Joana Faria

University of York

The largest gene families in eukaryotes are subject to allelic exclusion, but mechanisms underpinning single allele selection and inheritance remain unclear. Here, we describe a protein complex sustaining variant surface glycoprotein (VSG) allelic exclusion and antigenic variation in Trypanosoma brucei parasites. The VSG-exclusion-1 (VEX1) protein...

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections1. African trypanosomes—parasites that cause lethal diseases in humans and livestock—employ an antigenic variation mechanism that involves monogenic antigen expression...

Survival of the African trypanosome within its mammalian hosts, and hence transmission between hosts, relies upon antigenic variation, where stochastic changes in the composition of their protective variant-surface glycoprotein (VSG) coat thwart effective removal of the pathogen by adaptive immunity. Antigenic variation has evolved remarkable mecha...

Several persistent pathogens employ antigenic variation to continually evade mammalian host adaptive immune responses. African trypanosomes use variant surface glycoproteins (VSGs) for this purpose, transcribing one telomeric VSG expression-site at a time, and exploiting a reservoir of (sub)telomeric VSG templates to switch the active VSG. It has b...

UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation, and also in monogenic variant surface glycoprotein (VSG) expression via VSG exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. We show that VEX2 associates with chromatin specifically at the single active VSG expression si...

Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a m...

African trypanosomes are early divergent protozoan parasites responsible for high mortality and morbidity as well as a great economic burden among the world's poorest populations. Trypanosomes undergo antigenic variation in their mammalian hosts, a highly sophisticated immune evasion mechanism. Their nuclear organization and mechanisms for gene exp...

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections. African trypanosomes, parasites that cause lethal diseases in humans and livestock, employ an antigenic variation mechanism that involves monogenic antigen expression...

Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transp...

THT knockdown in bloodstream and insect stage cells. (A) The protein blot shows native tagged mNGTHT2 in bloodstream form cells following THT1 knockdown for 5 days. (B) The protein blot shows depletion of native tagged mNGTHT2 following THT1/THT2 knockdown in insect stage cells; see Fig 2C for depletion of mNGTHT1 by the same approach in bloodstrea...

Measurement of incorporation of 13C glycerol into the mannose and galactose residues of VSG. Total ion chromatograms of the methyl-glycoside TMS derivatives from wild-type (panels A-B) and THT1/THT2 knockdown trypanosomes (panels C-D) grown in the absence and presence of 13C glycerol, respectively. The peaks due to mannose (Man), galactose (Gal) an...

LC-MS metabolomics data with 13C3-glycerol. The samples are from WT cells, WT grown in 13C-glycerol, THT1/THT2 RNAi grown in 12C-glycerol and THT1/THT2 RNAi grown in 13C-glycerol, n = 4. (XLSX)

Additional metabolites detected by the LC-MS analysis. The size of the bars represents the total abundance, and coloured parts indicate 13C labelling as depicted in the legend. The samples are from WT cells, WT grown in 13C-glycerol, THT1/THT2 RNAi grown in 12C-glycerol and THT1/THT2 RNAi grown in 13C-glycerol. Natural abundance of 13C is 1%, hence...

Protein abundance differs from a few to millions of copies per cell. Trypanosoma brucei presents an excellent model for studies on codon bias and differential gene expression because transcription is broadly unregulated and uniform across the genome. T. brucei is also a major human and animal protozoal pathogen. Here, an experimental assessment, us...

The de novo crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1 (LiSir2rp1) has been solved at 1.99Å in complex with an acetyl-lysine peptide substrate. The structure is broadly commensurate with Hst2/SIRT2 proteins of yeast and human origin, reproducing many of the structural features common to these sirtu...

Trypsin hyper-sensitivity of the internal serine-rich region linking stable N and C-terminal regions provides experimental evidence for an intrinsically disordered region. Native trypsin digestion of the hexa histidine tagged LiSIR2rp1 protein was carried out as described for Fig 1 (see Methods). (A) Time resolved native digests showing (i) the rap...

LiSIR2rp1 human (A) and yeast (B) homologue based models. Models (i) and (ii) are based on hSIRT2 pdb 3zgo and model (iii) on hSIRT2 pdb 1j8f. Models (iv), (v) and (vi) are based on ScHst2 pdb 1q14, 1q17 and 1q1a, respectively. The large Rossmann-fold and small zinc-binding domains are colored in turquoise and pink respectively. The LiSIR2RP1 inser...

Structure-mapping of the stable fragments of native tryptic digests. The two proteolytically-stable domains are colored in the same way as Fig 1. The p53 peptide substrate is in grey with acetyl-lysine side chain depicted as sticks. (TIF)

Validation report for crystal structure PDB reference 5Ol0 from worldwide protein data bank. (PDF)

Oligonucleotide sequences used in the generation of deletion constructs for crystallography (primers 1 and 2 coupled with the corresponding 2 primers for each deletion mutant). (PDF)

Intrinsic disorder prediction for LiSIR2rp1 using the DisEmble Remark 465 algorithm. (PDF)

Superposition of LiSIR2rp1(ΔP253-E303)/p53 with various ScHst2 complex structures indicate conformational changes of LiSIR2rp1 upon substrate binding. LiSIR2rp1(ΔP253-E303)/p53 is colored as in Fig 3 while ScHst2 is shown in grey. The acetyl-lysine residue, substrate analogue (carba-NAD) and product (2’-O-acetyl-ADP-ribose) are shown as sticks. A....

