Yes / Background. Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes. Methodology. We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin. Principal Findings. The findings suggest that after surgical treatment—without antibiotics—the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse. Conclusions. To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development. / This work was supported by the UBS Optimus Foundation (Zurich, Switzerland) and the Department of Economy, Science and Innovation of the Flemish Government (Belgium). KV was supported by a VLADOC PhD scholarship of VLIRUOS (Belgium).

A inativação do cromossomo X (ICX) em fêmeas é um exemplo de regulação epigenética. O silenciamento de um dos cromossomos X leva à formação estável da heterocromatina facultativa através da aquisição de múltiplas modificações na cromatina que são mantidas nas subsequentes divisões celulares. Atualmente, algumas características epigenéticas associadas à manutenção da ICX têm sido descritas, contudo os mecanismos de ação e a identidade dos diferentes fatores envolvidos na manutenção da ICX ainda são desconhecidos ou pouco compreendidos. Nosso laboratório realizou uma triagem funcional genômica por bibliotecas de shRNAs (short harpin RNAs) para encontrar genes envolvidos na manutenção da ICX em humanos. A partir deste estudo foram identificados 20 novos genes candidatos a estarem envolvidos na manutenção da ICX. Assim, o objetivo deste trabalho foi validar o grau de envolvimento de dois destes genes candidatos (H3F3B e ASF1A) no processo de controle epigenético do cromossomo X. Para isto, foi realizado o silenciamento dos genes candidatos através da utilização de partículas lentivirais portando shRNAs específicos em fibroblastos primários femininos heterozigotos para uma mutação no gene HPRT e com desvio total de ICX, onde o único alelo normal do gene HPRT está no Xi. A reativação do Xi nestas células foi avaliada por cultivo das mesmas em meio HAT, que seleciona células HPRT+. Só sobreviveram os fibroblastos que foram silenciados para o gene H3F3B. Nestes, as células transduzidas com o shH3F3B.2 expressam o alelo selvagem do gene, presente no Xi, além do gene mutante. Ensaios de RNA-FISH e trimetilação de histonas foram feitos nessas células para avaliar a perda das marcas de cromatina inativa. Foi observada uma perda da nuvem de XIST nas células transduzidas com o shH3F3B.2 e selecionadas em HAT em passagens altas. Por último, análises de expressão alelo-específica de genes ligados ao X comprovaram que dois genes que são submetidos à ICX apresentaram expressão do alelo inativado (FLNA e FHL1). Porém, também foi observada uma mudança no padrão de expressão alelo-específica em genes autossômicos. Finalmente, as análises de expressão geral do cromossomo X mostraram que as células transduzidas com o shH3F3B.2 e selecionada em HAT tinham uma expressão gênica aumentada em relação ao controle. Em conclusão, nossos resultados sugerem uma descondensação da cromatina no cromossomo Xi e portanto um provável envolvimento do gene H3F3B na manutenção da ICX. / The X chromosome Inactivation (XCI) in females is an example of epigenetic regulation. Silencing of one of the X chromosomes leads to the stable formation of the facultative heterochromatin through the acquisition of multiple modifications in the chromatin that are maintained in the subsequent cell divisions. Currently, some epigenetic features associated with the maintenance of XCI have been described. Nonetheless, the mechanisms of action and the identity of the different factors involved in the maintenance of XCI are still unknown or poorly understood. Our laboratory performed a genomic functional screening by shRNA (short harpin RNAs) libraries to find genes involved in the maintenance of XCI in humans. From this study, we identified 20 new candidate genes to be involved in the maintenance of XCI. Thus, the objective of this work was to validate the degree of involvement of two of these candidate genes (H3F3B and ASF1A) in the epigenetic process control of the X chromosome. For this, the silencing of the candidate genes was performed in female heterozygous primary fibroblasts for a mutation of the HPRT gene and with a total XCI shift through the use of lentiviral particles carrying specific shRNAs, where the only normal allele of the HPRT gene is in the Xi (inactivated X). Xi reactivation was evaluated in these cells by culturing them in HAT medium, which selects HPRT + cells. Only the fibroblasts that were silenced for the H3F3B gene survived. Furthermore, the cells transduced with shH3F3B.2 express the HPRT wild gene allele, present in Xi, in addition to the mutant gene. RNA-FISH and histone trimethylation assays were performed on these cells to evaluate the loss of inactive chromatin marks. A loss of the XIST cloud was observed in cells transduced with shH3F3B.2 and selected in HAT at high passages. Finally, allele-specific expression analyzes of X-linked genes showed that two genes that undergo XCI showed expression of the inactivated allele (FLNA and FHL1). However, a change in allele-specific expression pattern was also observed in autosomal genes. Finally, the X chromosome general expression analyses showed that cells transduced with shH3F3B.2 and selected on HAT had increased gene expression relative to the control. In conclusion, our results suggest a decondensation of the chromatin in the Xi chromosome and therefore a probable involvement of the H3F3B gene in the maintenance of ICX.

