Publications by authors named "Álvaro Acosta-Serrano"

58 Publications

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes.

PLoS Negl Trop Dis 2021 Feb 2;15(2):e0009071. Epub 2021 Feb 2.

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.
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http://dx.doi.org/10.1371/journal.pntd.0009071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880456PMC
February 2021

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis.

PLoS Biol 2021 Jan 26;19(1):e3000796. Epub 2021 Jan 26.

Department of Vector Biology, Liverpool School of Tropical Medicine, United Kingdom.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.
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http://dx.doi.org/10.1371/journal.pbio.3000796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837477PMC
January 2021

Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays.

J Infect Dis 2020 10;222(9):1462-1467

Department of Vector Biology and Tropical Disease Biology, Centre for Neglected Tropical Disease, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

The scientific community has responded to the coronavirus disease 2019 (COVID-19) pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undertaken in high-containment laboratories; however, it is often desirable to work with samples at lower-containment levels. To facilitate the transfer of infectious samples from high-containment laboratories, we have tested methods commonly used to inactivate virus and prepare the sample for additional experiments. Incubation at 80°C, a range of detergents, Trizol reagents, and UV energies were successful at inactivating a high titer of SARS-CoV-2. Methanol and paraformaldehyde incubation of infected cells also inactivated the virus. These protocols can provide a framework for in-house inactivation of SARS-CoV-2 in other laboratories, ensuring the safe use of samples in lower-containment levels.
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http://dx.doi.org/10.1093/infdis/jiaa507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529010PMC
October 2020

Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis.

Sci Rep 2020 07 31;10(1):12903. Epub 2020 Jul 31.

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK.

During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host's immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with ManGlcNAc being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.
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http://dx.doi.org/10.1038/s41598-020-69753-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395719PMC
July 2020

Trypanosoma brucei colonizes the tsetse gut via an immature peritrophic matrix in the proventriculus.

Nat Microbiol 2020 07 20;5(7):909-916. Epub 2020 Apr 20.

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut. Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.
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http://dx.doi.org/10.1038/s41564-020-0707-zDOI Listing
July 2020

Association of Phlebotomus guggisbergi with Leishmania major and Leishmania tropica in a complex transmission setting for cutaneous leishmaniasis in Gilgil, Nakuru county, Kenya.

PLoS Negl Trop Dis 2019 10 18;13(10):e0007712. Epub 2019 Oct 18.

International Centre of Insect Physiology and Ecology, Nairobi, Kenya.

Background: Phlebotomus (Larroussius) guggisbergi is among the confirmed vectors for cutaneous leishmaniasis (CL) transmission in Kenya. This scarring and stigmatizing form of leishmaniasis accounts for over one million annual cases worldwide. Most recent CL epidemics in Kenya have been reported in Gilgil, Nakuru County, where the disease has become a public health issue. However, little is known about the factors that drive its transmission. Here, we sought to determine the occurrence, distribution and host blood feeding preference of the vectors, and to identify Leishmania species and infection rates in sandflies using molecular techniques. This information could lead to a better understanding of the disease transmission and improvement of control strategies in the area.

Methodology/ Principal Findings: An entomological survey of sandflies using CDC light traps was conducted for one week per month in April 2016, and in June and July 2017 from five villages of Gilgil, Nakuru county; Jaica, Sogonoi, Utut, Gitare and Njeru. Sandflies were identified to species level using morphological keys and further verified by PCR analysis of cytochrome c oxidase subunit I (COI) gene. Midguts of female sandflies found to harbour Leishmania were ruptured and the isolated parasites cultured in Novy-MacNeal-Nicolle (NNN) media overlaid with Schneider's insect media to identify the species. Leishmania parasite screening and identification in 198 randomly selected Phlebotomus females and parasite cultures was done by PCR-RFLP analysis of ITS1 gene, nested kDNA-PCR and real-time PCR-HRM followed by sequencing. Bloodmeal source identification was done by real-time PCR-HRM of the vertebrate cytochrome-b gene. A total of 729 sandflies (males: n = 310; females: n = 419) were collected from Utut (36.6%), Jaica (24.3%), Sogonoi (34.4%), Njeru (4.5%), and Gitare (0.1%). These were found to consist of nine species: three Phlebotomus spp. and six Sergentomyia spp. Ph. guggisbergi was the most abundant species (75.4%, n = 550) followed by Ph. saevus sensu lato (11.3%, n = 82). Sandfly species distribution across the villages was found to be significantly different (p<0.001) with Jaica recording the highest diversity. The overall Leishmania infection rate in sandflies was estimated at 7.07% (14/198). Infection rates in Ph. guggisbergi and Ph. saevus s.l. were 9.09% (12/132) and 3.57% (2/56) respectively. L. tropica was found to be the predominant parasite in Gilgil with an overall infection rate of 6.91% (13/188) in Ph. guggisbergi (n = 11) and Ph. saevus s.l. (n = 2) sandflies. However, PCR analysis also revealed L. major infection in one Ph. guggisbergi specimen. Bloodmeal analysis in the 74 blood-fed sandflies disclosed a diverse range of vertebrate hosts in Ph. guggisbergi bloodmeals, while Ph. saevus s.l. fed mainly on humans.

