A Conserved DNA Repeat Promotes Selection of a Diverse Repertoire of Trypanosoma brucei Surface Antigens from the Genomic Archive.

African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG) genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive

An ORF-based whole-genome gain-of-function library for Trypanosoma brucei

Despite decades of active discovery in all areas of trypanosome research, more than 50% of the Trypanosoma brucei genome is annotated as hypothetical genes of unknown function. Further progress in understanding both the pathogenesis and basic biology of Trypanosoma species requires the development of versatile approaches for genome-scale functional analysis. A whole-genome RNAi loss-of-function library has proved instrumental in the identification of new genetic functions and pathways in T. brucei. Previously, use of shotgun cloning and digested genomic DNA have been implemented in the formation of whole-genome over expression libraries with some success. To better capture the majority of T. brucei gene full open reading frames (ORFs), we have produced an ORF-based whole-genome gain-of-function library that contains \u3e90% of the targeted genes. The library is induced in the context of a genetic screen to isolate cells resulting in a desired phenotype. Following a genetic screen, next-generation sequencing libraries are enriched using a unique oligo that targets the gain-of-function library for PCR amplification and Illumina platform sequencing. Preliminary experiments returned insufficient read depth and suggested that further optimization of sequencing libraries was required. To this end, we developed a qPCR method for assessing the amount of gain-of-function specific DNA in each next-generation library preparation. Assessment of library quality by qPCR enabled further optimization of sequencing libraries, through modifications to the enrichment PCR, and more accurate pooling of samples for Illumina sequencing. This tool was then applied to a preliminary screen for the identification of genes associated with melarsoprol resistance, in which a specific melarsoprol resistant population was isolated following induction of the gain-of-function library. The tools and optimizations made for gain-of-function library implementation will be discussed as well as early findings from a melarsoprol genetic screen

Analysis of DNA break repair inhibition in Trypanosoma brucei

African trypanosomes (Trypanosoma brucei spp.), the cause of African Sleeping sickness, are masters of antigenic variation and can change their dense variant surface glycoprotein (VSG) coat to new variants, thereby escaping host immunity. Switching VSG gene expression is thought to occur predominantly following DNA break formation at naturally unstable telomeric ends. Trypanosomes appear to not have a classical non-homologous end-joining pathway (NHEJ), which predominates in mammalian DNA double-stranded break (DSB) repair. Rather, DSB repair in T. brucei occurs through homologous recombination (HR) and microhomology mediated end-joining (MMEJ). Mechanisms of HR predominate in DSB repair and are currently the only known mechanisms that can result in the activation of a new VSG gene, a process called “VSG switching”. Thus, tracking DNA break formation events that occur can elucidate whether the break will be repaired in a manner that results in blunt end-joining or the expression of a new VSG through HR. While T. brucei DNA break repair pathways are only partially known, the factors that regulate the outcome of a DNA break repair are unknown. Here, we investigate the consequences of inhibiting two subsequent steps of HR, end resection by MRE11 and the coating of single-stranded DNA filaments with RAD51, which then promotes homologous pairing. We sought to determine if known chemical inhibitors of mammalian DSB repair, developed in cancer research, function in T. brucei based on a collection of phenotypes, both previously published and novel to this investigation. We determined that both RAD51 inhibitor RI-1 and the MRE11 inhibitor mirin, have the anticipated effects on cell growth and sensitivity to DNA damage. However, we also observed distinct behaviors of each inhibitor on both the processing of DNA breaks and the progression of cell cycle. These data further support previous T. brucei literature that suggests MRE11 does not exclusively function upstream of RAD51. Furthermore, our findings indicate both MRE11 and RAD51 may integrate signals for non-canonical processes of DNA break repair and cell cycle progression checkpoints, whose identification could yield novel drug targets against African trypanosomiasis. Chemical inhibitors thus provide a new avenue for further studying the factors regulating VSG switching in T. brucei

New genetic tools to study gene function in fatal African sleeping sickness parasite

