Sam Alsford

High-throughput decoding of antitrypanosomal drug efficacy and resistance

The concept of disease-specific chemotherapy was developed a century ago. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work, and the drugs that emerged remain in use for treating human African trypanosomiasis (HAT). The importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies has been recognized, but these mechanisms have remained largely unknown. Here we use all five current HAT drugs for genome-scale RNA interference target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate antitrypanosomal drug action. RIT-seq profiling identifies both known drug importers and the only known pro-drug activator, and links more than fifty additional genes to drug action. A bloodstream stage-specific invariant surface glycoprotein (ISG75) family mediates suramin uptake, and the AP1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis, all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H+-ATPases to pentamidine action, and trypanothione and several putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of antitrypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented molecular insight into the mode of action of antitrypanosomal drugs.

A sirtuin in the African trypanosome is involved in both DNA repair and telomeric gene silencing but is not required for antigenic variation

Silent information regulator 2 (Sir2)‐related proteins or sirtuins function as NAD+‐dependent deacetylases or ADP ribosylases that target a range of substrates, thereby influencing chromatin structure and a diverse range of other biological functions. Genes encoding three Sir2‐related proteins (SIR2rp1–3) have been identified in the parasitic trypanosomatids, early branching protozoa with no previously reported transcriptional silencing machinery. Here we show that, in the mammalian‐infective bloodstream‐stage of the African trypanosome, Trypanosoma brucei, SIR2rp1 localizes to the nucleus while SIR2rp2 and SIR2rp3 are both mitochondrial proteins. The nuclear protein, SIR2rp1, controls DNA repair and repression of RNA polymerase I‐mediated expression immediately adjacent to telomeres. Antigenic variation, however, which involves the silencing and Pol I‐mediated transcriptional switching of subtelomeric variant surface glycoprotein genes, continues to operate independent of SIR2rp1.

Genome-wide RNAi screens in African trypanosomes identify the nifurtimox activator NTR and the eflornithine transporter AAT6.

Graphical abstract Genome-scale RNA interference (RNAi) library screens in bloodstream-form Trypanosoma brucei, using nifurtimox and benznidazole, revealed type I nitroreductase, NTR, knockdown. A similar screen with eflornithine, revealed amino-acid transporter, AAT6, knockdown. . Research highlights ▶ A genome-scale RNA interference library is used to identify loss-of-function resistance mechanisms in bloodstream-form Trypanosoma brucei. ▶ Screens for resistance to nifurtimox or benznidazole identified loss of nitroreductase function. ▶ A screen for resistance to eflornithine identified loss of amino-acid transporter function.

Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei.

A major obstacle to reproducible expression of recombinant transcripts lies in the epigenetic effects of the flanking chromatin following integration. We previously presented a strategy to overcome this problem in bloodstream form Trypanosoma brucei, using a reporter to identify a ribosomal-spacer locus that supports optimal expression and then marking that locus for subsequent targeting. Advantages include elimination of variable-expression position-effects and the easy confirmation of correct integration. We now report a set of validated constructs that exploit this system for expression of dsRNA or recombinant protein. The current construct-set allows expression of intramolecular dsRNA for RNA interference knockdown or expression of proteins that can incorporate c-Myc epitope(s) or a fluorescent-tag for subcellular localisation, interaction and/or other functional analysis. The constructs are integrated at a single, marked locus and deliver reliable and reproducible expression.

NUP-1 Is a Large Coiled-Coil Nucleoskeletal Protein in Trypanosomes with Lamin-Like Functions

NUP1, the first example of a nuclear lamin analog in nonmetazoans, performs roles similar to those of lamins in maintaining the structure and organization of the nucleus in Trypanosoma brucei.

Two essential MYST‐family proteins display distinct roles in histone H4K10 acetylation and telomeric silencing in trypanosomes

Chromatin modification is important for virtually all aspects of DNA metabolism but little is known about the consequences of such modification in trypanosomatids, early branching protozoa of significant medical and veterinary importance. MYST‐family histone acetyltransferases in other species function in transcription regulation, DNA replication, recombination and repair. Trypanosoma brucei HAT3 was recently shown to acetylate histone H4K4 and we now report characterization of all three T. brucei MYST acetyltransferases (HAT1–3). First, GFP‐tagged HAT1–3 all localize to the trypanosome nucleus. While HAT3 is dispensable, both HAT1 and HAT2 are essential for growth. Strains with HAT1 knock‐down display mitosis without nuclear DNA replication and also specific de‐repression of a telomeric reporter gene, a rare example of transcription control in an organism with widespread and constitutive polycistronic transcription. Finally, we show that HAT2 is responsible for H4K10 acetylation. By analogy to the situation in Saccharomyces cerevisiae, we discuss low‐level redundancy of acetyltransferase function in T. brucei and suggest that two MYST‐family acetyltransferases are essential due to the absence of a Gcn5 homologue. The results are also consistent with the idea that HAT1 contributes to establishing boundaries between transcriptionally active and repressed telomeric domains in T. brucei.

