Publications by authors named "Jeremy C Mottram"

157 Publications

Role of the inhibitor of serine peptidase 2 (ISP2) of Trypanosoma brucei rhodesiense in parasite virulence and modulation of the inflammatory responses of the host.

PLoS Negl Trop Dis 2021 06 21;15(6):e0009526. Epub 2021 Jun 21.

Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Centro de Ciências da Saúde, Ilha do Fundão, Rio de Janeiro, RJ, Brazil.

Trypanosoma brucei rhodesiense is one of the causative agents of Human African Trypanosomiasis (HAT), known as sleeping sickness. The parasite invades the central nervous system and causes severe encephalitis that is fatal if left untreated. We have previously identified ecotin-like inhibitors of serine peptidases, named ISPs, in trypanosomatid parasitic protozoa. Here, we investigated the role of ISP2 in bloodstream form T. b. rhodesiense. We generated gene-deficient mutants lacking ISP2 (Δisp2), which displayed a growth profile in vitro similar to that of wild-type (WT) parasites. C57BL/6 mice infected with Δisp2 displayed lower blood parasitemia, a delayed hind leg pathological phenotype and survived longer. The immune response was examined at two time-points that corresponded with two peaks of parasitemia. At 4 days, the spleens of Δisp2-infected mice had a greater percentage of NOS2+ myeloid cells, IFN-γ+-NK cells and increased TNF-α compared to those infected with WT and parasites re-expressing ISP2 (Δisp2:ISP2). By 13 days the increased NOS2+ population was sustained in Δisp2-infected mice, along with increased percentages of monocyte-derived dendritic cells, as well as CD19+ B lymphocytes, and CD8+ and CD4+ T lymphocytes. Taken together, these findings indicate that ISP2 contributes to T. b. rhodesiense virulence in mice and attenuates the inflammatory response during early infection.
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http://dx.doi.org/10.1371/journal.pntd.0009526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248637PMC
June 2021

The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis, cell division and virulence in the mammalian host.

PLoS Pathog 2021 06 18;17(6):e1009666. Epub 2021 Jun 18.

Research Unit "MiVEGEC", University of Montpellier, CNRS, IRD, Academic Hospital (CHU) of Montpellier, Montpellier, France.

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.
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http://dx.doi.org/10.1371/journal.ppat.1009666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8244899PMC
June 2021

A CLK1-KKT2 Signaling Pathway Regulating Kinetochore Assembly in Trypanosoma brucei.

mBio 2021 06 15;12(3):e0068721. Epub 2021 Jun 15.

York Biomedical Research Institute, Department of Biology, University of York, Heslington, United Kingdom.

During mitosis, eukaryotic cells must duplicate and separate their chromosomes in a precise and timely manner. The apparatus responsible for this is the kinetochore, which is a large protein structure that links chromosomal DNA and spindle microtubules to facilitate chromosome alignment and segregation. The proteins that comprise the kinetochore in the protozoan parasite Trypanosoma brucei are divergent from yeast and mammals and comprise an inner kinetochore complex composed of 24 distinct proteins (KKT1 to KKT23, KKT25) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2, and KKT3. We recently reported the identification of a specific trypanocidal inhibitor of T. brucei CLK1, an amidobenzimidazole, AB1. We now show that chemical inhibition of CLK1 with AB1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. Here, we show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 by CLK1 to be essential for KKT2 function and for kinetochore assembly. Additionally, KKT2 protein kinase activity is required for parasite proliferation but not for assembly of the inner kinetochore complex. We also show that chemical inhibition of the aurora kinase AUK1 does not affect CLK1 phosphorylation of KKT2, indicating that AUK1 and CLK1 are in separate regulatory pathways. We propose that CLK1 is part of a divergent signaling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. In eukaryotic cells, kinetochores are large protein complexes that link chromosomes to dynamic microtubule tips, ensuring proper segregation and genomic stability during cell division. Several proteins tightly coordinate kinetochore functions, including the protein kinase aurora kinase B. The kinetochore has diverse evolutionary roots. For example, trypanosomatids, single-cell parasitic protozoa that cause several neglected tropical diseases, possess a unique repertoire of kinetochore components whose regulation during the cell cycle remains unclear. Here, we shed light on trypanosomatid kinetochore biology by showing that the protein kinase CLK1 coordinates the assembly of the inner kinetochore by phosphorylating one of its components, KKT2, allowing the timely spatial recruitment of the rest of the kinetochore proteins and posterior attachment to microtubules in a process that is aurora kinase B independent.
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http://dx.doi.org/10.1128/mBio.00687-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262961PMC
June 2021

