Galactokinase-like protein from Leishmania donovani: Biochemical and structural characterization of a recombinant protein.

Leishmaniasis is a spectrum of conditions caused by infection with the protozoan Leishmania spp. parasites. Leishmaniasis is endemic in 98 countries around the world, and resistance to current anti-leishmanial drugs is rising. Our work has identified and characterised a previously unstudied galactokinase-like protein (GalK) in Leishmania donovani, which catalyses the MgATP-dependent phosphorylation of the C-1 hydroxyl group of d-galactose to galactose-1-phosphate. Here, we report the production of the catalytically active recombinant protein in E. coli, determination of its substrate specificity and kinetic constants, as well as analysis of its molecular envelope using in solution X-ray scattering. Our results reveal kinetic parameters in range with other galactokinases with an average apparent Km value of 76 µM for galactose, Vmax and apparent Kcat values with 4.46376 × 10-9 M/s and 0.021 s-1, respectively. Substantial substrate promiscuity was observed, with galactose being the preferred substrate, followed by mannose, fructose and GalNAc. LdGalK has a highly flexible protein structure suggestive of multiple conformational states in solution, which may be the key to its substrate promiscuity. Our data presents novel insights into the galactose salvaging pathway in Leishmania and positions this protein as a potential target for the development of pharmaceuticals seeking to interfere with parasite substrate metabolism.

Placing Leishmaniasis in the Limelight through the Communicable Disease Surveillance System: An Experience from Sri Lanka.

Having an effective surveillance system is imperative to take timely and appropriate actions for disease control and prevention. In Sri Lanka, leishmaniasis was declared as a notifiable disease in 2008. This paper presents a comprehensive compilation of the up-to-date documents on the communicable disease and leishmaniasis surveillance in Sri Lanka in order to describe the importance of the existing leishmaniasis surveillance system and to identify gaps that need to be addressed. The documents perused included circulars, reports, manuals, guidelines, ordinances, presentations, and published articles. The disease trends reported were linked to important landmarks in leishmaniasis surveillance. The findings suggest that there is a well-established surveillance system in Sri Lanka having a massive impact on increased case detection, resulting in im-proved attention on leishmaniasis. However, the system is not without its short comings and there is room for further improvements.

Current and future strategies against cutaneous parasites.

Cutaneous parasites are identified by their specific cutaneous symptoms which are elicited based on the parasite's interactions with the host. Standard anti-parasitic treatments primarily focus on the use of specific drugs to disrupt the regular function of the target parasite. In cases where secondary infections are induced by the parasite itself, antibiotics may also be used in tandem with the primary treatment to deal with the infection. Whilst drug-based treatments are highly effective, the development of resistance by bacteria and parasites, is increasingly prevalent in the modern day, thus requiring the development of non-drug based anti-parasitic strategies. Cutaneous parasites vary significantly in terms of the non-systemic methods that are required to deal with them. The main factors that need to be considered are the specifically elicited cutaneous symptoms and the relative cutaneous depth in which the parasites typically reside in. Due to the various differences in their migratory nature, certain cutaneous strategies are only viable for specific parasites, which then leads to the idea of developing an all-encompassing anti-parasitic strategy that works specifically against cutaneous parasites. The main benefit of this would be the overall time saved in regards to the period that is needed for accurate diagnosis of parasite, coupled with the prescription and application of the appropriate treatment based on the diagnosis. This review will assess the currently identified cutaneous parasites, detailing their life cycles which will allow for the identification of certain areas that could be exploited for the facilitation of cutaneous anti-parasitic treatment.

Community Engagement in Cutaneous Leishmaniasis Research in Brazil, Ethiopia, and Sri Lanka: A Decolonial Approach for Global Health.

Cutaneous leishmaniasis (CL) is a parasitic skin disease endemic in at least 88 countries where it presents an urgent, albeit often "neglected" public health problem. In this paper, we discuss our model of decolonial community engagement in the ECLIPSE global health research program, which aims to improve physical and mental health outcomes for people with CL. The ECLIPSE program has four interlinked phases and underpinning each of these phases is sustained and robust community engagement and involvement that guides and informs all activities in ECLIPSE. Our decolonial approach implies that the model for community engagement will be different in Brazil, Ethiopia and Sri Lanka. Indeed, we adopt a critical anthropological approach to engaging with community members and it is precisely this approach we evaluate in this paper. The data and material we draw on were collected through qualitative research methods during community engagement activities. We established 13 Community Advisory Groups (CAGs): in Brazil (n = 4), Ethiopia (n = 6), and Sri Lanka (n = 3). We identified four overarching themes during a thematic analysis of the data set: (1) Establishing community advisory groups, (2) CAG membership and community representation, (3) Culturally appropriate and context-bespoke engagement, and (4) Relationships between researchers and community members. During our first period of ECLIPSE community engagement, we have debunked myths (for instance about communities being "disempowered"), critiqued our own practices (changing approaches in bringing together CAG members) and celebrated successes (notably fruitful online engagement during a challenging COVID-19 pandemic context). Our evaluation revealed a gap between the exemplary community engagement frameworks available in the literature and the messy, everyday reality of working in communities. In the ECLIPSE program, we have translated ideal(istic) principles espoused by such community engagement guidance into the practical realities of "doing engagement" in low-resourced communities. Our community engagement was underpinned by such ideal principles, but adapted to local sociocultural contexts, working within certain funding and regulatory constraints imposed on researchers. We conclude with a set of lessons learned and recommendations for the conduct of decolonial community engagement in global health research.