Trypanosomatid histones: Trypanosomatid histones

The histones are responsible for packaging and regulating access to eukaryotic genomes. Trypanosomatids are flagellated protists that diverged early from the eukaryotic lineage and include parasites that cause disease in humans and other mammals. Here, we review the properties of histones in parasitic trypanosomatids, from gene organization and sequence to expression, post‐translational modification and function within chromatin. Phylogenetic and experimental analysis indicates that certain specifically conserved histone sequence motifs, particularly within the N‐terminal ‘tail’ domains, possibly represent functionally important modification substrates conserved throughout the eukaryotic lineage. For example, histone H3 contains a highly conserved methylation substrate. Trypanosomatids also possess at least three variant histones. Among these is an orthologue of H2A.Z, a histone involved in protecting ‘active’ chromatin from silencing in yeast. Histones provide docking platforms for a variety of regulatory factors. The presence of histone modification and variant histones in trypanosomatids therefore represents evidence for a network that provides the discrimination required to regulate transcription, recombination, repair and chromosome replication and segregation.

Segregation of minichromosomes in trypanosomes: implications for mitotic mechanisms

In addition to 11 pairs of housekeeping chromosomes, the genome of Trypanosoma brucei contains approximately 100 minichromosomes that are probably involved in the ability of the parasite to evade the hosts immune response. This minichromosomal population is segregated on the mitotic spindle. How this is achieved provides insight into potential segregation mechanisms for small DNA molecules in eukaryotic microorganisms.

DNA Break Site at Fragile Subtelomeres Determines Probability and Mechanism of Antigenic Variation in African Trypanosomes

Antigenic variation in African trypanosomes requires monoallelic transcription and switching of variant surface glycoprotein (VSG) genes. The transcribed VSG, always flanked by ‘70 bp’-repeats and telomeric-repeats, is either replaced through DNA double-strand break (DSB) repair or transcriptionally inactivated. However, little is known about the subtelomeric DSBs that naturally trigger antigenic variation in Trypanosoma brucei, the subsequent DNA damage responses, or how these responses determine the mechanism of VSG switching. We found that DSBs naturally accumulate close to both transcribed and non-transcribed telomeres. We then induced high-efficiency meganuclease-mediated DSBs and monitored DSB-responses and DSB-survivors. By inducing breaks at distinct sites within both transcribed and silent VSG transcription units and assessing local DNA resection, histone modification, G2/M-checkpoint activation, and both RAD51-dependent and independent repair, we reveal how breaks at different sites trigger distinct responses and, in ‘active-site’ survivors, different switching mechanisms. At the active site, we find that promoter-adjacent breaks typically failed to trigger switching, 70 bp-repeat-adjacent breaks almost always triggered switching through 70 bp-repeat recombination (∼60% RAD51-dependent), and telomere-repeat-adjacent breaks triggered switching through loss of the VSG expression site (25% of survivors). Expression site loss was associated with G2/M-checkpoint bypass, while 70 bp-repeat-recombination was associated with DNA-resection, γH2A-focus assembly and a G2/M-checkpoint. Thus, the probability and mechanism of antigenic switching are highly dependent upon the location of the break. We conclude that 70 bp-repeat-adjacent and telomere-repeat-adjacent breaks trigger distinct checkpoint responses and VSG switching pathways. Our results show how subtelomere fragility can generate the triggers for the major antigenic variation mechanisms in the African trypanosome.

Genetic dissection of drug resistance in trypanosomes

SUMMARY The trypanosomes cause two neglected tropical diseases, Chagas disease in the Americas and African trypanosomiasis in sub-Saharan Africa. Over recent years a raft of molecular tools have been developed enabling the genetic dissection of many aspects of trypanosome biology, including the mechanisms underlying resistance to some of the current clinical and veterinary drugs. This has led to the identification and characterization of key resistance determinants, including transporters for the anti-Trypanosoma brucei drugs, melarsoprol, pentamidine and eflornithine, and the activator of nifurtimox-benznidazole, the anti-Trypanosoma cruzi drugs. More recently, advances in sequencing technology, combined with the development of RNA interference libraries in the clinically relevant bloodstream form of T. brucei have led to an exponential increase in the number of proteins known to interact either directly or indirectly with the anti-trypanosomal drugs. In this review, we discuss these findings and the technological developments that are set to further revolutionise our understanding of drug-trypanosome interactions. The new knowledge gained should inform the development of novel interventions against the devastating diseases caused by these parasites.