Whole-Genome Sequencing Reveals the Presence of the blaCTX-M-65 Gene in Extended-Spectrum ß-Lactamase-Producing and Multi-Drug-Resistant Clones of Salmonella Serovar Infantis Isolated from Broiler Chicken Environments in the Galapagos Islands.

Salmonella Infantis, a common contaminant of poultry products, is known to harbor mobile genetic elements that confer multi-drug resistance (MDR) and have been detected in many continents. Here, we report four MDR S. Infantis strains recovered from poultry house environments in Santa Cruz Island of the Galapagos showing extended-spectrum ß-lactamase (ESBL) resistance and reduced fluoroquinolone susceptibility. Whole-genome sequencing (WGS) revealed the presence of the ESBL-conferring blaCTX-M-65 gene in an IncFIB-like plasmid in three S. Infantis isolates. Multi-locus sequence typing (MLST) and single nucleotide variant/polymorphism (SNP) SNVPhyl analysis showed that the S. Infantis isolates belong to sequence type ST32, likely share a common ancestor, and are closely related (1-3 SNP difference) to blaCTX-M-65-containing clinical and veterinary S. Infantis isolates from the United States and Latin America. Furthermore, phylogenetic analysis of SNPs following core-genome alignment (i.e., ParSNP) inferred close relatedness between the S. Infantis isolates from Galapagos and the United States. Prophage typing confirmed the close relationship among the Galapagos S. Infantis and was useful in distinguishing them from the United States isolates. This is the first report of MDR blaCTX-M-65-containing S. Infantis in the Galapagos Islands and highlights the need for increased monitoring and surveillance programs to determine prevalence, sources, and reservoirs of MDR pathogens.

Management of Coronavirus Disease 2019 (COVID-19) Pandemic: From Diagnosis to Treatment Strategies.

Following the emergence of severe acute respiratory syndrome (SARS) in 2002 and the Middle East respiratory syndrome (MERS) in 2012, the world is now combating a third large-scale outbreak caused by a coronavirus, the coronavirus disease 2019 (COVID-19). After the rapid spread of SARS-coronavirus (CoV)-2 (the virus causing COVID-19) from its origin in China, the World Health Organization (WHO) declared a Public Health Emergency of International Concern (PHEIC) on January 30, 2020. From the beginning of the COVID-19 pandemic, a significant number of studies have been conducted to better understand the biology and pathogenesis of the novel coronavirus, and to aid in developing effective treatment regimens, therapeutics, and vaccines. This review focuses on the recent advancements in the rapidly evolving areas of clinical care and management of COVID-19. The emerging strategies for the diagnosis and treatment of this disease are explored, and the development of effective vaccines is reviewed.

The host cell secretory pathway mediates the export of Leishmania virulence factors out of the parasitophorous vacuole.

To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV.

Leishmania donovani Lipophosphoglycan Increases Macrophage-Dependent Chemotaxis of CXCR6-Expressing Cells via CXCL16 Induction.

CXCL16 is a multifunctional chemokine that is highly expressed by macrophages and other immune cells in response to bacterial and viral pathogens; however, little is known regarding the role of CXCL16 during parasitic infections. The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. Even though chemokine production is a host defense mechanism during infection, subversion of the host chemokine system constitutes a survival strategy adopted by the parasite. Here, we report that L. donovani promastigotes upregulate CXCL16 synthesis and secretion by bone marrow-derived macrophages (BMDM). In contrast to wild-type parasites, a strain deficient in the virulence factor lipophosphoglycan (LPG) failed to induce CXCL16 production. Consistent with this, cell treatment with purified L. donovani LPG augmented CXCL16 expression and secretion. Notably, the ability of BMDM to promote migration of cells expressing CXCR6, the cognate receptor of CXCL16, was augmented upon L. donovani infection in a CXCL16- and LPG-dependent manner. Mechanistically, CXCL16 induction by L. donovani required the activity of AKT and the mechanistic target of rapamycin (mTOR) but was independent of Toll-like receptor signaling. Collectively, these data provide evidence that CXCL16 is part of the inflammatory response elicited by L. donovani LPG in vitro Further investigation using CXCL16 knockout mice is required to determine whether this chemokine contributes to the pathogenesis of visceral leishmaniasis and to elucidate the underlying molecular mechanisms.

Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties.