Comparison of the β8—β9 connector of the Rossmann-fold domain of SIR2 (ScHst2/hSIRT2) identifies a serine-rich peptide extension unique to SIR2rp1 proteins of kinetoplastid/trypanosomatid parasites. (PDF)

Oligonucleotide sequences used in the generation of constructs and quantitative PCR. (PDF)

Analysis of native tryptic digest of LiSIR2rp1 by LC/ ESI-TOF MS. (PDF)

Design of LiSIR2rp1 deleted mutants for structural studies. (PDF)

Phosphorylation site prediction in LiSIR2rp1 using the DISorder-enhanced PHOSphorylation predictor detects 13 potential sites all within the β8—β9 connector region. (PDF)

Sheet 1: Synthetic genes. Sequences of gLUC and GFP genes with high, medium or low proportions of GC3-codons. Sheet 2: T. brucei expression data. CAI values, proteome and transcriptome data and predicted expression levels are tabulated for the non-redundant gene sets analysed in Figures 5–6. Sheet 3: T. brucei gene cohorts. Data for the genes analy...

The trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp, are causative agents of important human diseases such African sleeping sickness, Chagas' disease and Leishmaniasis, respectively. The high impact of these diseases on human health and economy worldwide, the unsatisfactory available chemotherapeutic options and the absenc...

Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null muta...

A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A,...

Oligonucleotides sequences used to obtain gene replacement cassettes (P1-P8) and to confirm LiASA mutants’ genotype (P9-P20). (DOCX)

Semi-quantification of AS-A levels in LiASA mutants cultured in vitro in Asn replete or depleting conditions. (DOCX)

Cumulative in vitro growth of logarithmic LiASA mutants in normal or Asn depleted medium. A/D and B/E) L. infantum promastigotes growth curves of LiASA mutants (versus WT), cultured in cRPMI and cRPMI + L-asparaginase, respectively. Parasites were maintained in logarithmic phase by subculturing every 2 days. The results correspond to mean values of...

Oligonucleotides sequences used to obtain LiAS-A recombinant protein (P1-P2) and pSPαBLASTαLiASA (P3-P4). (DOCX)

Rabbit polyclonal anti-LiAS-A antibody validation. A) Western-blot analysis of WT and LiASA null mutant (clone A2) promastigotes extracts using rabbit polyclonal anti-LiAS-A (1:1000). B) Representative immunofluorescence images of different genotypes (WT, dKO clone A2 and OE) of mid-log L. infantum promastigotes, using rabbit polyclonal anti-LiAS-A...

Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme i...

Human African trypanosomiasis (HAT) is a vector-transmitted tropical disease caused by the protozoan parasite Trypanosoma brucei. High-throughput screening (HTS) of small-molecule libraries in whole-cell assays is one of the most frequently used approaches in drug discovery for infectious diseases. To aid in drug discovery efforts for HAT, the SYBR...

Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in h...

Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in h...

Gomphrena globosa L. is a popular edible plant used as food colourant and in traditional medicine. In this work, 24 phenolic compounds and eight betacyanins were determined by HPLC-DAD in three different extracts of G. globosa inflorescences. The decoction presented the highest amount of phenolic compounds, kaempferol-3-O-(6-rhamnosyl)hexoside plus...

The leaves of Catharanthus roseus constitute the only source of the well known indolomonoterpenic alkaloids vincristine and vinblastine. In this work we studied the biological potential of the roots, which are used in several countries as decocts or hot water extracts for the treatment of a number of conditions. The aqueous extract strongly inhibit...

Catharanthus roseus is one of the most important medicinal plants worldwide. The leaves of this species are the only source of the indolomonoterpenic alkaloids vincristin (leurocristine) and vinblastin (vincaleucoblastine), whose anticancer activity represents powerful therapeutics to many diseases, such as Hodgkin lymphoma. Usually, the remaining...

Catharanthus roseus is among the most important medicinal plants, mainly due to its anticancer alkaloids.Phenolics and organic acids were studied in this plant’s roots, using HPLC–MS and HPLC–UV, respectively. Neither phenolic acids nor flavonoids could be identified, but an organic acid profile composed of oxalic, cis-aconitic, citric, pyruvic, ma...

Boerhaavia diffusa is a plant which is extensively used in folk medicine. However, when it comes to its phytochemical characterization, little attention has been given to secondary metabolites other than rotenoids and alkaloids. A metabolite profiling and biological study was undertaken in this species' leaves and roots and substantial differences...