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).

Resolving the apparent transmission paradox of African sleeping sickness.

Human African trypanosomiasis (HAT), or African sleeping sickness, is a fatal disease found throughout sub-Saharan Africa. The disease is close to elimination in many areas, although it was similarly close to elimination once before and subsequently reemerged, despite seemingly low rates of transmission. Determining how these foci persisted and overcame an apparent transmission paradox is key to finally eliminating HAT. By assessing clinical, laboratory, and mathematical data, we propose that asymptomatic infections contribute to transmission through the presence of an overlooked reservoir of skin-dwelling parasites. Our assessment suggests that a combination of asymptomatic and parasitaemic cases is sufficient to maintain transmission at foci without animal reservoirs, and we argue that the current policy not to treat asymptomatic HAT should be reconsidered.

Extravascular Dermal Trypanosomes in Suspected and Confirmed Cases of gambiense Human African Trypanosomiasis.

BACKGROUND: The diagnosis of gambiense human African trypanosomiasis (gHAT) typically involves 2 steps: a serological screen, followed by the detection of living trypanosome parasites in the blood or lymph node aspirate. Live parasites can, however, remain undetected in some seropositive individuals, who, we hypothesize, are infected with Trypanosoma brucei gambiense parasites in their extravascular dermis. METHODS: To test this hypothesis, we conducted a prospective observational cohort study in the gHAT focus of Forecariah, Republic of Guinea. Of the 5417 subjects serologically screened for gHAT, 66 were enrolled into our study and underwent a dermatological examination. At enrollment, 11 seronegative, 8 unconfirmed seropositive, and 18 confirmed seropositive individuals had blood samples and skin biopsies taken and examined for trypanosomes by molecular and immunohistological methods. RESULTS: In seropositive individuals, dermatological symptoms were significantly more frequent, relative to seronegative controls. T.b. gambiense parasites were present in the blood of all confirmed cases (n = 18) but not in unconfirmed seropositive individuals (n = 8). However, T. brucei parasites were detected in the extravascular dermis of all unconfirmed seropositive individuals and all confirmed cases. Skin biopsies of all treated cases and most seropositive untreated individuals progressively became negative for trypanosomes 6 and 20 months later. CONCLUSIONS: Our results highlight the skin as a potential reservoir for African trypanosomes, with implications for our understanding of this disease's epidemiology in the context of its planned elimination and underlining the skin as a novel target for gHAT diagnostics.

Toxoplasma gondii Syntaxin 6 is required for vesicular transport between endosomal-like compartments and the Golgi complex.

Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post-Golgi and it has been speculated that they pass through endosomal-like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well-established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant-like secretory pathway.

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.

A co-evolutionary arms race: trypanosomes shaping the human genome, humans shaping the trypanosome genome.

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of several pathogens that cause the related veterinary disease Nagana. A complex co-evolution has occurred between these parasites and primates that led to the emergence of trypanosome-specific defences and counter-measures. The first line of defence in humans and several other catarrhine primates is the trypanolytic protein apolipoprotein-L1 (APOL1) found within two serum protein complexes, trypanosome lytic factor 1 and 2 (TLF-1 and TLF-2). Two sub-species of T. brucei have evolved specific mechanisms to overcome this innate resistance, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. In T. b. rhodesiense, the presence of the serum resistance associated (SRA) gene, a truncated variable surface glycoprotein (VSG), is sufficient to confer resistance to lysis. The resistance mechanism of T. b. gambiense is more complex, involving multiple components: reduction in binding affinity of a receptor for TLF, increased cysteine protease activity and the presence of the truncated VSG, T. b. gambiense-specific glycoprotein (TgsGP). In a striking example of co-evolution, evidence is emerging that primates are responding to challenge by T. b. gambiense and T. b. rhodesiense, with several populations of humans and primates displaying resistance to infection by these two sub-species.

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.

Multiple genetic mechanisms lead to loss of functional TbAT1 expression in drug-resistant trypanosomes.

The P2 aminopurine transporter, encoded by TbAT1 in African trypanosomes in the Trypanosoma brucei group, carries melaminophenyl arsenical and diamidine drugs into these parasites. Loss of this transporter contributes to drug resistance. We identified the genomic location of TbAT1 to be in the subtelomeric region of chromosome 5 and determined the status of the TbAT1 gene in two trypanosome lines selected for resistance to the melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), and in a Trypanosoma equiperdum clone selected for resistance to the diamidine, diminazene aceturate. In the Trypanosoma brucei gambiense STIB 386 melarsamine hydrochloride-resistant line, TbAT1 is deleted, while in the Trypanosoma brucei brucei STIB 247 melarsamine hydrochloride-resistant and T. equiperdum diminazene-resistant lines, TbAT1 is present, but expression at the RNA level is no longer detectable. Further characterization of TbAT1 in T. equiperdum revealed that a loss of heterozygosity at the TbAT1 locus accompanied loss of expression and that P2-mediated uptake of [(3)H]diminazene is lost in drug-resistant T. equiperdum. Adenine-inhibitable adenosine uptake is still detectable in a DeltaTbat1 T. b. brucei mutant, although at a greatly reduced capacity compared to that of the wild type, indicating that an additional adenine-inhibitable adenosine permease, distinct from P2, is present in these cells.

