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.MN is funded by grants from the Spanish Ministerio de Ciencia e Innovación, (SAF2012-40029), Junta de Andalucia (CTS-5841) and VI PN de I+D+I 2008–2011, Instituto de Salud Carlos III – Subdirección General de Redes y Centros de Investigación 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 Investigación 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.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/cmi.1245

Glossina palpalis palpalis populations from Equatorial Guinea belong to distinct allopatric clades.

BACKGROUND: Luba is one of the four historical foci of Human African Trypanosomiasis (HAT) on Bioko Island, in Equatorial Guinea. Although no human cases have been detected since 1995, T. b. gambiense was recently observed in the vector Glossina palpalis palpalis. The existence of cryptic species within this vector taxon has been previously suggested, although no data are available regarding the evolutionary history of tsetse flies populations in Bioko. METHODS: A phylogenetic analysis of 60 G. p. palpalis from Luba was performed sequencing three mitochondrial (COI, ND2 and 16S) and one nuclear (rDNA-ITS1) DNA markers. Phylogeny reconstruction was performed by Distance Based, Maximum Likelihood and Bayesian Inference methods. RESULTS: The COI and ND2 mitochondrial genes were concatenated and revealed 10 closely related haplotypes with a dominant one found in 61.1% of the flies. The sequence homology of the other 9 haplotypes compared to the former ranged from 99.6 to 99.9%. Phylogenetic analysis clearly clustered all island samples with flies coming from the Western African Clade (WAC), and separated from the flies belonging to the Central Africa Clade (CAC), including samples from Mbini and Kogo, two foci of mainland Equatorial Guinea. Consistent with mitochondrial data, analysis of the microsatellite motif present in the ITS1 sequence exhibited two closely related genotypes, clearly divergent from the genotypes previously identified in Mbini and Kogo. CONCLUSIONS: We report herein that tsetse flies populations circulating in Equatorial Guinea are composed of two allopatric subspecies, one insular and the other continental. The presence of these two G. p. palpalis cryptic taxa in Equatorial Guinea should be taken into account to accurately manage vector control strategy, in a country where trypanosomiasis transmission is controlled but not definitively eliminated yet

Glossina palpalis palpalis populations from Equatorial Guinea belong to distinct allopatric clades

[Background] Luba is one of the four historical foci of Human African Trypanosomiasis (HAT) on Bioko Island, in Equatorial Guinea. Although no human cases have been detected since 1995, T. b. gambiense was recently observed in the vector Glossina palpalis palpalis. The existence of cryptic species within this vector taxon has been previously suggested, although no data are available regarding the evolutionary history of tsetse flies populations in Bioko.[Methods] A phylogenetic analysis of 60 G. p. palpalis from Luba was performed sequencing three mitochondrial (COI, ND2 and 16S) and one nuclear (rDNA-ITS1) DNA markers. Phylogeny reconstruction was performed by Distance Based, Maximum Likelihood and Bayesian Inference methods.[Results] The COI and ND2 mitochondrial genes were concatenated and revealed 10 closely related haplotypes with a dominant one found in 61.1% of the flies. The sequence homology of the other 9 haplotypes compared to the former ranged from 99.6 to 99.9%. Phylogenetic analysis clearly clustered all island samples with flies coming from the Western African Clade (WAC), and separated from the flies belonging to the Central Africa Clade (CAC), including samples from Mbini and Kogo, two foci of mainland Equatorial Guinea. Consistent with mitochondrial data, analysis of the microsatellite motif present in the ITS1 sequence exhibited two closely related genotypes, clearly divergent from the genotypes previously identified in Mbini and Kogo.[Conclusions] We report herein that tsetse flies populations circulating in Equatorial Guinea are composed of two allopatric subspecies, one insular and the other continental. The presence of these two G. p. palpalis cryptic taxa in Equatorial Guinea should be taken into account to accurately manage vector control strategy, in a country where trypanosomiasis transmission is controlled but not definitively eliminated yet.This work has been supported by ‘Fondo de Investigacion Sanitaria (FIS)’ (PI10/01128) and by VI PN de I + D + I 2008–2011, ISCIII -Subdirección General de Redes y Centros de Investigación Cooperativa RD12/0018/0001 and RD12/0018/0015 (RICET). JMB is supported by Miguel Servet Fellowship CP09/00300.Peer Reviewe

Trypanosoma brucei gambiense Adaptation to Different Mammalian Sera Is Associated with VSG Expression Site Plasticity

