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.     

Genetic analysis of the human infective trypanosome <Emphasis Type="Italic">Trypanosoma brucei gambiense</Emphasis>: chromosomal segregation,crossing over,and the construction of a genetic map

Background  

Trypanosoma brucei is the causative agent of human sleeping sickness and animal trypanosomiasis in sub-Saharan Africa, and it has been subdivided into three subspecies: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause sleeping sickness in humans, and the nonhuman infective Trypanosoma brucei brucei. T. b. gambiense is the most clinically relevant subspecies, being responsible for more than 90% of all trypanosomal disease in humans. The genome sequence is now available, and a Mendelian genetic system has been demonstrated in T. brucei, facilitating genetic analysis in this diploid protozoan parasite. As an essential step toward identifying loci that determine important traits in the human-infective subspecies, we report the construction of a high-resolution genetic map of the STIB 386 strain of T. b. gambiense.     

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.     

African trypanosomes: intracellular trafficking of host defense molecules

Trypanosoma brucei brucei is the causative agent of Nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL), containing apolipoprotein A-I (APOA1), apolipoprotein L-I (APOL1) and haptoglobin-related protein (HPR) provides this innate protection against T. b. brucei infection. Both HPR and APOL1 are cytotoxic to T. b. brucei but their specific activities for killing increase several hundred-fold when assembled in the same HDL. This HDL is called trypanosome lytic factor (TLF) and kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosome lytic factor is activated in the acidic lysosome and facilitates lysosomal membrane disruption. Lysosomal localization is necessary for T. b. brucei killing by TLF. Trypanosoma brucei rhodesiense, which is indistinguishable from T. b. brucei, is resistant to TLF killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a human serum resistance associated protein (SRA) that is able to ablate TLF killing. When T. b. brucei is transfected with the SRA gene it becomes highly resistant to TLF and human serum. In the SRA transfected cells, intracellular trafficking of TLF is altered and TLF mainly localizes to a subset of SRA containing cytoplasmic vesicles but not to the lysosome. These findings indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine susceptibility.     

C-Terminal Mutants of Apolipoprotein L-I Efficiently Kill Both Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Apolipoprotein L-I (apoL1) is a human-specific serum protein that kills Trypanosoma brucei through ionic pore formation in endosomal membranes of the parasite. The T. brucei subspecies rhodesiense and gambiense resist this lytic activity and can infect humans, causing sleeping sickness. In the case of T. b. rhodesiense, resistance to lysis involves interaction of the Serum Resistance-Associated (SRA) protein with the C-terminal helix of apoL1. We undertook a mutational and deletional analysis of the C-terminal helix of apoL1 to investigate the linkage between interaction with SRA and lytic potential for different T. brucei subspecies. We confirm that the C-terminal helix is the SRA-interacting domain. Although in E. coli this domain was dispensable for ionic pore-forming activity, its interaction with SRA resulted in inhibition of this activity. Different mutations affecting the C-terminal helix reduced the interaction of apoL1 with SRA. However, mutants in the L370-L392 leucine zipper also lost in vitro trypanolytic activity. Truncating and/or mutating the C-terminal sequence of human apoL1 like that of apoL1-like sequences of Papio anubis resulted in both loss of interaction with SRA and acquired ability to efficiently kill human serum-resistant T. b. rhodesiense parasites, in vitro as well as in transgenic mice. These findings demonstrate that SRA interaction with the C-terminal helix of apoL1 inhibits its pore-forming activity and determines resistance of T. b. rhodesiense to human serum. In addition, they provide a possible explanation for the ability of Papio serum to kill T. b. rhodesiense, and offer a perspective to generate transgenic cattle resistant to both T. b. brucei and T. b. rhodesiense.     

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.     

Endocytosis of a cytotoxic human high density lipoprotein results in disruption of acidic intracellular vesicles and subsequent killing of African trypanosomes

The host range of Trypanosoma brucei brucei is restricted by the cytolytic effects of human serum high-density lipoprotein (HDL). The lytic activity is caused by a minor subclass of human serum HDL called trypanosome lytic factor (TLF). TLF binds in the flagellar pocket to specific TLF-binding sites. Internalization and localization of TLF to a population of endocytic vesicles, and ultimately large lysosome-like vesicles, precedes lysis of T. b. brucei. The membranes of these large vesicles are disrupted by the accumulation of TLF particles. Inhibitor studies with lysosomotropic amines have shown these large vesicles to be acidic in nature and that prevention of their rupture spares the cells from TLF-mediated lysis. Furthermore, leupeptin inhibition suggests that a thioprotease may be involved in the mechanism of TLF- mediated lysis of T. b. brucei. Based on these results, we propose a lytic mechanism involving cell surface binding, endocytosis and lysosomal targeting. This is followed by lysosomal disruption and subsequent autodigestion of the cell.     

Serum resistance-associated protein blocks lysosomal targeting of trypanosome lytic factor in Trypanosoma brucei

Trypanosoma brucei brucei is the causative agent of nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I (apoA-I), apolipoprotein L-I (apoL-I), and haptoglobin-related protein (Hpr) provides this innate protection against T. b. brucei infection. This HDL subfraction, called trypanosome lytic factor (TLF), kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosoma brucei rhodesiense, which is morphologically and physiologically indistinguishable from T. b. brucei, is resistant to TLF-mediated killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a resistance-associated protein (SRA) that is able to ablate TLF killing. To examine the mechanism of TLF resistance, we transfected T. b. brucei with an epitope-tagged SRA gene. Transfected T. b. brucei expressed SRA mRNA at levels comparable to those in T. b. rhodesiense and was highly resistant to TLF. In the SRA-transfected cells, intracellular trafficking of TLF was altered, with TLF being mainly localized to a subset of SRA-containing cytoplasmic vesicles but not to the lysosome. These results indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine the organism's susceptibility to TLF.     

