Differences between Trypanosoma brucei gambiense groups 1 and 2 in their resistance to killing by trypanolytic factor 1

Background

The three sub-species of Trypanosoma brucei are important pathogens of sub-Saharan Africa. T. b. brucei is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. T. b. rhodesiense and T. b. gambiense are able to resist lysis by TLF. There are two distinct sub-groups of T. b. gambiense that differ genetically and by human serum resistance phenotypes. Group 1 T. b. gambiense have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 T. b. gambiense are resistant to TLF-1 due in-part to reduced uptake of TLF-1 mediated by reduced expression of the TLF-1 receptor (the haptoglobin-hemoglobin receptor (HpHbR)) gene. Here we investigate if this is also true in group 2 parasites.

Methodology

Isogenic resistant and sensitive group 2 T. b. gambiense were derived and compared to other T. brucei parasites. Both resistant and sensitive lines express the HpHbR gene at similar levels and internalized fluorescently labeled TLF-1 similar fashion to T. b. brucei. Both resistant and sensitive group 2, as well as group 1 T. b. gambiense, internalize recombinant APOL1, but only sensitive group 2 parasites are lysed.

Conclusions

Our data indicate that, despite group 1 T. b. gambiense avoiding TLF-1, it is resistant to the main lytic component, APOL1. Similarly group 2 T. b. gambiense is innately resistant to APOL1, which could be based on the same mechanism. However, group 2 T. b. gambiense variably displays this phenotype and expression does not appear to correlate with a change in expression site or expression of HpHbR. Thus there are differences in the mechanism of human serum resistance between T. b. gambiense groups 1 and 2.     

Trypanosoma brucei gambiense group 1 is distinguished by a unique amino acid substitution in the HpHb receptor implicated in human serum resistance

Trypanosoma brucei rhodesiense (Tbr) and T. b. gambiense (Tbg), causative agents of Human African Trypanosomiasis (sleeping sickness) in Africa, have evolved alternative mechanisms of resisting the activity of trypanosome lytic factors (TLFs), components of innate immunity in human serum that protect against infection by other African trypanosomes. In Tbr, lytic activity is suppressed by the Tbr-specific serum-resistance associated (SRA) protein. The mechanism in Tbg is less well understood but has been hypothesized to involve altered activity and expression of haptoglobin haemoglobin receptor (HpHbR). HpHbR has been shown to facilitate internalization of TLF-1 in T.b. brucei (Tbb), a member of the T. brucei species complex that is susceptible to human serum. By evaluating the genetic variability of HpHbR in a comprehensive geographical and taxonomic context, we show that a single substitution that replaces leucine with serine at position 210 is conserved in the most widespread form of Tbg (Tbg group 1) and not found in related taxa, which are either human serum susceptible (Tbb) or known to resist lysis via an alternative mechanism (Tbr and Tbg group 2). We hypothesize that this single substitution contributes to reduced uptake of TLF and thus may play a key role in conferring serum resistance to Tbg group 1. In contrast, similarity in HpHbR sequence among isolates of Tbg group 2 and Tbb/Tbr provides further evidence that human serum resistance in Tbg group 2 is likely independent of HpHbR function.     

The TgsGP Gene Is Essential for Resistance to Human Serum in Trypanosoma brucei gambiense

Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense.     

Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei

Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N′-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.     

A Primate APOL1 Variant That Kills Trypanosoma brucei gambiense

Humans are protected against infection from most African trypanosomes by lipoprotein complexes present in serum that contain the trypanolytic pore-forming protein, Apolipoprotein L1 (APOL1). The human-infective trypanosomes, Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West Africa have separately evolved mechanisms that allow them to resist APOL1-mediated lysis and cause human African trypanosomiasis, or sleeping sickness, in man. Recently, APOL1 variants were identified from a subset of Old World monkeys, that are able to lyse East African T. b. rhodesiense, by virtue of C-terminal polymorphisms in the APOL1 protein that hinder that parasite’s resistance mechanism. Such variants have been proposed as candidates for developing therapeutic alternatives to the unsatisfactory anti-trypanosomal drugs currently in use. Here we demonstrate the in vitro lytic ability of serum and purified recombinant protein of an APOL1 ortholog from the West African Guinea baboon (Papio papio), which is able to lyse examples of all sub-species of T. brucei including T. b. gambiense group 1 parasites, the most common agent of human African trypanosomiasis. The identification of a variant of APOL1 with trypanolytic ability for both human-infective T. brucei sub-species could be a candidate for universal APOL1-based therapeutic strategies, targeted against all pathogenic African trypanosomes.     

