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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., Live trypanosomes can remain undetected in the blood of individuals seropositive for sleeping sickness. Here, we show that they could be infected with parasites in their extravascular dermis, highlighting the skin as a potential reservoir for trypanosomes.

La trypanosomiase humaine africaine ou maladie du sommeil est causée par plusieurs sous-espèces membres de l'espèce Trypanosoma brucei : Trypanosoma brucei rhodesiense (Tbr) et Trypanosoma brucei gambiense groupe 1 (Tbg1). Leur biologie a été étudiée en détail, en particulier en ce qui concerne les protéines impliquées dans les mécanismes de résistance au sérum humain (SH). Il existe un autre groupe également capable d'infecter l'homme nommé Trypanosoma brucei gambiense groupe 2 (Tbg2). Celui-ci se résume à une trentaine de souches répertoriées dans la littérature, ne pose pas de problème de santé publique et par conséquent a été très peu étudié, pour ne pas dire oublié. Après un rappel des mécanismes qui permettent à Tbr et Tbg1 de résister à l'action lytique du SH, nous résumerons les connaissances acquises sur Tbg2. Les phénotypes de résistance au sérum humain sont très variables et seulement une de ces souches a fait l'objet de quelques recherches. La résistance de cette dernière est conditionnelle et implique des mécanismes ayant une action directe sur ApoL-1. Cette revue sera donc l'occasion non seulement de rappeler l'état des connaissances concernant la « course à l'armement » menée entre les trypanosomes et l'homme au cours de l'évolution, mais nous permettra également d'entamer une réflexion sur ce groupe particulier et hétérogène qu'est Tbg2, un trypanosome pathogène pour l'homme mais dont le caractère zoonotique doit être pris en compte dans la perspective du contexte de post-élimination de la maladie du sommeil.

La trypanosomiase humaine africaine (THA) est l'une des maladies tropicales les plus négligées en Afrique subsaharienne. Avec moins de 1 000 cas déclarés en 2018 et 2019, elle a été ciblée comme maladie pouvant être éliminée d'ici 2030. Des réservoirs cryptiques de Trypanosoma brucei gambiense, tels que les animaux domestiques ou sauvages, apparaissent comme des déterminants pouvant compromettre cet objectif. Mieux comprendre l'impact d'un tel réservoir sur le maintien de la transmission de T. b gambiense nous permettra d'affiner les stratégies de lutte contre la THA. Dans cette revue, nous discutons des différents obstacles (techniques, biologiques, épidémiologiques) auxquels ont été confrontés les chercheurs qui se sont penchés sur cette problématique. Ces obstacles, qui perdurent aujourd'hui, expliquent en partie pourquoi, près d'un siècle après les premières études, l'impact du réservoir animal est toujours un sujet controversé.

Le trypanosome africain Trypanosoma brucei gambiense, parasite responsable de la trypanosomiase humaine africaine, est transmis à l'homme par la piqûre d'une mouche tsé-tsé infectée, puis il prolifère dans le sang et la lymphe et enfin dans le système nerveux central. Le diagnostic de trypanosomiase implique deux étapes : un dépistage sérologique suivi de la détection de parasites vivants dans un fluide biologique. Cependant, les parasites peuvent rester indétectables chez certains individus qualifiés de séropositifs non confirmés qui restent alors sans traitement. Dans différents modèles de laboratoire, des chercheurs ont récemment démontré l'existence de réservoirs anatomiques de parasites extravasculaires, en particulier la peau, qui pourraient expliquer les infections latentes. Récemment, la présence de trypanosomes extravasculaires dermiques a également été démontrée chez l'homme, soulignant l'importance des individus séropositifs non confirmés comme réservoir de parasites qui pourrait compromettre l'élimination de la maladie. Les impacts cliniques et épidémiologiques de ces trypanosomes dermiques sont ici discutés.

posté sur MedRxiv le 25 février 2020 : https://www.medrxiv.org/content/10.1101/2020.02.24.20026211v1; International audience; Background The diagnosis of Human African Trypanosomiasis (HAT) typically involves two 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 and findings To test this hypothesis, we conducted a prospective observational cohort study in the gambiense HAT (gHAT) focus of Forecariah, in the Republic of Guinea. 5,417 subjects in this disease foci underwent serological screening for gHAT. Of these individuals, 66 were enrolled into our study, of whom 40 were seronegative, 8 were seropositive but unconfirmed, and 18 confirmed gHAT cases. Enrolled individuals underwent a dermatological examination, and had blood samples and skin biopsies taken and examined for trypanosomes by molecular and immuno-histological methods. In confirmed cases, dermatological symptoms were significantly more frequent, relative to seronegative controls. T. b. gambiense parasites were present in the blood of all confirmed cases but not in unconfirmed seropositive individuals. However, trypanosomes were detected in the dermis of all unconfirmed seropositive individuals and confirmed cases. After 6 and 20 months of treatment, dermal trypanosome numbers in skin biopsies of confirmed cases progressively reduced. Conclusions Our results thus highlight the skin as a potential reservoir for trypanosomes, with implications for our understanding of this disease's epidemiology in the context of its planned elimination and highlighting the skin as a novel target for gHAT diagnostics.