La réplication et l’assemblage du virus de l’hépatite C (VHC) sont régulés finement dans le temps et l’espace par les interactions protéiques entre le virus avec l’hôte. La compréhension de la biologie du virus ainsi que sa pathogénicité passe par les connaissances relatives aux interactions virus/hôte. Afin d’identifier ces interactions, nous avons exploité une approche d’immunoprécipitation (IP) couplée à une détection par spectrométrie de masse (MS), pour ensuite évaluer le rôle des protéines identifiées dans le cycle viral par une technique de silençage génique. Les protéines virales Core, NS2, NS3/4A, NS4B, NS5A et NS5B ont été exprimées individuellement dans les cellules humaines 293T et immunoprécipitées afin d’isoler des complexes protéiques qui ont été soumis à l’analyse MS. Ainsi, 98 protéines de l’hôte ont été identifiées avec un enrichissement significatif et illustrant une spécificité d’interaction. L’enrichissement de protéines connues dans la littérature a démontré la force de l’approche, ainsi que la validation de 6 nouvelles interactions virus/hôte. Enfin, le rôle de ces interactants sur la réplication virale a été évalué dans un criblage génomique par ARN interférant (ARNi). Deux systèmes rapporteurs de la réplication virale ont été utilisés : le système de réplicon sous-génomique (Huh7-Con1-Fluc) et le système infectieux (J6/JFH-1/p7Rluc2a), ainsi qu’un essai de toxicité cellulaire (Alamar Blue). Parmi les protéines de l’hôte interagissant avec le VHC, 28 protéines ont démontré un effet significatif sans effet de toxicité cellulaire, suggérant fortement un rôle dans la réplication du VHC. Globalement, l’étude a mené à l’identification de nouvelles interactions virus/hôte et l’identification de nouvelles cibles thérapeutiques potentielles. / Hepatitis C virus (HCV) replication and assembly are tightly regulated in time and space within the cell, most likely due to protein interactions between virus and host. In order to better understand HCV biology and its pathogenesis, there is a need to unravel virus/host interaction network. We extended our knowledge of virus/host interactions by the identification of cellular proteins associated to HCV proteins using an immunoprecipitation (IP) technique coupled to mass spectrometry (MS), and further evaluate the role of retrieved interactors using gene knockdown. FLAG-tagged viral proteins Core, NS2, NS3/4A, NS4B, NS5A and NS5B have been expressed individually in 293T human cells, and immunoprecipitated protein complexes have been submitted to MS analysis for identification of host proteins. In this study, 98 proteins were significantly enriched and showed specific interaction to a viral protein. Retrieval of previously characterized interacting proteins proved the strength of the method. Six newly identified interactors by MS were individually confirmed using IP of viral proteins. We evaluated the role of identified interactors in HCV replication by performing a functional lentivirus-based RNA interference (RNAi) screen. Two reporter systems were used: the sub- genomic replicon (Huh7-Con1-Fluc) and a full length infectious clone (J6/JFH-1/p7Rluc2a), as well as the cellular toxicity assay Alamar blue. Of the identified host interactors, 28 proteins showed a significant effect on HCV replication upon gene knockdown and without cellular toxicity. Overall, the study led to the identification of novel virus/host interactions essential in HCV life cycle and provides novel potential drug targets.

La réplication et l’assemblage du virus de l’hépatite C (VHC) sont régulés finement dans le temps et l’espace par les interactions protéiques entre le virus avec l’hôte. La compréhension de la biologie du virus ainsi que sa pathogénicité passe par les connaissances relatives aux interactions virus/hôte. Afin d’identifier ces interactions, nous avons exploité une approche d’immunoprécipitation (IP) couplée à une détection par spectrométrie de masse (MS), pour ensuite évaluer le rôle des protéines identifiées dans le cycle viral par une technique de silençage génique. Les protéines virales Core, NS2, NS3/4A, NS4B, NS5A et NS5B ont été exprimées individuellement dans les cellules humaines 293T et immunoprécipitées afin d’isoler des complexes protéiques qui ont été soumis à l’analyse MS. Ainsi, 98 protéines de l’hôte ont été identifiées avec un enrichissement significatif et illustrant une spécificité d’interaction. L’enrichissement de protéines connues dans la littérature a démontré la force de l’approche, ainsi que la validation de 6 nouvelles interactions virus/hôte. Enfin, le rôle de ces interactants sur la réplication virale a été évalué dans un criblage génomique par ARN interférant (ARNi). Deux systèmes rapporteurs de la réplication virale ont été utilisés : le système de réplicon sous-génomique (Huh7-Con1-Fluc) et le système infectieux (J6/JFH-1/p7Rluc2a), ainsi qu’un essai de toxicité cellulaire (Alamar Blue). Parmi les protéines de l’hôte interagissant avec le VHC, 28 protéines ont démontré un effet significatif sans effet de toxicité cellulaire, suggérant fortement un rôle dans la réplication du VHC. Globalement, l’étude a mené à l’identification de nouvelles interactions virus/hôte et l’identification de nouvelles cibles thérapeutiques potentielles. / Hepatitis C virus (HCV) replication and assembly are tightly regulated in time and space within the cell, most likely due to protein interactions between virus and host. In order to better understand HCV biology and its pathogenesis, there is a need to unravel virus/host interaction network. We extended our knowledge of virus/host interactions by the identification of cellular proteins associated to HCV proteins using an immunoprecipitation (IP) technique coupled to mass spectrometry (MS), and further evaluate the role of retrieved interactors using gene knockdown. FLAG-tagged viral proteins Core, NS2, NS3/4A, NS4B, NS5A and NS5B have been expressed individually in 293T human cells, and immunoprecipitated protein complexes have been submitted to MS analysis for identification of host proteins. In this study, 98 proteins were significantly enriched and showed specific interaction to a viral protein. Retrieval of previously characterized interacting proteins proved the strength of the method. Six newly identified interactors by MS were individually confirmed using IP of viral proteins. We evaluated the role of identified interactors in HCV replication by performing a functional lentivirus-based RNA interference (RNAi) screen. Two reporter systems were used: the sub- genomic replicon (Huh7-Con1-Fluc) and a full length infectious clone (J6/JFH-1/p7Rluc2a), as well as the cellular toxicity assay Alamar blue. Of the identified host interactors, 28 proteins showed a significant effect on HCV replication upon gene knockdown and without cellular toxicity. Overall, the study led to the identification of novel virus/host interactions essential in HCV life cycle and provides novel potential drug targets.