Cathepsin-L Can Resist Lysis by Human Serum in Trypanosoma brucei brucei

Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence ‘hits’ were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.     

Haptoglobin-related protein mediates trypanosome lytic factor binding to trypanosomes

Trypanosome lytic factor (TLF-1) is an unusual high density lipoprotein (HDL) found in human serum that is toxic to Trypanosoma brucei brucei and may be critical in preventing human infections by this parasite. TLF-1 is composed of four major apolipoproteins: apolipoprotein AI, apolipoprotein AII, paraoxonase, and the primate-specific haptoglobin-related protein (Hpr). Hpr is greater than 90% homologous to haptoglobin (Hp), an abundant acute phase serum protein. Killing of trypanosomes by TLF-1 requires cell surface binding, endocytosis, and subsequent lysosomal targeting. Low temperature binding studies reveal two receptors for TLF-1: one that is high affinity/low capacity (K(d) approximately 12 nm, 350 receptors per cell) and another that binds with low affinity/high capacity (K(d) approximately 1 microm, 60,000 receptors per cell). The low affinity binding is competed by nonlytic human HDL and is likely to be apolipoprotein AI-mediated. Purified human Hpr and human Hp bind to trypanosomes, are internalized, and are targeted to the lysosome. Furthermore, Hpr shows competition for TLF-1 binding, and a monoclonal antibody against Hpr prevents both TLF-1 uptake and trypanosome killing. Based on these results, we propose that Hpr mediates the high affinity binding of TLF-1 to T. b. brucei through a haptoglobin-like receptor.     

Phylogeography and taxonomy of Trypanosoma brucei

Background

Characterizing the evolutionary relationships and population structure of parasites can provide important insights into the epidemiology of human disease.

Methodology/Principal Findings

We examined 142 isolates of Trypanosoma brucei from all over sub-Saharan Africa using three distinct classes of genetic markers (kinetoplast CO1 sequence, nuclear SRA gene sequence, eight nuclear microsatellites) to clarify the evolutionary history of Trypanosoma brucei rhodesiense (Tbr) and T. b. gambiense (Tbg), the causative agents of human African trypanosomosis (sleeping sickness) in sub-Saharan Africa, and to examine the relationship between Tbr and the non-human infective parasite T. b. brucei (Tbb) in eastern and southern Africa. A Bayesian phylogeny and haplotype network based on CO1 sequences confirmed the taxonomic distinctness of Tbg group 1. Limited diversity combined with a wide geographical distribution suggested that this parasite has recently and rapidly colonized hosts across its current range. The more virulent Tbg group 2 exhibited diverse origins and was more closely allied with Tbb based on COI sequence and microsatellite genotypes. Four of five COI haplotypes obtained from Tbr were shared with isolates of Tbb, suggesting a close relationship between these taxa. Bayesian clustering of microsatellite genotypes confirmed this relationship and indicated that Tbr and Tbb isolates were often more closely related to each other than they were to other members of the same subspecies. Among isolates of Tbr for which data were available, we detected just two variants of the SRA gene responsible for human infectivity. These variants exhibited distinct geographical ranges, except in Tanzania, where both types co-occurred. Here, isolates possessing distinct SRA types were associated with identical COI haplotypes, but divergent microsatellite signatures.

Conclusions/Significance

Our data provide strong evidence that Tbr is only a phenotypic variant of Tbb; while relevant from a medical perspective, Tbr is not a reproductively isolated taxon. The wide distribution of the SRA gene across diverse trypanosome genetic backgrounds suggests that a large amount of genetic diversity is potentially available with which human-infective trypanosomes may respond to selective forces such as those exerted by drugs.     