17-AAG-Induced Activation of the Autophagic Pathway in Is Associated with Parasite Death.

Microorganisms 2021 May 19;9(5). Epub 2021 May 19.

Laboratory of Parasite-Host Interaction and Epidemiology (LAIPHE), Gonçalo Moniz Institute-FIOCRUZ, Salvador 40296-710, Brazil.

The heat shock protein 90 (Hsp90) is thought to be an excellent drug target against parasitic diseases. The leishmanicidal effect of an Hsp90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), was previously demonstrated in both in vitro and in vivo models of cutaneous leishmaniasis. Parasite death was shown to occur in association with severe ultrastructural alterations in , suggestive of autophagic activation. We hypothesized that 17-AAG treatment results in the abnormal activation of the autophagic pathway, leading to parasite death. To elucidate this process, experiments were performed using transgenic parasites with GFP-ATG8-labelled autophagosomes. Mutant parasites treated with 17-AAG exhibited autophagosomes that did not entrap cargo, such as glycosomes, or fuse with lysosomes. ATG5-knockout (Δ) parasites, which are incapable of forming autophagosomes, demonstrated lower sensitivity to 17-AAG-induced cell death when compared to wild-type (WT) , further supporting the role of autophagy in 17-AAG-induced cell death. In addition, Hsp90 inhibition resulted in greater accumulation of ubiquitylated proteins in both WT- and Δ-treated parasites compared to controls, in the absence of proteasome overload. In conjunction with previously described ultrastructural alterations, herein we present evidence that treatment with 17-AAG causes abnormal activation of the autophagic pathway, resulting in the formation of immature autophagosomes and, consequently, incidental parasite death.
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http://dx.doi.org/10.3390/microorganisms9051089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158731PMC
May 2021

Divergent Cytochrome Maturation System in Kinetoplastid Protists.

mBio 2021 05 4;12(3). Epub 2021 May 4.

School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom

In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes and is catalyzed by either multisubunit cytochrome maturation system I or holocytochrome synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids and parasites, attach heme to mitochondrial -type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists. Cytochromes are essential proteins for respiratory and photosynthetic electron transfer. They are posttranslationally modified by covalent attachment of a heme cofactor. Kinetoplastids include important tropical disease-causing parasites; many aspects of their biology differ from other organisms, including their mammalian or plant hosts. Uniquely, kinetoplastids produce cytochromes with a type of heme attachment not seen elsewhere in nature and were the only cytochrome bearing taxa without evidence of protein machinery to attach heme to the apocytochrome. Using bioinformatics, biochemistry, and molecular genetics, we report how kinetoplastids make their cytochromes Unexpectedly, they use a highly diverged version of an enzyme used for heme-protein attachment in many eukaryotes. Mutations in the human enzyme lead to genetic disease. Identification of kinetoplastid cytochrome synthetase, thus, solves an evolutionary unknown, provides a possible target for antiparasite drug development, and an unanticipated resource for studying the mechanistic basis of a human genetic disease.
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http://dx.doi.org/10.1128/mBio.00166-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262978PMC
May 2021

Importance of Angomonas deanei KAP4 for kDNA arrangement, cell division and maintenance of the host-bacterium relationship.

Sci Rep 2021 04 28;11(1):9210. Epub 2021 Apr 28.

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, IBCCF, CCS, UFRJ, Cidade Universitária, Rio de Janeiro, RJ, CEP 21941-590, Brazil.