Conclusions/ Significance: The high infection rates of L. tropica and abundance of Ph. guggisbergi in this study confirms this sandfly as a vector of L. tropica in Kenya. Furthermore, isolation of live L. tropica parasites from Ph. saevus s.l. suggest that there are at least three potential vectors of this parasite species in Gilgil; Ph. guggisbergi, Ph. aculeatus and Ph. saevus s.l. Molecular identification of L. major infections in Ph. guggisbergi suggested this sandfly species as a potential permissive vector of L. major, which needs to be investigated further. Sandfly host preference analysis revealed the possibility of zoonotic transmissions of L. tropica in Gilgil since the main vector (Ph. guggisbergi) does not feed exclusively on humans but also other vertebrate species. Further investigations are needed to determine the potential role of these vertebrate species in L. tropica and L. major transmission in the area.
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http://dx.doi.org/10.1371/journal.pntd.0007712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821134PMC
October 2019

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

Genome Biol 2019 09 2;20(1):187. Epub 2019 Sep 2.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA.

Background: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity.

Results: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges.

Conclusions: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.
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http://dx.doi.org/10.1186/s13059-019-1768-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721284PMC
September 2019

Characterization of a novel glycosylated glutathione transferase of , closest relative of the human river blindness parasite.

Parasitology 2019 12 3;146(14):1773-1784. Epub 2019 Jul 3.

Liverpool School of Tropical Medicine, Liverpool, UK.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.
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http://dx.doi.org/10.1017/S0031182019000763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939172PMC
December 2019

Cutaneous leishmaniasis in north Lebanon: re-emergence of an important neglected tropical disease.

Trans R Soc Trop Med Hyg 2019 08;113(8):471-476

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, England, UK.

Background: Cutaneous leishmaniasis (CL) is the most prevalent neglected tropical disease among externally displaced people in the Middle East. In recent years, the Lebanese population has increased >30%, mainly due to a mass influx of Syrian migrants, thousands of them carrying CL, among other infectious diseases. Here we revisit the current CL prevalence among refugees in northern Lebanon.

Methods: This cohort study was conducted at the Al Bashaer Medical Center in north Lebanon between January and June 2017. A total of 48 randomly selected suspected CL patients were clinically diagnosed by dermatologists and samples were obtained for microscopic examination and molecular identification by polymerase chain reaction restriction fragment length polymorphism. The treatment response to antimonials was assessed each week and was followed for up 6 months.

Results: Leishmania tropica was the predominant species (91.7%) followed by Leishmania major (8.3%). Confirmed cases were treated with one to two courses of antimonials and healing was usually achieved after receiving a second course of treatment. Importantly, we show evidence of possible local CL transmission by indigenous sandflies in three separate patients who had no history of recent travel to Syria.

Conclusions: This highlights the urgent necessity to implement preventive disease strategies to avoid further dispersion of L. tropica CL in north Lebanon.
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http://dx.doi.org/10.1093/trstmh/trz030DOI Listing
August 2019

Old World cutaneous leishmaniasis treatment response varies depending on parasite species, geographical location and development of secondary infection.

Parasit Vectors 2019 May 2;12(1):195. Epub 2019 May 2.

Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool, UK.

Background: In the Kingdom of Saudi Arabia (KSA), Leishmania major and L. tropica are the main causative agents of Old World cutaneous leishmaniasis (CL). The national CL treatment regimen consists of topical 1% clotrimazole/2% fusidic acid cream followed by 1-2 courses of intralesional sodium stibogluconate (SSG); however, treatment efficacy is highly variable and the reasons for this are not well understood. In this study, we present a complete epidemiological map of CL and determined the efficacy of the standard CL treatment regime in several endemic regions of KSA.