Haptoglobin (HP) and Haptoglobin-related protein (HPR) copy number variation, natural selection, and trypanosomiasis

Haptoglobin, coded by the HP gene, is a plasma protein that acts as a scavenger for free heme, and haptoglobin-related protein (coded by the HPR gene) forms part of the trypanolytic factor TLF-1, together with apolipoprotein L1 (ApoL1). We analyse the polymorphic small intragenic duplication of the HP gene, with alleles Hp1 and Hp2, in 52 populations, and find no evidence for natural selection either from extended haplotype analysis or from correlation with pathogen richness matrices. Using fiber-FISH, the paralog ratio test, and array-CGH data, we also confirm that the HPR gene is copy number variable, with duplication of the whole HPR gene at polymorphic frequencies in west and central Africa, up to an allele frequency of 15 %. The geographical distribution of the HPR duplication allele overlaps the region where the pathogen causing chronic human African trypanosomiasis, Trypanosoma brucei gambiense, is endemic. The HPR duplication has occurred on one SNP haplotype, but there is no strong evidence of extended homozygosity, a characteristic of recent natural selection. The HPR duplication shows a slight, non-significant undertransmission to human African trypanosomiasis-affected children of unaffected parents in the Democratic Republic of Congo. However, taken together with alleles of APOL1, there is an overall significant undertransmission of putative protective alleles to human African trypanosomiasis-affected children.     

Trypanosome alternative oxidase, a potential therapeutic target for sleeping sickness, is conserved among Trypanosoma brucei subspecies

Trypanosoma brucei rhodesiense and T. b. gambiense are known causes of human African trypanosomiasis (HAT), or “sleeping sickness,” which is deadly if untreated. We previously reported that a specific inhibitor of trypanosome alternative oxidase (TAO), ascofuranone, quickly kills African trypanosomes in vitro and cures mice infected with another subspecies, non-human infective T. b. brucei, in in vivo trials. As an essential factor for trypanosome survival, TAO is a promising drug target due to the absence of alternative oxidases in the mammalian host. This study found TAO expression in HAT-causing trypanosomes; its amino acid sequence was identical to that in non-human infective T. b. brucei. The biochemical understanding of the TAO including its 3 dimensional structure and inhibitory compounds against TAO could therefore be applied to all three T. brucei subspecies in search of a cure for HAT. Our in vitro study using T. b. rhodesiense confirmed the effectiveness of ascofuranone (IC₅₀ value: 1 nM) to eliminate trypanosomes in human infective strain cultures.     

Aquaporin 2 Mutations in Trypanosoma brucei gambiense Field Isolates Correlate with Decreased Susceptibility to Pentamidine and Melarsoprol

The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.     

Epidemiology of Sleeping Sickness in Boffa (Guinea): Where Are the Trypanosomes?

Human African Trypanosomiasis (HAT) in West Africa is a lethal, neglected disease caused by Trypanosoma brucei gambiense transmitted by the tsetse Glossina palpalis gambiensis. Although the littoral part of Guinea with its typical mangrove habitat is the most prevalent area in West Africa, very few data are available on the epidemiology of the disease in such biotopes. As part of a HAT elimination project in Guinea, we carried a cross-sectional study of the distribution and abundance of people, livestock, tsetse and trypanosomes in the focus of Boffa. An exhaustive census of the human population was done, together with spatial mapping of the area. Entomological data were collected, a human medical survey was organized together with a survey in domestic animals. In total, 45 HAT cases were detected out of 14445 people who attended the survey, these latter representing 50.9% of the total population. Potential additional carriers of T. b. gambiense were also identified by the trypanolysis test (14 human subjects and two domestic animals). No trypanosome pathogenic to animals were found, neither in the 874 tsetse dissected nor in the 300 domestic animals sampled. High densities of tsetse were found in places frequented by humans, such as pirogue jetties, narrow mangrove channels and watering points. The prevalence of T. b. gambiense in humans, combined to low attendance of the population at risk to medical surveys, and to an additional proportion of human and animal carriers of T. b. gambiense who are not treated, highlights the limits of strategies targeting HAT patients only. In order to stop T. b. gambiense transmission, vector control should be added to the current strategy of case detection and treatment. Such an integrated strategy will combine medical surveillance to find and treat cases, and vector control activities to protect people from the infective bites of tsetse.     