African trypanosomes evade the host immune response by periodically changing their protein coat, constituted by a single Variant Surface Glycoprotein (VSG), allowing for chronic infections. We have previously published that Cohesin complex regulates in situ transcriptional VSG switching, as partial depletion of Cohesin subunits increases the frequency of antigenic variation. Condensin complex, structurally similar to the cohesin complex, has recently emerged in eukaryotes as a mayor regulator of chromosome architecture, chromatin compaction during interphase, and is greatly enriched near highly expressed genes. Previous results suggest that SUMOylation of chromatin at the active VSG locus may function to nucleate factors to the nuclear body ESB, where VSG transcription occurs. Furthermore, we found by proteomic analysis that TbSMC4, a subunit of the condensin complex, is a consistent and abundant SUMO target. Therefore, it seems probable that Condensin functions in the regulation of VSG monoallelic expression and/or transcriptional switching. Co-IP experiments showed that trypanosome condensin includes well-known subunits CND1 & 2 and SMC4, suggesting a conserved multiprotein complex that localizes in the nucleus, as described in other eukaryotes. In addition, we found distinct SUMOylation sites in the condensin subunits of the infective bloodstream forms. Interestingly, partial depletion of condensin subunits resulted in a significant increase of VSG221 switching off events, reaching up to 10% of the population, a switching frequency higher than previously described for cohesin depletion. Isolated switches corresponded to in situ transcriptional activation events of independent telomeric VSG-ESs. All data suggest that condensin complex has a key role in establishing and/or maintaining the transcriptional state of VSG-ES chromatin.

Supplementary Data are available at NAR Online., The nuclear lamina is a filamentous structure subtending the nuclear envelope and required for chromatin organization, transcriptional regulation and maintaining nuclear structure. The trypanosomatid coiled-coil NUP-1 protein is a lamina component functionally analogous to lamins, the major lamina proteins of metazoa. There is little evidence for shared ancestry, suggesting the presence of a distinct lamina system in trypanosomes. To find additional trypanosomatid lamina components we identified NUP-1 interacting proteins by affinity capture and mass-spectrometry. Multiple components of the nuclear pore complex (NPC) and a second coiled-coil protein, which we termed NUP-2, were found. NUP-2 has a punctate distribution at the nuclear periphery throughout the cell cycle and is in close proximity to NUP-1, the NPCs and telomeric chromosomal regions. RNAi-mediated silencing of NUP-2 leads to severe proliferation defects, gross alterations to nuclear structure, chromosomal organization and nuclear envelope architecture. Further, transcription is altered at telomere-proximal variant surface glycoprotein (VSG) expression sites (ESs), suggesting a role in controlling ES expression, although NUP-2 silencing does not increase VSG switching. Transcriptome analysis suggests specific alterations to Pol I-dependent transcription. NUP-1 is mislocalized in NUP-2 knockdown cells and vice versa, implying that NUP-1 and NUP-2 form a co-dependent network and identifying NUP-2 as a second trypanosomatid nuclear lamina component., This work was supported by the Wellcome Trust (program grant 082813 to MCF, 093010 and 100320 to DH), the Gates Cambridge Trust (studentship to LM) and the NIH (R21 AI096069, U54 GM103511 to BTC, JA and MPR, U01 GM098256, P41 GM109824 to MPR, R01 GM112108 to JA and MPR, P50 GM076547 to JA and P41 GM103314 to BTC). Funding for open access charge: Wellcome Trust

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 approximate to 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., MN is funded by grants from the Spanish Ministerio de Ciencia e Innovacion, (SAF2012-40029), Junta de Andalucia (CTS-5841) and VI PN de I+D+I 2008-2011, Instituto de Salud Carlos III - Subdireccion General de Redes y Centros de Investigacion Cooperativa (RICET) RD12/0018/0001 and RD12/0018/0015. J-MB is supported by a Miguel Servet Fellowship (CP09/00300) and funded by 'Fondo de Investigacion Sanitaria' PI10/01128. JR and MC were funded by a Wellcome Trust Project Grant 093008/Z/10/Z. Work in the Dundee laboratory was funded by the Wellcome Trust (program grant 093008/Z/10/Z) and the Medical Research Council.