Results: Overall, three quarters of patients in all CL-endemic areas studied responded satisfactorily to the current treatment regime, with the remaining requiring only an extra course of SSG. The majority of unresponsive cases were infected with L. tropica. Furthermore, the development of secondary infections (SI) around or within the CL lesion significantly favoured the treatment response of L. major patients but had no effect on L. tropica cases.

Conclusions: The response of CL patients to a national treatment protocol appears to depend on several factors, including Leishmania parasite species, geographical location and occurrences of SI. Our findings suggest there is a need to implement alternative CL treatment protocols based on these parameters.
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http://dx.doi.org/10.1186/s13071-019-3453-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498568PMC
May 2019

Cutaneous leishmaniasis and co-morbid major depressive disorder: A systematic review with burden estimates.

PLoS Negl Trop Dis 2019 02 25;13(2):e0007092. Epub 2019 Feb 25.

Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, United Kingdom.

Background: Major depressive disorder (MDD) associated with chronic neglected tropical diseases (NTDs) has been identified as a significant and overlooked contributor to overall disease burden. Cutaneous leishmaniasis (CL) is one of the most prevalent and stigmatising NTDs, with an incidence of around 1 million new cases of active CL infection annually. However, the characteristic residual scarring (inactive CL) following almost all cases of active CL has only recently been recognised as part of the CL disease spectrum due to its lasting psychosocial impact.

Methods And Findings: We performed a multi-language systematic review of the psychosocial impact of active and inactive CL. We estimated inactive CL (iCL) prevalence for the first time using reported WHO active CL (aCL) incidence data that were adjusted for life expectancy and underreporting. We then quantified the disability (YLD) burden of co-morbid MDD in CL using MDD disability weights at three severity levels. Overall, we identified 29 studies of CL psychological impact from 5 WHO regions, representing 11 of the 50 highest burden countries for CL. We conservatively calculated the disability burden of co-morbid MDD in CL to be 1.9 million YLDs, which equalled the overall (DALY) disease burden (assuming no excess mortality in depressed CL patients). Thus, upon inclusion of co-morbid MDD alone in both active and inactive CL, the DALY burden was seven times higher than the latest 2016 Global Burden of Disease study estimates, which notably omitted both psychological impact and inactive CL.

Conclusions: Failure to include co-morbid MDD and the lasting sequelae of chronic NTDs, as exemplified by CL, leads to large underestimates of overall disease burden.
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http://dx.doi.org/10.1371/journal.pntd.0007092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405174PMC
February 2019

Venezuela's humanitarian crisis, resurgence of vector-borne diseases, and implications for spillover in the region.

Lancet Infect Dis 2019 05 21;19(5):e149-e161. Epub 2019 Feb 21.

Instituto de Medicina Tropical, Universidad Central de Venezuela, Caracas, Venezuela.

In the past 5-10 years, Venezuela has faced a severe economic crisis, precipitated by political instability and declining oil revenue. Public health provision has been affected particularly. In this Review, we assess the impact of Venezuela's health-care crisis on vector-borne diseases, and the spillover into neighbouring countries. Between 2000 and 2015, Venezuela witnessed a 359% increase in malaria cases, followed by a 71% increase in 2017 (411 586 cases) compared with 2016 (240 613). Neighbouring countries, such as Brazil, have reported an escalating trend of imported malaria cases from Venezuela, from 1538 in 2014 to 3129 in 2017. In Venezuela, active Chagas disease transmission has been reported, with seroprevalence in children (<10 years), estimated to be as high as 12·5% in one community tested (n=64). Dengue incidence increased by more than four times between 1990 and 2016. The estimated incidence of chikungunya during its epidemic peak is 6975 cases per 100 000 people and that of Zika virus is 2057 cases per 100 000 people. The re-emergence of many vector-borne diseases represents a public health crisis in Venezuela and has the possibility of severely undermining regional disease elimination efforts. National, regional, and global authorities must take action to address these worsening epidemics and prevent their expansion beyond Venezuelan borders.
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http://dx.doi.org/10.1016/S1473-3099(18)30757-6DOI Listing
May 2019

Variant antigen repertoires in populations and experimental infections can be profiled from deep sequence data using universal protein motifs.

Genome Res 2018 09 13;28(9):1383-1394. Epub 2018 Jul 13.

Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, United Kingdom.

African trypanosomes are vector-borne hemoparasites of humans and animals. In the mammal, parasites evade the immune response through antigenic variation. Periodic switching of the variant surface glycoprotein (VSG) coat covering their cell surface allows sequential expansion of serologically distinct parasite clones. Trypanosome genomes contain many hundreds of genes, subject to rapid changes in nucleotide sequence, copy number, and chromosomal position. Thus, analyzing, or even quantifying, VSG diversity over space and time presents an enormous challenge to conventional techniques. Indeed, previous population genomic studies have overlooked this vital aspect of pathogen biology for lack of analytical tools. Here we present a method for analyzing population-scale VSG diversity in from deep sequencing data. Previously, we suggested that VSGs segregate into defined "phylotypes" that do not recombine. In our data set comprising 41 genome sequences from across Africa, these phylotypes are universal and exhaustive. Screening sequence contigs with diagnostic protein motifs accurately quantifies relative phylotype frequencies, providing a metric of VSG diversity, called the "variant antigen profile." We applied our metric to VSG expression in the tsetse fly, showing that certain, rare VSG phylotypes may be preferentially expressed in infective, metacyclic-stage parasites. Hence, variant antigen profiling accurately and rapidly determines the gene and transcript repertoire from sequence data, without need for manual curation or highly contiguous sequences. It offers a tractable approach to measuring VSG diversity across strains and during infections, which is imperative to understanding the host-parasite interaction at population and individual scales.
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http://dx.doi.org/10.1101/gr.234146.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120623PMC
September 2018

Anti-α-Gal antibodies detected by novel neoglycoproteins as a diagnostic tool for Old World cutaneous leishmaniasis caused by Leishmania major.

Parasitology 2018 11 14;145(13):1758-1764. Epub 2018 Jun 14.

Department of Parasitology,Liverpool School of Tropical Medicine,Pembroke Place,Liverpool L3 5QA,UK.

Outbreaks of Old World cutaneous leishmaniasis (CL) have significantly increased due to the conflicts in the Middle East, with most of the cases occurring in resource-limited areas such as refugee settlements. The standard methods of diagnosis include microscopy and parasite culture, which have several limitations. To address the growing need for a CL diagnostic that can be field applicable, we have identified five candidate neoglycoproteins (NGPs): Galα (NGP3B), Galα(1,3)Galα (NGP17B), Galα(1,3)Galβ (NGP9B), Galα(1,6)[Galα(1,2)]Galβ (NGP11B), and Galα(1,3)Galβ(1,4)Glcβ (NGP1B) that are differentially recognized in sera from individuals with Leishmania major infection as compared with sera from heterologous controls. These candidates contain terminal, non-reducing α-galactopyranosyl (α-Gal) residues, which are known potent immunogens to humans. Logistic regression models found that NGP3B retained the best diagnostic potential (area under the curve from receiver-operating characteristic curve = 0.8). Our data add to the growing body of work demonstrating the exploitability of the human anti-α-Gal response in CL diagnosis.
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http://dx.doi.org/10.1017/S0031182018000860DOI Listing
November 2018

Variations in the Peritrophic Matrix Composition of Heparan Sulphate from the Tsetse Fly, Glossina morsitans morsitans.

Pathogens 2018 Mar 19;7(1). Epub 2018 Mar 19.

Molecular & Structural Bioscience, School of Life Sciences, Keele University, Keele ST5 5BG, UK.

Tsetse flies are the principal insect vectors of African -sleeping sickness in humans and Nagana in cattle. One of the tsetse fly species, , is host to the parasite, , a major cause of African trypanosomiasis. Precise details of the life cycle have yet to be established, but the parasite life cycle involves crossing the insect peritrophic matrix (PM). The PM consists of the polysaccharide chitin, several hundred proteins, and both glycosamino- and galactosaminoglycan (GAG) polysaccharides. Owing to the technical challenges of detecting small amounts of GAG polysaccharides, their conclusive identification and composition have not been possible until now. Following removal of PMs from the insects and the application of heparinases (bacterial lyase enzymes that are specific for heparan sulphate (HS) GAG polysaccharides), dot blots with a HS-specific antibody showed heparan sulphate proteoglycans (HSPGs) to be present, consistent with genome analysis, as well as the likely expression of the HSPGs syndecan and perlecan. Exhaustive HS digestion with heparinases, fluorescent labeling of the resulting disaccharides with BODIPY fluorophore, and separation by strong anion exchange chromatography then demonstrated the presence of HS for the first time and provided the disaccharide composition. There were no significant differences in the type of disaccharide species present between genders or between ages (24 vs 48 h post emergence), although the HS from female flies was more heavily sulphated overall. Significant differences, which may relate to differences in infection between genders or ages, were evident, however, in overall levels of 2--sulphation between sexes and, for females, between 24 and 48 h post-emergence, implying a change in expression or activity for the 2--sulphotransferase enzyme. The presence of significant quantities of disaccharides containing the monosaccharide GlcNAc6S contrasts with previous findings in and suggests subtle differences in HS fine structure between species of the .
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http://dx.doi.org/10.3390/pathogens7010032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874758PMC
March 2018