Angomonas deanei coevolves in a mutualistic relationship with a symbiotic bacterium that divides in synchronicity with other host cell structures. Trypanosomatid mitochondrial DNA is contained in the kinetoplast and is composed of thousands of interlocked DNA circles (kDNA). The arrangement of kDNA is related to the presence of histone-like proteins, known as KAPs (kinetoplast-associated proteins), that neutralize the negatively charged kDNA, thereby affecting the activity of mitochondrial enzymes involved in replication, transcription and repair. In this study, CRISPR-Cas9 was used to delete both alleles of the A. deanei KAP4 gene. Gene-deficient mutants exhibited high compaction of the kDNA network and displayed atypical phenotypes, such as the appearance of a filamentous symbionts, cells containing two nuclei and one kinetoplast, and division blocks. Treatment with cisplatin and UV showed that Δkap4 null mutants were not more sensitive to DNA damage and repair than wild-type cells. Notably, lesions caused by these genotoxic agents in the mitochondrial DNA could be repaired, suggesting that the kDNA in the kinetoplast of trypanosomatids has unique repair mechanisms. Taken together, our data indicate that although KAP4 is not an essential protein, it plays important roles in kDNA arrangement and replication, as well as in the maintenance of symbiosis.
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http://dx.doi.org/10.1038/s41598-021-88685-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080567PMC
April 2021

Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice.

Nat Commun 2021 02 16;12(1):1052. Epub 2021 Feb 16.

Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh, UK.

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.
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http://dx.doi.org/10.1038/s41467-021-21273-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887271PMC
February 2021

Chromosomal assembly of the nuclear genome of the endosymbiont-bearing trypanosomatid Angomonas deanei.

G3 (Bethesda) 2021 01;11(1)

Department of Biology, University of York, York YO10 5DD, UK.

Angomonas deanei is an endosymbiont-bearing trypanosomatid with several highly fragmented genome assemblies and unknown chromosome number. We present an assembly of the A. deanei nuclear genome based on Oxford Nanopore sequence that resolves into 29 complete or close-to-complete chromosomes. The assembly has several previously unknown special features; it has a supernumerary chromosome, a chromosome with a 340-kb inversion, and there is a translocation between two chromosomes. We also present an updated annotation of the chromosomal genome with 10,365 protein-coding genes, 59 transfer RNAs, 26 ribosomal RNAs, and 62 noncoding RNAs.
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http://dx.doi.org/10.1093/g3journal/jkaa018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022732PMC
January 2021

Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex.

PLoS Pathog 2020 10 27;16(10):e1008784. Epub 2020 Oct 27.

York Biomedical Research Institute and Department of Biology, University of York, United Kingdom.

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.
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http://dx.doi.org/10.1371/journal.ppat.1008784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647121PMC
October 2020

Role for the flagellum attachment zone in Leishmania anterior cell tip morphogenesis.

PLoS Pathog 2020 10 22;16(10):e1008494. Epub 2020 Oct 22.

Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, United Kingdom.

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.
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http://dx.doi.org/10.1371/journal.ppat.1008494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608989PMC
October 2020

Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors.

Nat Microbiol 2020 10 13;5(10):1207-1216. Epub 2020 Jul 13.

Novartis Institute for Tropical Diseases, Emeryville, CA, USA.

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei, the proteins that make up the kinetochore are highly divergent; the inner kinetochore comprises at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases-CLK1 (also known as KKT10), CLK2 (also known as KKT19), KKT2 and KKT3. Here, we report the identification and characterization of the amidobenzimidazoles (AB) protein kinase inhibitors that show nanomolar potency against T. brucei bloodstream forms, Leishmania and Trypanosoma cruzi. We performed target deconvolution analysis using a selection of 29 T. brucei mutants that overexpress known essential protein kinases, and identified CLK1 as a primary target. Biochemical studies and the co-crystal structure of CLK1 in complex with AB1 show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with Cys 215 in the ATP-binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell-cycle progression, leading to cell death. This research highlights a unique drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.
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http://dx.doi.org/10.1038/s41564-020-0745-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610364PMC
October 2020

Essential roles for deubiquitination in Leishmania life cycle progression.