Free-ranging pigs identified as a multi-reservoir of Trypanosoma brucei and Trypanosoma congolense in the Vavoua area,a historical sleeping sickness focus of Côte d’Ivoire

BackgroundThe existence of an animal reservoir of Trypanosoma brucei gambiense (T. b. gambiense), the agent of human African trypanosomiasis (HAT), may compromise the interruption of transmission targeted by World Health Organization. The aim of this study was to investigate the presence of trypanosomes in pigs and people in the Vavoua HAT historical focus where cases were still diagnosed in the early 2010’s.MethodsFor the human survey, we used the CATT, mini-anion exchange centrifugation technique and immune trypanolysis tests. For the animal survey, the buffy coat technique was also used as well as the PCR using Trypanosoma species specific, including the T. b. gambiense TgsGP detection using single round and nested PCRs, performed from animal blood samples and from strains isolated from subjects positive for parasitological investigations.ResultsNo HAT cases were detected among 345 people tested. A total of 167 pigs were investigated. Free-ranging pigs appeared significantly more infected than pigs in pen. Over 70% of free-ranging pigs were positive for CATT and parasitological investigations and 27–43% were positive to trypanolysis depending on the antigen used. T. brucei was the most prevalent species (57%) followed by T. congolense (24%). Blood sample extracted DNA of T. brucei positive subjects were negative to single round TgsGP PCR. However, 1/22 and 6/22 isolated strains were positive with single round and nested TgsGP PCRs, respectively.DiscussionFree-ranging pigs were identified as a multi-reservoir of T. brucei and/or T. congolense with mixed infections of different strains. This trypanosome diversity hinders the easy and direct detection of T. b. gambiense. We highlight the lack of tools to prove or exclude with certainty the presence of T. b. gambiense. This study once more highlights the need of technical improvements to explore the role of animals in the epidemiology of HAT.     

Diversity of trypanosomes in humans and cattle in the HAT foci Mandoul and Maro,Southern Chad—A matter of concern for zoonotic potential?

BackgroundAfrican trypanosomes are parasites mainly transmitted by tsetse flies. They cause trypanosomiasis in humans (HAT) and animals (AAT). In Chad, HAT/AAT are endemic. This study investigates the diversity and distribution of trypanosomes in Mandoul, an isolated area where a tsetse control campaign is ongoing, and Maro, an area bordering the Central African Republic (CAR) where the control had not started.Methods717 human and 540 cattle blood samples were collected, and 177 tsetse flies were caught. Trypanosomal DNA was detected using PCR targeting internal transcribed spacer 1 (ITS1) and glycosomal glyceraldehyde-3 phosphate dehydrogenase (gGAPDH), followed by amplicon sequencing.ResultsTrypanosomal DNA was identified in 14 human samples, 227 cattle samples, and in tsetse. Besides T. b. gambiense, T. congolense was detected in human in Maro. In Mandoul, DNA from an unknown Trypanosoma sp.-129-H was detected in a human with a history of a cured HAT infection and persisting symptoms. In cattle and tsetse samples from Maro, T. godfreyi and T. grayi were detected besides the known animal pathogens, in addition to T. theileri (in cattle) and T. simiae (in tsetse). Furthermore, in Maro, evidence for additional unknown trypanosomes was obtained in tsetse. In contrast, in the Mandoul area, only T. theileri, T. simiae, and T. vivax DNA was identified in cattle. Genetic diversity was most prominent in T. vivax and T. theileri.ConclusionTsetse control activities in Mandoul reduced the tsetse population and thus the pathogenic parasites. Nevertheless, T. theileri, T. vivax, and T. simiae are frequent in cattle suggesting transmission by other insect vectors. In contrast, in Maro, transhumance to/from Central African Republic and no tsetse control may have led to the high diversity and frequency of trypanosomes observed including HAT/AAT pathogenic species. Active HAT infections stress the need to enforce monitoring and control campaigns. Additionally, the diverse trypanosome species in humans and cattle indicate the necessity to investigate the infectivity of the unknown trypanosomes regarding their zoonotic potential. Finally, this study should be widened to other trypanosome hosts to capture the whole diversity of circulating trypanosomes.     