Temperate Zone Plant Natural Products-A Novel Resource for Activity against Tropical Parasitic Diseases.

The use of plant-derived natural products for the treatment of tropical parasitic diseases often has ethnopharmacological origins. As such, plants grown in temperate regions remain largely untested for novel anti-parasitic activities. We describe here a screen of the PhytoQuest Phytopure library, a novel source comprising over 600 purified compounds from temperate zone plants, against in vitro culture systems for Plasmodium falciparum, Leishmania mexicana, Trypanosoma evansi and T. brucei. Initial screen revealed 6, 65, 15 and 18 compounds, respectively, that decreased each parasite's growth by at least 50% at 1-2 µM concentration. These initial hits were validated in concentration-response assays against the parasite and the human HepG2 cell line, identifying hits with EC50 < 1 µM and a selectivity index of >10. Two sesquiterpene glycosides were identified against P. falciparum, four sterols against L. mexicana, and five compounds of various scaffolds against T. brucei and T. evansi. An L. mexicana resistant line was generated for the sterol 700022, which was found to have cross-resistance to the anti-leishmanial drug miltefosine as well as to the other leishmanicidal sterols. This study highlights the potential of a temperate plant secondary metabolites as a novel source of natural products against tropical parasitic diseases.

Genetic validation of Leishmania genes essential for amastigote survival in vivo using N-myristoyltransferase as a model.

BACKGROUND: Proving that specific genes are essential for the intracellular viability of Leishmania parasites within macrophages remains a challenge for the identification of suitable targets for drug development. This is especially evident in the absence of a robust inducible expression system or functioning RNAi machinery that works in all Leishmania species. Currently, if a target gene of interest in extracellular parasites can only be deleted from its genomic locus in the presence of ectopic expression from a wild type copy, it is assumed that this gene will also be essential for viability in disease-promoting intracellular parasites. However, functional essentiality must be proven independently in both life-cycle stages for robust validation of the gene of interest as a putative target for chemical intervention. METHODS: Here, we have used plasmid shuffle methods in vivo to provide supportive genetic evidence that N-myristoyltransferase (NMT) is essential for Leishmania viability throughout the parasite life-cycle. Following confirmation of NMT essentiality in vector-transmitted promastigotes, a range of mutant parasites were used to infect mice prior to negative selection pressure to test the hypothesis that NMT is also essential for parasite viability in an established infection. RESULTS: Ectopically-expressed NMT was only dispensable under negative selection in the presence of another copy. Total parasite burdens in animals subjected to negative selection were comparable to control groups only if an additional NMT copy, not affected by the negative selection, was expressed. CONCLUSIONS: NMT is an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development.

Nanoparticle-mediated magnetic hyperthermia is an effective method for killing the human-infective protozoan parasite Leishmania mexicana in vitro.

Cutaneous leishmaniasis is a neglected tropical disease characterized by disfiguring skin lesions. Current chemotherapeutic options depend on toxic, expensive drugs that are both difficult to administer and becoming less effective due to increasing levels of resistance. In comparison, thermotherapy displays greater patient compliance and less adverse systemic effects, but there are still significant issues associated with this. The procedure is painful, requiring local anaesthetic, and is less effective against large lesions. Using nanoparticles to controllably generate heat in a localized manner may provide an alternative solution. Here we evaluate magnetic hyperthermia, using iron oxide magnetic nanoparticles, as a localized, heat-based method to kill the human-infective parasite in vitro. We assessed the effectiveness of this method against the differentiated, amastigote form of the parasite using three distinct viability assays: PrestoBlue, Live/Dead stain and a novel luciferase-based assay. Changes in amastigote morphology and ultrastructure were assessed by immunofluorescence, scanning and transmission electron microscopy. Our findings show that magnetic hyperthermia is an effective method to kill host-infective amastigotes, with morphological changes consistent with heat treatment. This method has the potential to be a step-change for research into new therapeutic options that moves away from the expensive chemotherapeutics currently dominating the research climate.

Development of NanoLuc-PEST expressing Leishmania mexicana as a new drug discovery tool for axenic- and intramacrophage-based assays.