Parasitic worms have a remarkable ability to modulate host immune responses through several mechanisms including excreted/secreted proteins (ESP), yet the exact nature of these proteins and their targets often remains elusive. Here, we performed mass spectrometry analyses of ESP (TsESP) from larval and adult stages of the pig whipworm Trichuris suis (Ts) and identified ~350 proteins. Transcriptomic analyses revealed large subsets of differentially expressed genes in the various life cycle stages of the parasite. Exposure of bone marrow-derived macrophages and dendritic cells to TsESP markedly diminished secretion of the pro-inflammatory cytokines TNFα and IL-12p70. Conversely, TsESP exposure strongly induced release of the anti-inflammatory cytokine IL-10, and also induced high levels of nitric oxide (NO) and upregulated arginase activity in macrophages. Interestingly, TsESP failed to directly induce CD4+ CD25+ FoxP3+ regulatory T cells (Treg cells), while OVA-pulsed TsESP-treated dendritic cells suppressed antigen-specific OT-II CD4+ T cell proliferation. Fractionation of TsESP identified a subset of proteins that promoted anti-inflammatory functions, an activity that was recapitulated using recombinant T. suis triosephosphate isomerase (TPI) and nucleoside diphosphate kinase (NDK). Our study helps illuminate the intricate balance that is characteristic of parasite-host interactions at the immunological interface, and further establishes the principle that specific parasite-derived proteins can modulate immune cell functions.

Vesicle-based secretion in schistosomes: Analysis of protein and microRNA (miRNA) content of exosome-like vesicles derived from Schistosoma mansoni.

Exosomes are small vesicles of endocytic origin, which are released into the extracellular environment and mediate a variety of physiological and pathological conditions. Here we show that Schistosoma mansoni releases exosome-like vesicles in vitro. Vesicles were purified from culture medium by sucrose gradient fractionation and fractions containing vesicles verified by western blot analyses and electron microscopy. Proteomic analyses of exosomal contents unveiled 130 schistosome proteins. Among these proteins are common exosomal markers such as heat shock proteins, energy-generating enzymes, cytoskeletal proteins, and others. In addition, the schistosome extracellular vesicles contain proteins of potential importance for host-parasite interaction, notably peptidases, signaling proteins, cell adhesion proteins (e.g., integrins) and previously described vaccine candidates, including glutathione-S-transferase (GST), tetraspanin (TSP-2) and calpain. S. mansoni exosomes also contain 143 microRNAs (miRNA), of which 25 are present at high levels, including miRNAs detected in sera of infected hosts. Quantitative PCR analysis confirmed the presence of schistosome-derived miRNAs in exosomes purified from infected mouse sera. The results provide evidence of vesicle-mediated secretion in these parasites and suggest that schistosome-derived exosomes could play important roles in host-parasite interactions and could be a useful tool in the development of vaccines and therapeutics.

Proteomic Profiling of Leishmania donovani Promastigote Subcellular Organelles.

To facilitate a greater understanding of the biological processes in the medically important Leishmania donovani parasite, a combination of differential and density-gradient ultracentrifugation techniques were used to achieve a comprehensive subcellular fractionation of the promastigote stage. An in-depth label-free proteomic LC-MS/MS analysis of the density gradients resulted in the identification of ∼50% of the Leishmania proteome (3883 proteins detected), which included ∼645 integral membrane proteins and 1737 uncharacterized proteins. Clustering and subcellular localization of proteins was based on a subset of training Leishmania proteins with known subcellular localizations that had been determined using biochemical, confocal microscopy, or immunoelectron microscopy approaches. This subcellular map will be a valuable resource that will help dissect the cell biology and metabolic processes associated with specific organelles of Leishmania and related kinetoplastids.

The hydrophobic region of the Leishmania peroxin 14: requirements for association with a glycosome mimetic membrane.

Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.

IRF-8 regulates expansion of myeloid-derived suppressor cells and Foxp3+ regulatory T cells and modulates Th2 immune responses to gastrointestinal nematode infection.