An α-Gal-containing neoglycoprotein-based vaccine partially protects against murine cutaneous leishmaniasis caused by Leishmania major.

PLoS Negl Trop Dis 2017 Oct 25;11(10):e0006039. Epub 2017 Oct 25.

Department of Biological Sciences, Border Biomedical Research Center, the University of Texas at El Paso, El Paso, Texas, United States of America.

Background: Protozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniases, which affect millions of people worldwide. Cutaneous leishmaniasis (CL), caused by L. major, is one the most common forms of the disease in the Old World. There is no preventive or therapeutic human vaccine available for L. major CL, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic interventions against the disease are necessary. Terminal, non-reducing, and linear α-galactopyranosyl (α-Gal) epitopes are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids. The absence of these α-Gal epitopes in human cells makes these glycans highly immunogenic and thus potential targets for vaccine development against CL.

Methodology/principal Findings: Here, we evaluated three neoglycoproteins (NGPs), containing synthetic α-Gal epitopes covalently attached to bovine serum albumin (BSA), as vaccine candidates against L. major, using α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice. These transgenic mice, similarly to humans, do not express nonreducing, linear α-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-α-Gal antibodies. We observed that Galα(1,6)Galβ-BSA (NGP5B), but not Galα(1,4)Galβ-BSA (NGP12B) or Galα(1,3)Galα-BSA (NGP17B), was able to significantly reduce the size of footpad lesions by 96% in comparison to control groups. Furthermore, we observed a robust humoral and cellular immune response with production of high levels of protective lytic anti-α-Gal antibodies and induction of Th1 cytokines.

Conclusions/significance: We propose that NGP5B is an attractive candidate for the study of potential synthetic α-Gal-neoglycoprotein-based vaccines against L. major infection.
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http://dx.doi.org/10.1371/journal.pntd.0006039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673233PMC
October 2017

Venezuela and its rising vector-borne neglected diseases.

PLoS Negl Trop Dis 2017 Jun 29;11(6):e0005423. Epub 2017 Jun 29.

Laboratorio de Biología de Vectores y Parásitos, Instituto de Zoología y Ecología Tropical, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela.

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http://dx.doi.org/10.1371/journal.pntd.0005423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490936PMC
June 2017

Identification of potential protein partners that bind to the variant surface glycoprotein in Trypanosoma equiperdum.

Parasitology 2017 Jun 10;144(7):923-936. Epub 2017 Feb 10.

Departamento de Biología Celular,Universidad Simón Bolívar,Caracas,Venezuela.

Trypanosoma equiperdum possesses a dense coat of a variant surface glycoprotein (VSG) that is used to evade the host immune response by a process known as antigenic variation. Soluble and membrane forms of the predominant VSG from the Venezuelan T. equiperdum TeAp-N/D1 strain (sVSG and mVSG, respectively) were purified to homogeneity; and antibodies against sVSG and mVSG were raised, isolated, and employed to produce anti-idiotypic antibodies that structurally mimic the VSG surface. Prospective VSG-binding partners were initially detected by far-Western blots, and then by immunoblots using the generated anti-idiotypic antibodies. Polypeptides of ~80 and 55 kDa were isolated when anti-idiotypic antibodies-Sepharose affinity matrixes were used as baits. Mass spectrometry sequencing yielded hits with various proteins from Trypanosoma brucei such as heat-shock protein 70, tryparedoxin peroxidase, VSG variants, expression site associated gene product 6, and two hypothetical proteins. In addition, a possible interaction with a protein homologous to the glutamic acid/alanine-rich protein from Trypanosoma congolense was also found. These results indicate that the corresponding orthologous gene products are candidates for VSG-interacting proteins in T. equiperdum.
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http://dx.doi.org/10.1017/S003118201700004XDOI Listing
June 2017

Proline Metabolism is Essential for Trypanosoma brucei brucei Survival in the Tsetse Vector.