PLoS Pathog 2020 06 16;16(6):e1008455. Epub 2020 Jun 16.

York Biomedical Research Institute and Department of Biology, University of York, United Kingdom.

The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.
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http://dx.doi.org/10.1371/journal.ppat.1008455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319358PMC
June 2020

In Vivo Bioluminescence Imaging to Assess Compound Efficacy Against Trypanosoma brucei.

Methods Mol Biol 2020 ;2116:801-817

York Biomedical Research Institute, Hull York Medical School, University of York, York, UK.

Traditional animal models for human African trypanosomiasis rely on detecting Trypanosoma brucei brucei parasitemia in the blood. Testing the efficacy of new compounds in these models is cumbersome because it may take several months after treatment before surviving parasites become detectable in the blood. To expedite compound screening, we have used a Trypanosoma brucei brucei GVR35 strain expressing red-shifted firefly luciferase to monitor parasite distribution in infected mice through noninvasive whole-body bioluminescence imaging. This protocol describes the infection and in vivo bioluminescence imaging of mice to assess compound efficacy against T. brucei during the two characteristic stages of disease, the hemolymphatic phase (stage 1) and the encephalitic or central nervous system phase (stage 2).
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http://dx.doi.org/10.1007/978-1-0716-0294-2_48DOI Listing
February 2021

New Drugs for Human African Trypanosomiasis: A Twenty First Century Success Story.

Trop Med Infect Dis 2020 Feb 19;5(1). Epub 2020 Feb 19.

Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK.

The twentieth century ended with human African trypanosomiasis (HAT) epidemics raging across many parts of Africa. Resistance to existing drugs was emerging, and many programs aiming to contain the disease had ground to a halt, given previous success against HAT and the competing priorities associated with other medical crises ravaging the continent. A series of dedicated interventions and the introduction of innovative routes to develop drugs, involving Product Development Partnerships, has led to a dramatic turnaround in the fight against HAT caused by . The World Health Organization have been able to optimize the use of existing tools to monitor and intervene in the disease. A promising new oral medication for stage 1 HAT, pafuramidine maleate, ultimately failed due to unforeseen toxicity issues. However, the clinical trials for this compound demonstrated the possibility of conducting such trials in the resource-poor settings of rural Africa. The Drugs for Neglected Disease initiative (DNDi), founded in 2003, has developed the first all oral therapy for both stage 1 and stage 2 HAT in fexinidazole. DNDi has also brought forward another oral therapy, acoziborole, potentially capable of curing both stage 1 and stage 2 disease in a single dosing. In this review article, we describe the remarkable successes in combating HAT through the twenty first century, bringing the prospect of the elimination of this disease into sight.
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http://dx.doi.org/10.3390/tropicalmed5010029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157223PMC
February 2020

Anti-Trypanosomal Proteasome Inhibitors Cure Hemolymphatic and Meningoencephalic Murine Infection Models of African Trypanosomiasis.

Trop Med Infect Dis 2020 Feb 17;5(1). Epub 2020 Feb 17.

Novartis Institute for Tropical Diseases, 5300 Chiron Way, Emeryville, CA 94608, USA.

Current anti-trypanosomal therapies suffer from problems of longer treatment duration, toxicity and inadequate efficacy, hence there is a need for safer, more efficacious and 'easy to use' oral drugs. Previously, we reported the discovery of the triazolopyrimidine (TP) class as selective kinetoplastid proteasome inhibitors with in vivo efficacy in mouse models of leishmaniasis, Chagas Disease and African trypanosomiasis (HAT). For the treatment of HAT, development compounds need to have excellent penetration to the brain to cure the meningoencephalic stage of the disease. Here we describe detailed biological and pharmacological characterization of triazolopyrimidine compounds in HAT specific assays. The TP class of compounds showed single digit nanomolar potency against and strains. These compounds are trypanocidal with concentration-time dependent kill and achieved relapse-free cure in vitro. Two compounds, GNF6702 and a new analog NITD689, showed favorable in vivo pharmacokinetics and significant brain penetration, which enabled oral dosing. They also achieved complete cure in both hemolymphatic (blood) and meningoencephalic (brain) infection of human African trypanosomiasis mouse models. Mode of action studies on this series confirmed the 20S proteasome as the target in . These proteasome inhibitors have the potential for further development into promising new treatment for human African trypanosomiasis.
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http://dx.doi.org/10.3390/tropicalmed5010028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157554PMC
February 2020