Trypanosoma brucei gambiense infection is widely considered an anthroponosis, although it has also been found in wild and domestic animals. Thus, fauna could act as reservoir, constraining the elimination of the parasite in hypo-endemic foci. To better understand the possible maintenance of T. b. gambiense in local fauna and investigate the molecular mechanisms underlying adaptation, we generated adapted cells lines (ACLs) by in vitro culture of the parasites in different mammalian sera. Using specific antibodies against the Variant Surface Glycoproteins (VSGs) we found that serum ACLs exhibited different VSG variants when maintained in pig, goat or human sera. Although newly detected VSGs were independent of the sera used, the consistent appearance of different VSGs suggested remodelling of the co-transcribed genes at the telomeric Expression Site (VSG-ES). Thus, Expression Site Associated Genes (ESAGs) sequences were analysed to investigate possible polymorphism selection. ESAGs 6 and 7 genotypes, encoding the transferrin receptor (TfR), expressed in different ACLs were characterised. In addition, we quantified the ESAG6/7 mRNA levels and analysed transferrin (Tf) uptake. Interestingly, the best growth occurred in pig and human serum ACLs, which consistently exhibited a predominant ESAG7 genotype and higher Tf uptake than those obtained in calf and goat sera. We also detected an apparent selection of specific ESAG3 genotypes in the pig and human serum ACLs, suggesting that other ESAGs could be involved in the host adaptation processes. Altogether, these results suggest a model whereby VSG-ES remodelling allows the parasite to express a specific set of ESAGs to provide selective advantages in different hosts. Finally, pig serum ACLs display phenotypic adaptation parameters closely related to human serum ACLs but distinct to parasites grown in calf and goat sera. These results suggest a better suitability of swine to maintain T. b. gambiense infection supporting previous epidemiological results. Figures 12 Citation: Cordon-Obras C, Cano J, González-Pacanowska D, Benito A, Navarro M, et al. (2013) Trypanosoma brucei gambiense Adaptation to Different Mammalian Sera Is Associated with VSG Expression Site Plasticity. PLoS ONE 8(12): e85072. doi:10.1371/journal.pone.0085072 Editor: Mauricio Martins Rodrigues, Federal University of São Paulo, Brazil Received: July 31, 2013; Accepted: November 21, 2013; Published: December 23, 2013 Copyright: © 2013 Cordon-Obras et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación, Plan Nacional (SAF2012-40029), Junta de Andalucia (CTS-5841), Fondo de Investigación Sanitaria (FIS) PI10/01128 and VI PN de I+D+I 2008-2011, ISCIII -Subdirección General de Redes y Centros de Investigación Cooperativa (RICET) RD12/0018/0001 and RD12/0018/0015. JMB is supported by a Miguel Servet Fellowship CP09/00300. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.Peer reviewe

CRISPR Interference of a Clonally Variant GC-Rich Noncoding RNA Family Leads to General Repression of var Genes in Plasmodium falciparum

International audienceThe human malaria parasite Plasmodium falciparum uses mutually exclusive expression of the PfEMP1-encoding var gene family to evade the host immune system. Despite progress in the molecular understanding of the default silencing mechanism, the activation mechanism of the uniquely expressed var member remains elusive. A GC-rich noncoding RNA (ncRNA) gene family has coevolved with Plasmodium species that express var genes. Here, we show that this ncRNA family is transcribed in a clonally variant manner, with predominant transcription of a single member occurring when the ncRNA is located adjacent to and upstream of an active var gene. We developed a specific CRISPR interference (CRISPRi) strategy that allowed for the transcriptional repression of all GC-rich members. A lack of GC-rich ncRNA transcription led to the downregulation of the entire var gene family in ring-stage parasites. Strikingly, in mature blood-stage parasites, the GC-rich ncRNA CRISPRi affected the transcription patterns of other clonally variant gene families, including the downregulation of all Pfmc-2TM members. We provide evidence for the key role of GC-rich ncRNA transcription in var gene activation and discovered a molecular link between the transcriptional control of various clonally variant multigene families involved in parasite virulence. This work opens new avenues for elucidating the molecular processes that control immune evasion and pathogenesis in P. falciparum IMPORTANCE Plasmodium falciparum is the deadliest malaria parasite species, accounting for the vast majority of disease cases and deaths. The virulence of this parasite is reliant upon the mutually exclusive expression of cytoadherence proteins encoded by the 60-member var gene family. Antigenic variation of this multigene family serves as an immune evasion mechanism, ultimately leading to chronic infection and pathogenesis. Understanding the regulation mechanism of antigenic variation is key to developing new therapeutic and control strategies. Our study uncovers a novel layer in the epigenetic regulation of transcription of this family of virulence genes by means of a multigene-targeting CRISPR interference approach

Alignment of Tf binding sites (ESAG6/7) and polymorphic ESAG3 regions.

<p>Amino acid alignment of polymorphic regions of ESAG6/7 (A) and ESAG3 (B). Tf binding sites are highlighted with red squares. Other polymorphic regions of ESAG6/7 are marked with black boxes. Polymorphic regions of ESAG3 are shown. I: S103-E119; II: L171-K189; III: A/G215-D/E244; IV: A309-R/E321. <i>T</i>. <i>b. brucei</i> ESAGs 6, 7 and 3 are shown in red.</p

VSG expression in ACLs.