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.     

The trypanolytic factor of human serum: many ways to enter the parasite,a single way to kill

Humans have developed a particular innate immunity system against African trypanosomes, and only two Trypanosoma brucei clones (T. b. gambiense, T. b. rhodesiense) can resist this defence and cause sleeping sickness. The main players of this immunity are the primate‐specific apolipoprotein L‐I (apoL1) and haptoglobin‐related protein (Hpr). These proteins are both associated with two serum complexes, a minor subfraction of HDLs and an IgM/apolipoprotein A‐I (apoA1) complex, respectively, termed trypanosome lytic factor (TLF) 1 and TLF2. Although the two complexes appear to lyse trypanosomes by the same mechanism, they enter the parasite through various modes of uptake. In case of TLF1 one uptake process was characterized. When released in the circulation, haemoglobin (Hb) binds to Hpr, hence to TLF1. In turn the TLF1–Hpr–Hb complex binds to the trypanosome haptoglobin (Hp)–Hb receptor, whose original function is to ensure haem uptake for optimal growth of the parasite. This binding triggers efficient uptake of TLF1 and subsequent trypanosome lysis. While Hpr is involved as TLF ligand, the lytic activity is due to apoL1, a Bcl‐2‐like pore‐forming protein. We discuss the in vivo relevance of this uptake pathway in the context of other potentially redundant delivery routes.     

The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense

Targett G. A. T. and Wilson V. C. L. C. 1973. The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense. International Journal for Parasitology, 3: 5–11. A simple test for distinguishing between the morphologically identical subspecies Trypanosoma brucei rhodesiense, which is infective to man, and T. brucei brucei, which by definition is not, has been described. This test, the blood incubation infectivity test (BIIT), is based on absolute differences in the infectivity to rats of the subspecies after exposure to human blood, and was applied to strains which are preserved in the laboratory as stabilates. Five T. brucei brucei strains were BIIT negative since their infectivity was destroyed by incubation in normal human blood but only five of the nine T. brucei rhodesiense strains tested were consistently BIIT positive. The other four gave equivocal results, indicating that the resistance of T. brucei rhodesiense strains to the trypanocidal effect of human blood can change, probably as a result of maintenance in the laboratory.     

Development of multiplex serological assay for the detection of human African trypanosomiasis

Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance.We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance.     

Purine metabolism in the bloodstream forms of trypanosoma gambiense and Trypanosoma rhodesiense

Bloodstream forms of Trypanosoma brucie gambiense and Trypanosoma brucei rhodesiense are incapable of de novo purine synthesis. Purine bases are converted directly to ribonucleotides and with the exception of guanine, are stable. Guanine is incorporated directly into ribonucleotides and also deaminated to xanthine. Purine ribonucleosides are hydrolyzed rapidly; these reactions may limit their incorporation since purine bases label the nucleotide pools more efficiently than do ribonucleosides. The apparent order of salvage efficiency for ribonucleosides is adenosine>inosine>guanosine>xanthosine for both organisms. T. b. gambiense salvages purine bases in the same order, while T. b. rhodesiense salvages purine bases in the order hypoxanthine>adenine>guanine>xanthine.     

Identification of Compounds with Anti-Proliferative Activity against Trypanosoma brucei brucei Strain 427 by a Whole Cell Viability Based HTS Campaign

Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC₅₀ value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC₅₀ values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill.     

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.     

Trypanosoma brucei gambiense: HMI-9 medium containing methylcellulose and human serum supports the continuous axenic in vitro propagation of the bloodstream form

Trypanosoma brucei (T.b.) gambiense causes the chronic form of human African trypanosomiasis or sleeping sickness. One of the major problems with studying T.b. gambiense is the difficulty to isolate it from its original host and the difficult adaptation to in vivo and in vitro mass propagation. The objective of this study was to evaluate if an established method for axenic culture of pleomorphic bloodstream form T.b. brucei strains, based on methylcellulose containing HMI-9 medium, also facilitated the continuous in vitro propagation of other bloodstream form Trypanozoon strains, in particular of T.b. gambiense. Bloodstream form trypanosomes from one T.b. brucei, two T.b. rhodesiense, one T. evansi and seven T.b. gambiense strains were isolated from mouse blood and each was concurrently cultivated in liquid and methylcellulose-containing HMI-9 based medium, either with or without additional human serum supplementation, for over 10 consecutive sub passages. Although HMI-9 based medium supplemented with 1.1% (w/v) methylcellulose supported the continuous cultivation of all non-gambiense strains better than liquid media could, the in vitro cultivation of all gambiense strains was only achieved in HMI-9 based medium containing 1.1% (w/v) methylcellulose, 15% (v/v) fetal calf serum and 5% (v/v) heat-inactivated human serum.     

The Genome Sequence of Trypanosoma brucei gambiense,Causative Agent of Chronic Human African Trypanosomiasis

Background

Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.

Methods and Findings

An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.

Conclusions

This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.     

A Panel of Trypanosoma brucei Strains Tagged with Blue and Red-Shifted Luciferases for Bioluminescent Imaging in Murine Infection Models

Background

Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients.

Methodology/Principal findings

We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT.

Conclusions/Significance

We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better kinetics of their substrate D-luciferin.     

Localization of serum resistance‐associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human‐infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance‐associated protein (SRA protein), protects against ApoL1‐mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA‐mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well‐characterized endosomal markers. By three‐dimensional deconvolved immunofluorescence single‐cell analysis, combined with double‐labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.     

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.