Tissue-specific transcriptomic changes associated with AmBisome® treatment of BALB/c mice with experimental visceral leishmaniasis.

Wellcome Open Res 2019 10;4:198. Epub 2019 Dec 10.

York Biomedical Research Institute, University of York, York, YO10 5DD, UK.

Liposomal amphotericin B (AmBisome®) as a treatment modality for visceral leishmaniasis (VL) has had significant impact on patient care in some but not all regions where VL is endemic.  As the mode of action of AmBisome® is poorly understood, we compared the tissue-specific transcriptome in drug-treated vs untreated mice with experimental VL.    BALB/c mice infected with ere treated with 8mg/kg AmBisome®, resulting in parasite elimination from liver and spleen over a 7-day period. At day 1 and day 7 post treatment (R +1 and R +7), transcriptomic profiling was performed on spleen and liver tissue from treated and untreated mice and uninfected mice.  BALB/c mice infected with BCG (an organism resistant to amphotericin B) were analysed to distinguish between direct effects of AmBisome® and those secondary to parasite death.   AmBisome® treatment lead to rapid parasitological clearance.  At R +1, spleen and liver displayed only 46 and 88 differentially expressed (DE) genes (P<0.05; 2-fold change) respectively. In liver, significant enrichment was seen for pathways associated with TNF, fatty acids and sterol biosynthesis.  At R +7, the number of DE genes was increased (spleen, 113; liver 400).  In spleen, these included many immune related genes known to be involved in anti-leishmanial immunity. In liver, changes in transcriptome were largely accounted for by loss of granulomas.   PCA analysis indicated that treatment only partially restored homeostasis.  Analysis of BCG-infected mice treated with AmBisome® revealed a pattern of immune modulation mainly targeting macrophage function.   Our data indicate that the tissue response to AmBisome® treatment varies between target organs and that full restoration of homeostasis is not achieved at parasitological cure.  The pathways required to restore homeostasis deserve fuller attention, to understand mechanisms associated with treatment failure and relapse and to promote more rapid restoration of immune competence.
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http://dx.doi.org/10.12688/wellcomeopenres.15606.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961418PMC
December 2019

Trypanosoma brucei ATR Links DNA Damage Signaling during Antigenic Variation with Regulation of RNA Polymerase I-Transcribed Surface Antigens.

Cell Rep 2020 01;30(3):836-851.e5

The Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity, and Inflammation, University of Glasgow, Sir Graeme Davis Building, 120 University Place, Glasgow G12 8TA, UK. Electronic address:

Trypanosoma brucei evades mammalian immunity by using recombination to switch its surface-expressed variant surface glycoprotein (VSG), while ensuring that only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have been implicated in the catalysis of VSG switching by recombination, not transcriptional control. How VSG switching is signaled to guide the appropriate reaction or to integrate switching into parasite growth is unknown. Here, we show that the loss of ATR, a DNA damage-signaling protein kinase, is lethal, causing nuclear genome instability and increased VSG switching through VSG-localized damage. Furthermore, ATR loss leads to the increased transcription of silent VSG expression sites and expression of mixed VSGs on the cell surface, effects that are associated with the altered localization of RNA polymerase I and VEX1. This work shows that ATR acts in antigenic variation both through DNA damage signaling and surface antigen expression control.
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http://dx.doi.org/10.1016/j.celrep.2019.12.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988115PMC
January 2020

Evaluation of clan CD C11 peptidase PNT1 and other Leishmania mexicana cysteine peptidases as potential drug targets.