Association of APOL1 renal disease risk alleles with Trypanosoma brucei rhodesiense infection outcomes in the northern part of Malawi.

Trypanosoma brucei (T.b.) rhodesiense is the cause of the acute form of human African trypanosomiasis (HAT) in eastern and southern African countries. There is some evidence that there is diversity in the disease progression of T.b. rhodesiense in different countries. HAT in Malawi is associated with a chronic haemo-lymphatic stage infection compared to other countries, such as Uganda, where the disease is acute with more marked neurological impairment. This has raised the question of the role of host genetic factors in infection outcomes. A candidate gene association study was conducted in the northern region of Malawi. This was a case-control study involving 202 subjects, 70 cases and 132 controls. All individuals were from one area; born in the area and had been exposed to the risk of infection since birth. Ninety-six markers were genotyped from 17 genes: IL10, IL8, IL4, HLA-G, TNFA, IL6, IFNG, MIF, APOL, HLA-A, IL1B, IL4R, IL12B, IL12R, HP, HPR, and CFH. There was a strong significant association with APOL1 G2 allele (p = 0.0000105, OR = 0.14, CI95 = [0.05-0.41], BONF = 0.00068) indicating that carriers of the G2 allele were protected against T.b. rhodesiense HAT. SNP rs2069845 in IL6 had raw p < 0.05, but did not remain significant after Bonferroni correction. There were no associations found with the other 15 candidate genes. Our finding confirms results from other studies that the G2 variant of APOL1 is associated with protection against T.b. rhodesiense HAT.

Macrophage migrating inhibitory factor expression is associated with Trypanosoma brucei gambiense infection and is controlled by trans-acting expression quantitative trait loci in the Guinean population.

Infection by Trypanosoma brucei gambiense is characterized by a wide array of clinical outcomes, ranging from asymptomatic to acute disease and even spontaneous cure. In this study, we investigated the association between macrophage migrating inhibitory factor (MIF), an important pro-inflammatory cytokine that plays a central role in both innate and acquired immunity, and disease outcome during T. b. gambiense infection. A comparative expression analysis of patients, individuals with latent infection and controls found that MIF had significantly higher expression in patients (n = 141; 1.25 ±â€¯0.07; p < .0001) and latent infections (n = 25; 1.23 ±â€¯0.13; p = .0005) relative to controls (n = 46; 0.94 ±â€¯0.11). Furthermore, expression decreased significantly after treatment (patients before treatment n = 33; 1.40 ±â€¯0.18 versus patients after treatment n = 33; 0.99 ±â€¯0.10, p = .0001). We conducted a genome wide eQTL analysis on 29 controls, 128 cases and 15 latently infected individuals for whom expression and genotype data were both available. Four loci, including one containing the chemokine CXCL13, were found to associate with MIF expression. Genes at these loci are candidate regulators of increased expression of MIF after infection. Our study is the first data demonstrating that MIF expression is elevated in T. b. gambiense-infected human hosts but does not appear to contribute to pathology.

Loss of SEC-23 in Caenorhabditis elegans causes defects in oogenesis, morphogenesis, and extracellular matrix secretion.

SEC-23 is a component of coat protein complex II (COPII)-coated vesicles involved in the endoplasmic reticulum-to-Golgi transport pathway of eukaryotes. During postembryonic life, Caenorhabditis elegans is surrounded by a collagenous exoskeleton termed the cuticle. From a screen for mutants defective in cuticle secretion, we identified and characterized a sec-23 mutant of C. elegans. By sequence homology, C. elegans has only the single sec-23 gene described herein. In addition to the cuticle secretion defect, mutants fail to complete embryonic morphogenesis. However, they progress through the earlier stages of embryogenesis, including gastrulation, and achieve substantial morphogenesis before death. We demonstrated a maternal component of SEC-23 function sufficient for progression through the earlier stages of embryogenesis and explaining the limited phenotype of the zygotic mutant. By RNA-mediated interference, we investigated the effects of perturbing COPII function during various postembryonic stages. During larval stages, major defects in cuticle synthesis and molting were observed. In the adult hermaphrodite, reduction of SEC-23 function by RNA-mediated interference caused a rapid onset of sterility, with defects in oogenesis including early maturation of the germline nuclei, probably a result of the observed loss of the GLP-1 receptor from the membrane surfaces adjacent to the developing germline nuclei.