The protozoan parasite Leishmania causes leishmaniasis; a spectrum of diseases of which there are an estimated 1 million new cases each year. Current treatments are toxic, expensive, difficult to administer, and resistance to them is emerging. New therapeutics are urgently needed, however, screening the infective amastigote form of the parasite is challenging. Only certain species can be differentiated into axenic amastigotes, and compound activity against these does not always correlate with efficacy against the parasite in its intracellular niche. Methods used to assess compound efficacy on intracellular amastigotes often rely on microscopy-based assays. These are laborious, require specialist equipment and can only determine parasite burden, not parasite viability. We have addressed this clear need in the anti-leishmanial drug discovery process by producing a transgenic L. mexicana cell line that expresses the luciferase NanoLuc-PEST. We tested the sensitivity and versatility of this transgenic strain, in comparison with strains expressing NanoLuc and the red-shifted firefly luciferase. We then compared the NanoLuc-PEST luciferase to the current methods in both axenic and intramacrophage amastigotes following treatment with a supralethal dose of Amphotericin B. NanoLuc-PEST was a more dynamic indicator of cell viability due to its high turnover rate and high signal:background ratio. This, coupled with its sensitivity in the intramacrophage assay, led us to validate the NanoLuc-PEST expressing cell line using the MMV Pathogen Box in a two-step process: i) identify hits against axenic amastigotes, ii) screen these hits using our bioluminescence-based intramacrophage assay. The data obtained from this highlights the potential of compounds active against M. tuberculosis to be re-purposed for use against Leishmania. Our transgenic L. mexicana cell line is therefore a highly sensitive and dynamic system suitable for Leishmania drug discovery in axenic and intramacrophage amastigote models.

The role of heparan sulfate in host macrophage infection by Leishmania species.

The leishmaniases are a group of neglected tropical diseases caused by parasites from the Leishmania genus. More than 20 Leishmania species are responsible for human disease, causing a broad spectrum of symptoms ranging from cutaneous lesions to a fatal visceral infection. There is no single safe and effective approach to treat these diseases and resistance to current anti-leishmanial drugs is emerging. New drug targets need to be identified and validated to generate novel treatments. Host heparan sulfates (HSs) are abundant, heterogeneous polysaccharides displayed on proteoglycans that bind various ligands, including cell surface proteins expressed on Leishmania promastigote and amastigote parasites. The fine chemical structure of HS is formed by a plethora of specific enzymes during biosynthesis, with various positions (N-, 2-O-, 6-O- and 3-O-) on the carbon sugar backbone modified with sulfate groups. Post-biosynthesis mechanisms can further modify the sulfation pattern or size of the polysaccharide, altering ligand affinity to moderate biological functions. Chemically modified heparins used to mimic the heterogeneous nature of HS influence the affinity of different Leishmania species, demonstrating the importance of specific HS chemical sequences in parasite interaction. However, the endogenous structures of host HSs that might interact with Leishmania parasites during host invasion have not been elucidated, nor has the role of HSs in host-parasite biology. Decoding the structure of HSs on target host cells will increase understanding of HS/parasite interactions in leishmaniasis, potentiating identification of new opportunities for the development of novel treatments.

Schistosoma mansoni cercarial elastase (SmCE): differences in immunogenic properties of native and recombinant forms.

The Schistosoma mansoni cercarial elastase (SmCE) has previously been shown to be poorly immunogenic in mice. However, a minority of mice were able to produce antibodies against SmCE after multiple immunizations with crude preparations containing the enzyme. These mice were partially protected against challenge infections of S. mansoni. In the present study, we show that in contrast to the poor immunogenicity of the enzymatically active native form of SmCE derived from a crude preparation (cercarial transformation fluid), immunization of CBA/Ca mice with two enzymatically inactive forms, namely purified native SmCE or a recombinant SmCE fused to recombinant Schistosoma japonicum glutathione S-transferase (rSmCE-SjGST), after adsorption onto aluminum hydroxide adjuvant, induced specific anti-SmCE immunoglobulin G (IgG) in all mice within 2 weeks of the second immunization. The IgG antibody response to rSmCE-SjGST was mainly of the IgG1 subclass. These results suggest that inactive forms of the antigen could be used to obtain the optimum immunogenic effects as a vaccine candidate against schistosomiasis. Mice immunized with the rSmCE-SjGST on alum had smaller mean worm burdens and lower tissue egg counts when compared with adjuvant alone- and recombinant SjGST-injected controls. The native SmCE was antigenically cross-reactive with homologous enzymes of Schistosoma haematobium and Schistosoma margrebowiei.

Global Profiling and Inhibition of Protein Lipidation in Vector and Host Stages of the Sleeping Sickness Parasite Trypanosoma brucei.