Biochimie 2019 Nov 28;166:150-160. Epub 2019 Aug 28.

York Biomedical Research Institute, Department of Biology, University of York, Wentworth Way, Heslington, York, YO10 5DD, UK. Electronic address:

Leishmania mexicana is one of the causative agents of cutaneous leishmaniasis in humans. There is an urgent need to identify new drug targets to combat the disease. Cysteine peptidases play crucial role in pathogenicity and virulence in Leishmania spp. and are promising targets for developing new anti-leishmanial drugs. Genetic drug target validation has been performed on a number of cysteine peptidases, but others have yet to be characterized. We targeted 16 L. mexicana cysteine peptidases for gene deletion and tagging using CRISPR-Cas9 in order to identify essential genes and ascertain their cellular localization. Our analysis indicates that two clan CA, family C2 calpains (LmCAL27.1, LmCAL31.6) and clan CD, family C11 PNT1 are essential for survival in the promastigote stage. The other peptidases analysed, namely calpains LmCAL4.1, LmCAL25.1, and members of clan CA C51, C78, C85 and clan CP C97 were found to be non-essential. We generated a gene deletion mutant (Δpnt1) which was severely compromised in its cell growth and a conditional gene deletion mutant of PNT1 (Δpnt1: PNT1/Δ pnt1:HYG [SSU DiCRE]). PNT1 localizes to distinct foci on the flagellum and on the surface of the parasite. The conditional gene deletion of PNT1 induced blebs and pits on the cell surface and eventual cell death. Over-expression of PNT1, but not an active site mutant PNT1, was lethal, suggesting that active PNT1 peptidase is required for parasite survival. Overall, our data suggests that PNT1 is an essential gene and one of a number of cysteine peptidases that are potential drug targets in Leishmania.
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http://dx.doi.org/10.1016/j.biochi.2019.08.015DOI Listing
November 2019

Natural Resistance of to Miltefosine Contributes to the Low Efficacy in the Treatment of Visceral Leishmaniasis in Brazil.

Am J Trop Med Hyg 2019 10;101(4):789-794

Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Brazil.

In India, visceral leishmaniasis (VL) caused by has been successfully treated with miltefosine with a cure rate of > 90%. To assess the efficacy and safety of oral miltefosine against Brazilian VL, which is caused by , a phase II, open-label, dose-escalation study of oral miltefosine was conducted in children (aged 2-12 years) and adolescent-adults (aged 13-60 years). Definitive cure was assessed at a 6-month follow-up visit. The cure rate was only 42% (6 of 14 patients) with a recommended treatment of 28 days and 68% (19 of 28 patients) with an extended treatment of 42 days. The in vitro miltefosine susceptibility profile of intracellular amastigote stages of the pretreatment isolates, from cured and relapsed patients, showed a positive correlation with the clinical outcome. The IC mean (SEM) of eventual cures was 5.1 (0.4) µM, whereas that of eventual failures was 12.8 (1.9) µM ( = 0.0002). An IC above 8.0 µM predicts failure with 82% sensitivity and 100% specificity. The finding of amastigotes resistant to miltefosine in isolates from patients who eventually failed treatment strongly suggests natural resistance to this drug, as miltefosine had never been used in Brazil before this trial was carried out.
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http://dx.doi.org/10.4269/ajtmh.18-0949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779219PMC
October 2019

Neutrophil elastase promotes infection interferon-β.

FASEB J 2019 10 5;33(10):10794-10807. Epub 2019 Jul 5.

Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Visceral leishmaniasis is a deadly illness caused by that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of , named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF-α and IFN-β production, and parasite survival. We report poor intracellular growth of in macrophages from knockout mice for NE (), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of mice were reduced and accompanied by increased NO and decreased TGF-β production. Expression of ISP2 was not detected in , and a transgenic line constitutively expressing 2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected macrophages displayed significantly lower IFN-β mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN-β. We propose that utilizes the host NE-TLR machinery to induce IFN-β necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in is necessary to preserve the activation of the NE-TLR pathway.-Dias, B. T., Dias-Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari-Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes infection interferon-β.
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http://dx.doi.org/10.1096/fj.201900524RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766642PMC
October 2019

DiCre-Based Inducible Gene Expression.