Interferon regulatory factor-8 (IRF-8) is critical for Th1 cell differentiation and negatively regulates myeloid cell development including myeloid-derived suppressor cells (MDSC). MDSC expand during infection with various pathogens including the gastrointestinal (GI) nematode Heligmosomoides polygyrus bakeri (Hpb). We investigated if IRF-8 contributes to Th2 immunity to Hpb infection. Irf8 expression was down-regulated in MDSC from Hpb-infected C57BL/6 (B6) mice. IRF-8 deficient Irf8-/- and BXH-2 mice had significantly higher adult worm burdens than B6 mice after primary or challenge Hpb infection. During primary infection, MDSC expanded to a significantly greater extent in mesenteric lymph nodes (MLN) and spleens of Irf8-/- and BXH-2 than B6 mice. CD4+GATA3+ T cells numbers were comparable in MLN of infected B6 and IRF-8 deficient mice, but MLN cells from infected IRF-8 deficient mice secreted significantly less parasite-specific IL-4 ex vivo. The numbers of alternatively activated macrophages in MLN and serum levels of Hpb-specific IgG1 and IgE were also significantly less in infected Irf8-/- than B6 mice. The frequencies of antigen-experienced CD4+CD11ahiCD49dhi cells that were CD44hiCD62L- were similar in MLN of infected Irf8-/- and B6 mice, but the proportions of CD4+GATA3+ and CD4+IL-4+ T cells were lower in infected Irf8-/- mice. CD11b+Gr1+ cells from naïve or infected Irf8-/- mice suppressed CD4+ T cell proliferation and parasite-specific IL-4 secretion in vitro albeit less efficiently than B6 mice. Surprisingly, there were significantly more CD4+ T cells in infected Irf8-/- mice, with a higher frequency of CD4+CD25+Foxp3+ T (Tregs) cells and significantly higher numbers of Tregs than B6 mice. In vivo depletion of MDSC and/or Tregs in Irf8-/- mice did not affect adult worm burdens, but Treg depletion resulted in higher egg production and enhanced parasite-specific IL-5, IL-13, and IL-6 secretion ex vivo. Our data thus provide a previously unrecognized role for IRF-8 in Th2 immunity to a GI nematode.

Excretory/secretory products from the gastrointestinal nematode Trichuris muris.

To better control gastrointestinal nematode infections in humans and animals, it is important to understand the strategies used by these parasites to modulate the host immune system. In this regard, molecules released by parasites have been attributed crucially important roles in host-parasite negotiations. We characterized the excretory/secretory (E/S) microRNA (miRNA) and protein profiles from the mouse gastrointestinal nematode parasite Trichuris muris. Released miRNAs were subjected to miRNA sequencing and E/S proteins were analysed by mass spectrometry. Fourteen miRNAs were identified in T. muris exosome-like vesicles, as well as 73 proteins of nematode origin, 11 of which were unique to this study. Comparison with published nematode protein secretomes revealed high conservation at the functional level.

Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells.

Chagas disease, caused by Trypanosoma cruzi, although endemic in many parts of Central and South America, is emerging as a global health threat through the potential contamination of blood supplies. Consequently, in the absence of a gold standard assay for the diagnosis of Chagas disease, additional antigens or strategies are needed. A proteomic analysis of the trypomastigote excreted-secreted antigens (TESA) associated with exosomal vesicles shed by T. cruzi identified ∼80 parasite proteins, with the majority being trans-sialidases. Mass spectrometry analysis of immunoprecipitation products performed using Chagas immune sera showed a marked enrichment in a subset of TESA proteins. Of particular relevance for diagnostic applications were the retrotransposon hot spot (RHS) proteins, which are absent in Leishmania spp., parasites that often confound diagnosis of Chagas disease. Interestingly, serological screens using recombinant RHS showed a robust immunoreactivity with sera from patients with clinical stages of Chagas ranging from asymptomatic to advance cardiomyopathy and this immunoreactivity was comparable to that of crude TESA. More importantly, no cross-reactivity with RHS was detected with sera from patients with malaria, leishmaniasis, toxoplasmosis, or African sleeping sickness, making this protein an attractive reagent for diagnosis of Chagas disease.

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies.

Multiplexed panel of precisely quantified salivary proteins for biomarker assessment.

An increasingly popular "absolute" quantitative technique involves the SRM or MRM approach with stable isotope-labeled standards (SIS). Using this approach, many proteins in human plasma/serum have been quantified for biomarker assessment and disease stratification. Due to the complexity of plasma and the invasive nature of its collection, alternative biosamples are currently being explored. Here, we present the broadest panel of multiplexed MRM assays with SIS peptides for saliva proteins developed to date. The validated panel consists of 158 candidate human saliva protein biomarkers, inferred from 244 interference-free peptides. The resulting concentrations were reproducibly quantified over a 6 order-of-magnitude concentration range (from 218 µg/mL to 88 pg/mL; average CVs of 12% over analytical triplicates). All concentrations were determined from reverse standard curves, which were generated using a constant concentration of endogenous material with varying concentrations of spiked-in SIS peptides. The large-scale screening of the soluble and membrane-associated proteins contained within the 158-plex assay could present new opportunities for biomarker assessment and clinical diagnostics.

Downregulation of the Syk Signaling Pathway in Intestinal Dendritic Cells Is Sufficient To Induce Dendritic Cells That Inhibit Colitis.