The enzyme N-myristoyltransferase (NMT) catalyzes the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localization of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labeling with an alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogue to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins, phosphatases, and many uncharacterized proteins as substrates of NMT in the parasite, providing a global view of the scope of this important protein modification and further evidence for the crucial and pleiotropic role of NMT in the cell.

The Leishmania major†BBSome subunit BBS1 is essential for parasite virulence in the mammalian host.

Bardet-Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole-like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild-type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.

Crystal structure of the small GTPase Arl6/BBS3 from Trypanosoma brucei.

Arl6/BBS3 is a small GTPase, mutations in which are implicated in the human ciliopathy Bardet-Biedl Syndrome (BBS). Arl6 is proposed to facilitate the recruitment of a large protein complex known as the BBSome to the base of the primary cilium, mediating specific trafficking of molecules to this important sensory organelle. Orthologues of Arl6 and the BBSome core subunits have been identified in the genomes of trypanosomes. Flagellum function and motility are crucial to the survival of Trypanosoma brucei, the causative agent of human African sleeping sickness, in the human bloodstream stage of its lifecycle and so the function of the BBSome proteins in trypanosomes warrants further study. RNAi knockdown of T. brucei Arl6 (TbArl6) has recently been shown to result in shortening of the trypanosome flagellum. Here we present the crystal structure of TbArl6 with the bound non-hydrolysable GTP analog GppNp at 2.0 Å resolution and highlight important differences between the trypanosomal and human proteins. Analysis of the TbArl6 active site confirms that it lacks the key glutamine that activates the nucleophile during GTP hydrolysis in other small GTPases. Furthermore, the trypanosomal proteins are significantly shorter at their N-termini suggesting a different method of membrane insertion compared to humans. Finally, analysis of sequence conservation suggests two surface patches that may be important for protein-protein interactions. Our structural analysis thus provides the basis for future biochemical characterisation of this important family of small GTPases.

A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei.

The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet-Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.

The orthologue of Sjögren's syndrome nuclear autoantigen 1 (SSNA1) in Trypanosoma brucei is an immunogenic self-assembling molecule.

Primary Sjögren's Syndrome (PSS) is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögren's Syndrome nuclear autoantigen 1 (SSNA1 or NA14) is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13) and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle.

Leishmania amazonensis META2 protein confers protection against heat shock and oxidative stress.

The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L. amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock.

The stage-regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector, Phlebotomus papatasi.

The stage-regulated HASPB and SHERP proteins of Leishmania major are predominantly expressed in cultured metacyclic parasites that are competent for macrophage uptake and survival. The role of these proteins in parasite development in the sand fly vector has not been explored, however. Here, we confirm that expression of HASPB is detected only in vector metacyclic stages, correlating with the expression of metacyclic-specific lipophosphoglycan and providing the first definitive protein marker for this infective sand fly stage. Similarly, SHERP is expressed in vector metacyclics but is also detected at low levels in the preceding short promastigote stage. Using genetically modified parasites lacking or complemented for the LmcDNA16 locus on chromosome 23 that contains the HASP and SHERP genes, we further show that the presence of this locus is essential for parasite differentiation to the metacyclic stage in Phlebotomus papatasi. While wild-type and complemented parasites transform normally in late-stage infections, generating metacyclic promastigotes and colonizing the sand fly stomodeal valve, null parasites accumulate at the earlier elongated nectomonad stage of development within the abdominal and thoracic midgut of the sand fly. Complementation with HASPB or SHERP alone suggests that HASPB is the dominant effector molecule in this process.

The small GTPase ARL2 is required for cytokinesis in Trypanosoma brucei.

The Arf-like (Arl) small GTPases have a diverse range of functions in the eukaryotic cell. Metazoan Arl2 acts as a regulator of microtubule biogenesis, binding to the tubulin-specific chaperone cofactor D. Arl2 also has a mitochondrial function through its interactions with BART and ANT-1, the only member of the Ras superfamily to be found in this organelle to date. In the present study, we describe characterization of the Arl2 orthologue in the protozoan parasite Trypanosoma brucei. Modulation of TbARL2 expression in bloodstream form parasites by RNA interference (RNAi) causes inhibition of cleavage furrow formation, resulting in a severe defect in cytokinesis and the accumulation of multinucleated cells. RNAi of TbARL2 also results in loss of acetylated alpha-tubulin but not of total -tubulin from cellular microtubules. While overexpression of TbARL2(myc) also leads to a defect in cytokinesis, an excess of untagged protein has no effect on cell division, demonstrating the importance of the extreme C-terminus in correct function. TbARL2 overexpressing cells (either myc-tagged or untagged) have an increase in acetylated -tubulin. Our data indicate that Arl2 has a fundamentally conserved role in trypanosome microtubule biogenesis that correlates with -tubulin acetylation.

N-myristoyltransferase inhibitors as new leads to treat sleeping sickness.

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.