Methods Mol Biol 2019 ;1971:225-235

Department of Biology, York Biomedical Research Institute, University of York, York, UK.

Induction of gene expression is a valuable approach for functional studies since it allows for the assessment of phenotypes without the need for clonal selection. Inducible expression can find a wide range of applications, from the study of essential genes to the characterization of overexpression of genes of interest. Here, we describe a detailed protocol for the use of the DiCre-based inducible gene expression system in Leishmania parasites. This is a tightly regulated induction system that allows for time- and dose-controlled expression of gene products, as rapidly as within 12 h.
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http://dx.doi.org/10.1007/978-1-4939-9210-2_11DOI Listing
August 2019

DiCre-Based Inducible Disruption of Leishmania Genes.

Methods Mol Biol 2019 ;1971:211-224

Department of Biology, York Biomedical Research Institute, University of York, York, UK.

Conditional gene deletion using dimerizable Cre recombinase (DiCre) is so far the best developed system for the phenotypic analysis of essential genes in Leishmania species. Here, we describe a protocol for the generation of a conditional gene deletion mutant and the subsequent inducible deletion of a target gene. Leishmania parasites are genetically modified to express two inactive Cre subunits (DiCre) and a single LoxP-flanked version of a target gene in a context where both endogenous copies of the gene have been deleted. Treatment with rapamycin dimerizes the DiCre subunits, resulting in activation of the enzyme, recombination between the LoxP sites, and excision of the LoxP-flanked target gene. Subsequent phenotyping allows for the analysis of essential gene function.
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http://dx.doi.org/10.1007/978-1-4939-9210-2_10DOI Listing
August 2019

flagellum attachment zone is critical for flagellar pocket shape, development in the sand fly, and pathogenicity in the host.

Proc Natl Acad Sci U S A 2019 03 8;116(13):6351-6360. Epub 2019 Mar 8.

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.

kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for pathogenicity. The FP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.
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http://dx.doi.org/10.1073/pnas.1812462116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442623PMC
March 2019

Tissue and host species-specific transcriptional changes in models of experimental visceral leishmaniasis.

Wellcome Open Res 2018 2;3:135. Epub 2019 Jan 2.

Centre for Immunology and Infection, University of York, York, YO10 5DD, UK.

: Human visceral leishmaniasis, caused by infection with or is a potentially fatal disease affecting 50,000-90,000 people yearly in 75 disease endemic countries, with more than 20,000 deaths reported. Experimental models of infection play a major role in understanding parasite biology, host-pathogen interaction, disease pathogenesis, and parasite transmission. In addition, they have an essential role in the identification and pre-clinical evaluation of new drugs and vaccines. However, our understanding of these models remains fragmentary. Although the immune response to infection in mice has been extensively characterized, transcriptomic analysis capturing the tissue-specific evolution of disease has yet to be reported. : We provide an analysis of the transcriptome of spleen, liver and peripheral blood of BALB/c mice infected with . Where possible, we compare our data in murine experimental visceral leishmaniasis with transcriptomic data in the public domain obtained from the study of -infected hamsters and patients with human visceral leishmaniasis. Digitised whole slide images showing the histopathology in spleen and liver are made available via a dedicated website, www.leishpathnet.org. Our analysis confirms marked tissue-specific alterations in the transcriptome of infected mice over time and identifies previously unrecognized parallels and differences between murine, hamster and human responses to infection. We show commonality of interferon-regulated genes whilst confirming a greater activation of type 2 immune pathways in infected hamsters compared to mice. Cytokine genes and genes encoding immune checkpoints were markedly tissue specific and dynamic in their expression, and pathways focused on non-immune cells reflected tissue specific immunopathology. Our data also addresses the value of measuring peripheral blood transcriptomics as a potential window into underlying systemic disease.  Our transcriptomic data, coupled with histopathologic analysis of the tissue response, provide an additional resource to underpin future mechanistic studies and to guide clinical research.
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http://dx.doi.org/10.12688/wellcomeopenres.14867.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6248268PMC
January 2019

TLR2 Signaling in Skin Nonhematopoietic Cells Induces Early Neutrophil Recruitment in Response to Leishmania major Infection.