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.

Population genomics reveals the origin and asexual evolution of human infective trypanosomes.

Evolutionary theory predicts that the lack of recombination and chromosomal re-assortment in strictly asexual organisms results in homologous chromosomes irreversibly accumulating mutations and thus evolving independently of each other, a phenomenon termed the Meselson effect. We apply a population genomics approach to examine this effect in an important human pathogen, Trypanosoma brucei gambiense. We determine that T.b. gambiense is evolving strictly asexually and is derived from a single progenitor, which emerged within the last 10,000 years. We demonstrate the Meselson effect for the first time at the genome-wide level in any organism and show large regions of loss of heterozygosity, which we hypothesise to be a short-term compensatory mechanism for counteracting deleterious mutations. Our study sheds new light on the genomic and evolutionary consequences of strict asexuality, which this pathogen uses as it exploits a new biological niche, the human population.

The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes.

The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology.

Exploiting genetic variation to discover genes involved in important disease phenotypes.

Elucidating the underlying genetic determinants of disease pathology is still in the early stages for many pathogenic parasites. There have, however, been a number of advances in which natural genetic diversity has been successfully utilized to untangle the often complex interactions between parasite and host. In this chapter we discuss various methods capable of exploiting this natural genetic variation to determine genes involved in phenotypes of interest, using virulence in the pathogenic parasite Trypanosoma brucei as a case study. This species is an ideal system to benefit from such an approach as there are several well-characterized laboratory strains; the parasite undergoes genetic exchange in both the field and the laboratory, and is amenable to efficient reverse genetics and RNAi.

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin.

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell division.

Fate specification and tissue-specific cell cycle control of the Caenorhabditis elegans intestine.

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein beta-transducin repeat-containing protein (beta-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans beta-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant.

Searching for novel cell cycle regulators in Trypanosoma brucei with an RNA interference screen.

BACKGROUND: The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis. Its cell cycle is complex and not fully understood at the molecular level. The T. brucei genome contains over 6000 protein coding genes with >50% having no predicted function. A small scale RNA interference (RNAi) screen was carried out in Trypanosoma brucei to evaluate the prospects for identifying novel cycle regulators. RESULTS: Procyclic form T. brucei were transfected with a genomic RNAi library and 204 clones isolated. However, only 76 RNAi clones were found to target a protein coding gene of potential interest. These clones were screened for defects in proliferation and cell cycle progression following RNAi induction. Sixteen clones exhibited proliferation defects upon RNAi induction, with eight clones displaying potential cell cycle defects. To confirm the phenotypes, new RNAi cell lines were generated and characterised for five genes targeted in these clones. While we confirmed that the targeted genes are essential for proliferation, we were unable to unambiguously classify them as cell cycle regulators. CONCLUSION: Our study identified genes essential for proliferation, but did not, as hoped, identify novel cell cycle regulators. Screening of the RNAi library for essential genes was extremely labour-intensive, which was compounded by the suboptimal quality of the library. For such a screening method to be viable for a large scale or genome wide screen, a new, significantly improved RNAi library will be required, and automated phenotyping approaches will need to be incorporated.

Oncogenic potential of a C.elegans cdc25 gene is demonstrated by a gain-of-function allele.

In multicellular organisms, developmental programmes must integrate with central cell cycle regulation to co-ordinate developmental decisions with cell proliferation. Hyperplasia caused by deregulated proliferation without significant change to other aspects of developmental behaviour is a probable step towards full oncogenesis in many malignancies. CDC25 phosphatase promotes progression through the eukaryotic cell cycle by dephosphorylation of cyclin-dependent kinase and, in humans, different cdc25 family members have been implicated as potential oncogenes. Demonstrating the direct oncogenic potential of a cdc25 gene, we identify a gain-of-function mutant allele of the Caenorhabditis elegans gene cdc-25.1 that causes a deregulated proliferation of intestinal cells resulting in hyperplasia, while other aspects of intestinal cell function are retained. Using RNA-mediated interference, we demonstrate modulation of the oncogenic behaviour of this mutant, and show that a reduction of the wild-type cdc-25.1 activity can cause a failure of proliferation of intestinal and other cell types. That gain and loss of CDC-25.1 activity has opposite effects on cellular proliferation indicates its critical role in controlling C.elegans cell number.