PLoS Pathog 2017 01 23;13(1):e1006158. Epub 2017 Jan 23.

Laboratory of Biochemistry of Tryps - LaBTryps, Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly's midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut.
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http://dx.doi.org/10.1371/journal.ppat.1006158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289646PMC
January 2017

RFT1 Protein Affects Glycosylphosphatidylinositol (GPI) Anchor Glycosylation.

J Biol Chem 2017 01 7;292(3):1103-1111. Epub 2016 Dec 7.

From the Institute of Biochemistry and Molecular Medicine and

The membrane protein RFT1 is essential for normal protein N-glycosylation, but its precise function is not known. RFT1 was originally proposed to translocate the glycolipid ManGlcNAc-PP-dolichol (needed to synthesize N-glycan precursors) across the endoplasmic reticulum membrane, but subsequent studies showed that it does not play a direct role in transport. In contrast to the situation in yeast, RFT1 is not essential for growth of the parasitic protozoan Trypanosoma brucei, enabling the study of its function in a null background. We now report that lack of T. brucei RFT1 (TbRFT1) not only affects protein N-glycosylation but also glycosylphosphatidylinositol (GPI) anchor side-chain modification. Analysis by immunoblotting, metabolic labeling, and mass spectrometry demonstrated that the major GPI-anchored proteins of T. brucei procyclic forms have truncated GPI anchor side chains in TbRFT1 null parasites when compared with wild-type cells, a defect that is corrected by expressing a tagged copy of TbRFT1 in the null background. In vivo and in vitro labeling experiments using radiolabeled GPI precursors showed that GPI underglycosylation was not the result of decreased formation of the GPI precursor lipid or defective galactosylation of GPI intermediates in the endoplasmic reticulum, but rather due to modifications that are expected to occur in the Golgi apparatus. Unexpectedly, immunofluorescence microscopy localized TbRFT1 to both the endoplasmic reticulum and the Golgi, consistent with the proposal that TbRFT1 plays a direct or indirect role in GPI anchor glycosylation in the Golgi apparatus. Our results implicate RFT1 in a wider range of glycosylation processes than previously appreciated.
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http://dx.doi.org/10.1074/jbc.M116.758367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247644PMC
January 2017

Skin deep.

Elife 2016 10 14;5. Epub 2016 Oct 14.

Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

Trypanosome parasites are hiding in human skin, a discovery that may undermine efforts to eliminate sleeping sickness by 2020.
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http://dx.doi.org/10.7554/eLife.21506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065310PMC
October 2016

Old World Cutaneous Leishmaniasis and Refugee Crises in the Middle East and North Africa.

PLoS Negl Trop Dis 2016 05 26;10(5):e0004545. Epub 2016 May 26.

Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, England, United Kingdom.

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http://dx.doi.org/10.1371/journal.pntd.0004545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882064PMC
May 2016

Cutaneous Leishmaniasis: The Truth about the 'Flesh-Eating Disease' in Syria.

Trends Parasitol 2016 06 19;32(6):432-435. Epub 2016 Apr 19.

Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK; Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK. Electronic address:

Recent news headlines claimed that corpses thrown into Syrian streets are causing cutaneous leishmaniasis (CL) outbreaks. However, leishmaniasis is only transmitted by blood-feeding sandflies, not through human remains. High CL prevalence in Syria may instead be attributed to the absence of disease control programs due to the disruption of health services.
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http://dx.doi.org/10.1016/j.pt.2016.03.003DOI Listing
June 2016

Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India.

Parasit Vectors 2016 Jan 27;9:25. Epub 2016 Jan 27.

Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.