J Invest Dermatol 2019 06 27;139(6):1318-1328. Epub 2018 Dec 27.

Department of Biochemistry, Faculty of Biology and Medicine, University of Lausanne, Epalinges, Switzerland; World Health Organization Immunology Research and Training Center, Faculty of Biology and Medicine, University of Lausanne, Epalinges, Switzerland. Electronic address:

Neutrophils are rapidly recruited to the mammalian skin in response to infection with the cutaneous Leishmania pathogen. The parasites use neutrophils to establish the disease; however, the signals driving early neutrophil recruitment are poorly known. Here, we identified the functional importance of TLR2 signaling in this process. Using bone marrow chimeras and immunohistology, we identified the TLR2-expressing cells involved in this early neutrophil recruitment to be of nonhematopoietic origin. Keratinocytes are damaged and briefly in contact with the parasites during infection. We show that TLR2 triggering by Leishmania major is required for their secretion of neutrophil-attracting chemokines. Furthermore, TLR2 triggering by L. major phosphoglycans is critical for neutrophil recruitment to negatively affect disease development, as shown by better control of lesion size and parasite load in Tlr2 compared with wild-type infected mice. Conversely, restoring early neutrophil presence in Tlr2 mice through injection of wild-type neutrophils or CXCL1 at the onset of infection resulted in delayed disease resolution comparable to that observed in wild-type mice. Taken together, our data show a crucial role for TLR2-expressing nonhematopoietic skin cells in the recruitment of the first wave of neutrophils after L. major infection, a process that delays disease control.
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http://dx.doi.org/10.1016/j.jid.2018.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024985PMC
June 2019

Drug Discovery for Kinetoplastid Diseases: Future Directions.

ACS Infect Dis 2019 02 13;5(2):152-157. Epub 2018 Dec 13.

Novartis Institute for Tropical Diseases (NITD) , 5300 Chiron Way , Emeryville , California 94608 , United States.

Kinetoplastid parasites have caused human disease for millennia. Significant achievements have been made toward developing new treatments for leishmaniasis (particularly on the Indian subcontinent) and for human African trypanosomiasis (HAT). Moreover, the sustained decrease in the incidence of HAT has made the prospect of elimination a tantalizing reality. Despite the gains, no new chemical or biological entities to treat kinetoplastid diseases have been registered in more than three decades, and more work is needed to discover safe and effective therapies for patients with Chagas disease and leishmaniasis. Advances in tools for drug discovery and novel insights into the biology of the host-parasite interaction may provide opportunities for accelerated progress. Here, we summarize the output from a gathering of scientists and physicians who met to discuss the current status and future directions in drug discovery for kinetoplastid diseases.
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http://dx.doi.org/10.1021/acsinfecdis.8b00298DOI Listing
February 2019

Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

mBio 2018 11 6;9(6). Epub 2018 Nov 6.

Unité de Parasitologiemoléculaire et Signalisation, Institut Pasteur, Paris, France

Protozoan parasites of the genus adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast growth. Together our data draw a complex picture of genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain. Protozoan parasites of the genus cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the , , and complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.
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http://dx.doi.org/10.1128/mBio.01399-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222132PMC
November 2018

Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9-1-1 complex in the maintenance of the plastic genome of Leishmania.

Nucleic Acids Res 2018 12;46(22):11835-11846

Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14049-900, SP, Brazil.

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9-RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome-wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryotes.
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http://dx.doi.org/10.1093/nar/gky1017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294564PMC
December 2018
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