Helminthic infections modulate host immunity and may protect people in less-developed countries from developing immunological diseases. In a murine colitis model, the helminth Heligmosomoides polygyrus bakeri prevents colitis via induction of regulatory dendritic cells (DCs). The mechanism driving the development of these regulatory DCs is unexplored. There is decreased expression of the intracellular signaling pathway spleen tyrosine kinase (Syk) in intestinal DCs from H. polygyrus bakeri-infected mice. To explore the importance of this observation, it was shown that intestinal DCs from DC-specific Syk(-/-) mice were powerful inhibitors of murine colitis, suggesting that loss of Syk was sufficient to convert these cells into their regulatory phenotype. DCs sense gut flora and damaged epithelium via expression of C-type lectin receptors, many of which signal through the Syk signaling pathway. It was observed that gut DCs express mRNA encoding for C-type lectin (CLEC) 7A, CLEC9A, CLEC12A, and CLEC4N. H. polygyrus bakeri infection downmodulated CLEC mRNA expression in these cells. Focusing on CLEC7A, which encodes for the dectin-1 receptor, flow analysis showed that H. polygyrus bakeri decreases dectin-1 expression on the intestinal DC subsets that drive Th1/Th17 development. DCs become unresponsive to the dectin-1 agonist curdlan and fail to phosphorylate Syk after agonist stimulation. Soluble worm products can block CLEC7A and Syk mRNA expression in gut DCs from uninfected mice after a brief in vitro exposure. Thus, downmodulation of Syk expression and phosphorylation in intestinal DCs could be important mechanisms through which helminths induce regulatory DCs that limit colitis.

α,ß-Dicarbonyl reduction is mediated by the Saccharomyces Old Yellow Enzyme.

The undesirable flavor compounds diacetyl and 2,3-pentanedione are vicinal diketones (VDKs) formed by extracellular oxidative decarboxylation of intermediate metabolites of the isoleucine, leucine and valine (ILV) biosynthetic pathway. These VDKs are taken up by Saccharomyces and enzymatically converted to acetoin and 3-hydroxy-2-pentanone, respectively. Purification of a highly enriched diacetyl reductase fraction from Saccharomyces cerevisiae in conjunction with mass spectrometry identified Old Yellow Enzyme (Oye) as an enzyme capable of catalyzing VDK reduction. Kinetic analysis of recombinant Oye1p, Oye2p and Oye3p isoforms confirmed that all three isoforms reduced diacetyl and 2,3-pentanedione in an NADPH-dependent reaction. Transcriptomic analysis of S. cerevisiae (ale) and S. pastorianus (lager) yeast during industrial fermentations showed that the transcripts for OYE1, OYE2, arabinose dehydrogenase (ARA1), α-acetolactate synthase (ILV2) and α-acetohydroxyacid reductoisomerase (ILV5) were differentially regulated in a manner that correlated with changes in extracellular levels of VDKs. These studies provide insights into the mechanism for reducing VDKs and decreasing maturation times of beer which are of commercial importance.

GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites.

Purine acquisition is an essential nutritional process for Leishmania. Although purine salvage into adenylate nucleotides has been investigated in detail, little attention has been focused on the guanylate branch of the purine pathway. To characterize guanylate nucleotide metabolism in Leishmania and create a cell culture model in which the pathways for adenylate and guanylate nucleotide synthesis can be genetically uncoupled for functional studies in intact cells, we created and characterized null mutants of L. donovani that were deficient in either GMP reductase alone (Δgmpr) or in both GMP reductase and its paralog IMP dehydrogenase (Δgmpr/Δimpdh). Whereas wild type parasites were capable of utilizing virtually any purine nucleobase/nucleoside, the Δgmpr and Δgmpr/Δimpdh null lines exhibited highly restricted growth phenotypes. The Δgmpr single mutant could not grow in xanthine, guanine, or their corresponding nucleosides, while no purine on its own could support the growth of Δgmpr/Δimpdh cells. Permissive growth conditions for the Δgmpr/Δimpdh necessitated both xanthine, guanine, or the corresponding nucleosides, and additionally, a second purine that could serve as a source for adenylate nucleotide synthesis. Interestingly, GMPR, like its paralog IMPDH, is compartmentalized to the leishmanial glycosome, a process mediated by its COOH-terminal peroxisomal targeting signal. The restricted growth phenotypes displayed by the L. donovani Δgmpr and Δgmpr/Δimpdh null mutants confirms the importance of GMPR in the purine interconversion processes of this parasite.

Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors.

Parasitic diseases cause ∼ 500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase) and TRACK (Trypanosoma receptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.

An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion.

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

A Protein Complex Map of Trypanosoma brucei.

The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org.

Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management.

Reactive oxygen and nitrogen species (ROS and RNS) produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS) plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL). There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed environments encountered during infection and can be targeted for chemotherapeutic purpose to treat visceral leishmaniasis.