Visceral Leishmaniasis (VL) is a neglected vector-borne disease. In India, it is transmitted to humans by Leishmania donovani-infected Phlebotomus argentipes sand flies. In 2005, VL was targeted for elimination by the governments of India, Nepal and Bangladesh by 2015. The elimination strategy consists of rapid case detection, treatment of VL cases and vector control using indoor residual spraying (IRS). However, to achieve sustained elimination of VL, an appropriate post elimination surveillance programme should be designed, and crucial knowledge gaps in vector bionomics, human infection and transmission need to be addressed. This review examines the outstanding knowledge gaps, specifically in the context of Bihar State, India.The knowledge gaps in vector bionomics that will be of immediate benefit to current control operations include better estimates of human biting rates and natural infection rates of P. argentipes, with L. donovani, and how these vary spatially, temporally and in response to IRS. The relative importance of indoor and outdoor transmission, and how P. argentipes disperse, are also unknown. With respect to human transmission it is important to use a range of diagnostic tools to distinguish individuals in endemic communities into those who: 1) are to going to progress to clinical VL, 2) are immune/refractory to infection and 3) have had past exposure to sand flies.It is crucial to keep in mind that close to elimination, and post-elimination, VL cases will become infrequent, so it is vital to define what the surveillance programme should target and how it should be designed to prevent resurgence. Therefore, a better understanding of the transmission dynamics of VL, in particular of how rates of infection in humans and sand flies vary as functions of each other, is required to guide VL elimination efforts and ensure sustained elimination in the Indian subcontinent. By collecting contemporary entomological and human data in the same geographical locations, more precise epidemiological models can be produced. The suite of data collected can also be used to inform the national programme if supplementary vector control tools, in addition to IRS, are required to address the issues of people sleeping outside.
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http://dx.doi.org/10.1186/s13071-016-1309-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729074PMC
January 2016

Kinetoplastid Phylogenomics Reveals the Evolutionary Innovations Associated with the Origins of Parasitism.

Curr Biol 2016 Jan 24;26(2):161-172. Epub 2015 Dec 24.

Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

The evolution of parasitism is a recurrent event in the history of life and a core problem in evolutionary biology. Trypanosomatids are important parasites and include the human pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., which in humans cause African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. Genome comparison between trypanosomatids reveals that these parasites have evolved specialized cell-surface protein families, overlaid on a well-conserved cell template. Understanding how these features evolved and which ones are specifically associated with parasitism requires comparison with related non-parasites. We have produced genome sequences for Bodo saltans, the closest known non-parasitic relative of trypanosomatids, and a second bodonid, Trypanoplasma borreli. Here we show how genomic reduction and innovation contributed to the character of trypanosomatid genomes. We show that gene loss has "streamlined" trypanosomatid genomes, particularly with respect to macromolecular degradation and ion transport, but consistent with a widespread loss of functional redundancy, while adaptive radiations of gene families involved in membrane function provide the principal innovations in trypanosomatid evolution. Gene gain and loss continued during trypanosomatid diversification, resulting in the asymmetric assortment of ancestral characters such as peptidases between Trypanosoma and Leishmania, genomic differences that were subsequently amplified by lineage-specific innovations after divergence. Finally, we show how species-specific, cell-surface gene families (DGF-1 and PSA) with no apparent structural similarity are independent derivations of a common ancestral form, which we call "bodonin." This new evidence defines the parasitic innovations of trypanosomatid genomes, revealing how a free-living phagotroph became adapted to exploiting hostile host environments.
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http://dx.doi.org/10.1016/j.cub.2015.11.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728078PMC
January 2016

Parasite Glycobiology: A Bittersweet Symphony.

PLoS Pathog 2015 12;11(11):e1005169. Epub 2015 Nov 12.

ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.

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http://dx.doi.org/10.1371/journal.ppat.1005169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642930PMC
April 2016

Characterization of an African trypanosome mutant refractory to lectin-induced death.

Biochem Biophys Rep 2015 Dec;4:33-38

Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University.

Incubation of African trypanosomes with the lectin concanavalin A (conA) leads to alteration in cellular DNA content, DNA degradation, and surface membrane blebbing. Here, we report the generation and characterization of a conA-refractory line. These insect stage parasites were resistant to conA killing, with a mediun lethal dose at least 50-fold greater than the parental line. Fluorescence-based experiments revealed that the resistant cells bound less lectin when compared to the parental line. Western blotting and mass spectrometry confirmed that the resistant line lacked an N-glycan required for conA binding on the cellular receptors, EP procyclin proteins. The failure to N-glycosylate the EP procyclins was not the consequence of altered N-glycan precursor biosynthesis, as another glycosylated protein (Fla1p) was normally modified. These findings support the likelihood that resistance to conA was a consequence of failure to bind the lectin trigger.
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http://dx.doi.org/10.1016/j.bbrep.2015.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574508PMC
December 2015