Trypanosoma brucei brucei and T. b. gambiense: Stumpy Bloodstream Forms Express More CB1 Epitope in Endosomes and Lysosomes than Slender Forms

CB1-glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1-glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1-glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1-glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle.     

Raman spectroscopic analysis of skin as a diagnostic tool for Human African Trypanosomiasis

Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT).     

TLTF in Cerebrospinal Fluid for Detection and Staging of T. b. gambiense Infection

Background

Trypanosome-derived lymphocyte triggering factor (TLTF) is a molecule released by African trypanosomes that interacts with the host immune system, resulting in increased levels of IFN-γ production.

Methodology/Principal findings

TLTF and anti-TLTF antibodies were assessed in sera and cerebrospinal fluid (CSF) from patients infected with Trypanosoma brucei gambiense (T. b. gambiense) in an attempt to identify alternative markers for diagnosis and stage determination of human African trypanosomiasis or sleeping sickness. Seventy-four serum and sixty-one CSF samples from patients with parasitologically confirmed infection and known disease stage along with 13 sera and CSF from uninfected controls were tested. In serum the levels of anti-TLTF antibodies were unrelated to the disease stage. In contrast, levels of anti-TLTF antibodies in CSF were higher in intermediate/late stages than in early stage disease patients. Specificity of the detected antibodies was assessed by inhibition of TLTF bioactivity as represented by its ability to induce IFN-γ production. Additionally, TLTF was detected in CSF from late stage patients by Western blotting with the anti-TLTF specific monoclonal antibody MO3.

Conclusions/Significance

These findings suggest a new possibility for disease diagnosis with focus on involvement of the CNS through detection of TLTF and anti-TLTF antibodies in the CSF.     

Human and Animal Trypanosomes in C?te d'Ivoire Form a Single Breeding Population

Background

Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and contributes to the related veterinary disease, Nagana. T. brucei is segregated into three subspecies based on host specificity, geography and pathology. T. b. brucei is limited to animals (excluding some primates) throughout sub-Saharan Africa and is non-infective to humans due to trypanolytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species. T. b. gambiense is the more prevalent human, causing over 97% of human cases. Study of T. b. gambiense is complicated in that there are two distinct groups delineated by genetics and phenotype. The relationships between the two groups and local T. b. brucei are unclear and may have a bearing on the evolution of the human infectivity traits.

Methodology/Principal Findings

A collection of sympatric T. brucei isolates from Côte d’Ivoire, consisting of T. b. brucei and both groups of T. b. gambiense have previously been categorized by isoenzymes, RFLPs and Blood Incubation Infectivity Tests. These samples were further characterized using the group 1 specific marker, TgSGP, and seven microsatellites. The relationships between the T. b. brucei and T. b. gambiense isolates were determined using principal components analysis, neighbor-joining phylogenetics, STRUCTURE, F_ST, Hardy-Weinberg equilibrium and linkage disequilibrium.

Conclusions/Significance

Group 1 T. b. gambiense form a clonal genetic group, distinct from group 2 and T. b. brucei, whereas group 2 T. b. gambiense are genetically indistinguishable from local T. b. brucei. There is strong evidence for mating within and between group 2 T. b. gambiense and T. b. brucei. We found no evidence to support the hypothesis that group 2 T. b. gambiense are hybrids of group 1 and T. b. brucei, suggesting that human infectivity has evolved independently in groups 1 and 2 T. b. gambiense.     

Identification of mimotopes with diagnostic potential for Trypanosoma brucei gambiense variant surface glycoproteins using human antibody fractions

Background

At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests.

Methodology/Principal Findings

A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79.

Conclusions/Significance

We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.