of the functional Ld1590 gene in L.major may have been associated with reduced virulence in visceral organs in its animal reservoir and could have contributed to L.major's tropism for cutaneous infections.

Evidence that a naturally occurring single nucleotide polymorphism in the RagC gene of Leishmania donovani contributes to reduced virulence.

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.