<p>(A) Schematic representation of a canonical <i>VSG</i>-Expression Site and the two main mechanisms of antigenic variation in <i>T. brucei</i>. Black arrows indicate the promoter sequence and blue arrows the active start transcription site. The thick black line mirrors the transcribed genes. The striped boxes are the 70-base-pair repeats upstream the <i>VSG</i> gene and white triangles represent telomeric repeats. Coloured boxes denote <i>ESAG</i> and <i>VSG</i> genes. Active VSG-ES can be silenced and another telomeric VSG-ES becomes active by <i>in </i><i>situ</i> switch (above). Alternatively recombination events in any part (or parts) of the active VSG-ES can occur by homologous recombination, inserting new gene/s from another VSG-ES or non-telomeric locations. (B) Ponceau staining of total protein extract from ACLs showing different VSGs (marked with an arrow). Equivalent amount of 5 x10⁶ parasites was loaded per well. Molecular weight is expressed in kDa. (C) WB analysis of VSGs expressed by different ACLs using anti-LiTat 2.1, anti-LiTat 3.1 and anti-221 antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085072#B25" target="_blank">25</a>]. Anti-Tubulin was used as loading control. Equivalent amount of 1 x 10⁵ parasites was loaded per well. ACL: adapted cell line. FCS: foetal calf serum, GS1/2/3: goat serum (adaptation experiments 1, 2 or 3), HS1/2/3/4: human serum (adaptation experiments 1, 2, 3 or 4), PS1/2/3: pig serum (adaptation experiments 1, 2 or 3), WB: Western Blot.</p

Identification of elusive sequence-specific promoters of RNA polymerase II polycistronic transcription in African trypanosomes

Kinetoplastids have evolved in isolation for one billion years resulting in several divergent molecular and cellular processes. One example is protein-coding genes transcribed polycistronically by a typical RNA polymerase II (RNA pol II). Transcription most likely starts at divergent Strand Switch Regions (dSSRs), long sequences between divergently oriented polycistronic transcription units (PTUs). The lack of regulation in trypanosome transcription has become the paradigm in our field. Previous work suggests that changes in chromatin structure over broad SSR regions drives unregulated and dispersed transcription initiation. We investigate such an exceptional feature in trypanosomes by first identifying RNA pol II-enriched regions using ChIP-Seq, as potential promoter sequences. The high resolution of this technique allowed us to accurately determine peaks of RNA pol II accumulation in the dSSRs. To functionally investigate pol II-enriched sequences unbiasedly, the peaks on chromosome VII were assayed for their ability to direct transcription using transient transfection. This analysis suggests that two unidirectional short sequence specific promoters within each dSSR make up the general structure. Primer extension analysis of nascent RNA allowed us to identify precise transcription start sites (TSS) of promoters inserted in a chromosome. Detailed analysis of one of these promoters defined 75bp as sufficient to fully drive transcription and identified essential nucleotides for precise initiation around the TSS. In addition, mutations to internal and downstream boxes led to dramatic decreased activity. In summary, we show that sequence-specific unidirectional RNA pol II promoters with proper TSS transcription initiation are present in the T. brucei genome. Our results challenge the currently accepted hypothesis that trypanosomes lack true promoters with transcription initiation control

Tf uptake and mRNA <i>ESAG6/7</i> expression levels.

<p>(A) Tf uptake of different ACLs. Mean ± SD of three (goat and pig) or four (human) sera independent adaptation experiments and three independent measures of calf serum ACL are shown. Uptake is expressed in FAU (fluorescence arbitrary units). One-way ANOVA test was performed (* p< 0.05; ** p<0.01). (B) Normalized Tf uptake of different ACLs. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085072#pone-0085072-g006" target="_blank">Figure 6A</a> were normalized with dextran uptake at 10 minutes of incubation (data not shown). One-way ANOVA test was performed (* p< 0.05; ** p<0.01). Human holo-Tf (iron saturated) was used at final concentration of 20 μg/ml. (C) Histogram showing relative expression of mRNA measured by qRT-PCR of <i>ESAG6</i>, <i>ESAG7</i> and <i>TbHpHbR</i> in all ACLs. Mean ± SD of two different measures is shown. Expression values are plotted in relative expression units, calculated relative to <i>ATM</i> (<i>PI3Kinase-</i>like -Tbb927.2.2260-) expression. <i>Myosin</i> housekeeping gene is also shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085072#B25" target="_blank">25</a>]. ACL: adapted cell line, <i>TbHpHbR</i>: Haptoglobin-Hemoglobin receptor, FCS: foetal calf serum, GS1/2/3: goat serum (adaptation experiments 1, 2 or 3), HS1/2/3/4: human serum (adaptation experiments 1, 2, 3 or 4), PS1/2/3: pig serum (adaptation experiments 1, 2 or 3).</p

<i>ESAG</i> diversity in ACLs.

<p>Graphic representation of <i>ESAG</i> genotype diversity. Each ring is divided in 2-4 inner circles representing each independent experiment. Unless otherwise is indicated, ACLs distribution in each ring is as indicated in <i>ESAG7</i> row. Each genotype is represented in a different colour according to indicated in the graph. Partial ORF was used for DNA genotyping (673-676 bp in <i>ESAG6/7</i> and 853 bp in <i>ESAG3</i>). FCS: foetal calf serum, GS1/2/3: goat serum (adaptation experiments 1, 2 or 3), HS1/2/3/4: human serum (adaptation experiments 1, 2, 3 or 4), PS1/2/3: pig serum (adaptation experiments 1, 2 or 3). </p