African trypanosomes are unicellular parasites that result in death if left untreated. Treatment of African trypanosomiasis relies on six effective drug treatments, which can be highly toxic and difficult to administer. While there are on-going studies to develop new drugs, resistance to existing drugs is a persistent threat. Trypanosomes are an early branching eukaryote with only limited homology to highly studied genomes; more than 60% of the Trypanosoma brucei genome remains annotated as hypothetical genes of unknown function. To better understand basic biology, pathogenesis, and resistance of this parasite, we need to uncover gene functions and regulations. To accomplish this goal, we have developed a whole-genome gain-of-function library to apply in forward genetic screens and identify genes associated with a wide range of phenotypes. Gene expression regulation in Trypanosomes required that we build an ORF-based (PCR amplified) library, which was cloned into a Gateway library for introduction into the Trypanosoma brucei genome by transfection. Preliminary genetic screens demonstrated that we were able to successfully incorporate 90% of the intended gene products into the genome with greater than 10-fold coverage. However, we encountered two challenges: 1) individual transfections result in different sets of genes represented and 2) inconsistent gene expression among transfections. Here, we have implemented steps for the overall normalization and consistent usage of our cloned overexpression library. Primarily, we undertook a process to produce a cell line that will permit the consistent, highly inducible expression of our library among multiple transfections. In addition, we have streamlined our transfection method to focus on producing a consistent population of cells that all harbor the same subset of library genes with the desired level of coverage. To further normalize the variance, we made our library cell line and expanded it to create a large bank of library cells for future genetic screens. With these steps now completed, we have prepared next-generation sequencing libraries to fully assess the expression and consistency. With this forward genetic tool in hand, we will be able to conduct genetic screens to uncover pathways of drug resistance, pathogenesis, and other key biological features of this devastating human parasite

CONTENT ALERTS

This article cites 47 articles, 23 of which can be accessed fre

ArgR-Regulated Genes Are Derepressed in the Legionella-Containing Vacuole▿ †

Legionella pneumophila is an intracellular pathogen that infects protozoa in aquatic environments and when inhaled by susceptible human hosts replicates in alveolar macrophages and can result in the often fatal pneumonia called Legionnaires' disease. The ability of L. pneumophila to replicate within host cells requires the establishment of a specialized compartment that evades normal phagolysosome fusion called the Legionella-containing vacuole (LCV). Elucidation of the biochemical composition of the LCV and the identification of the regulatory signals sensed during intracellular replication are inherently challenging. l-Arginine is a critical nutrient in the metabolism of both prokaryotic and eukaryotic organisms. We showed that the L. pneumophila arginine repressor homolog, ArgR, is required for maximal intracellular growth in the unicellular host Acanthamoeba castellanii. In this study, we present evidence that the concentration of l-arginine in the LCV is sensed by ArgR to produce an intracellular transcriptional response. We characterized the L. pneumophila ArgR regulon by global gene expression analysis, identified genes highly affected by ArgR, showed that ArgR repression is dependent upon the presence of l-arginine, and demonstrated that ArgR-regulated genes are derepressed during intracellular growth. Additional targets of ArgR that may account for the argR mutant's intracellular multiplication defect are discussed. These results suggest that l-arginine availability functions as a regulatory signal during Legionella intracellular growth

Evaluation of mechanisms that may generate DNA lesions triggering antigenic variation in African trypanosomes.

Antigenic variation by variant surface glycoprotein (VSG) coat switching in African trypanosomes is one of the most elaborate immune evasion strategies found among pathogens. Changes in the identity of the transcribed VSG gene, which is always flanked by 70-bp and telomeric repeats, can be achieved either by transcriptional or DNA recombination mechanisms. The major route of VSG switching is DNA recombination, which occurs in the bloodstream VSG expression site (ES), a multigenic site transcribed by RNA polymerase I. Recombinogenic VSG switching is frequently catalyzed by homologous recombination (HR), a reaction normally triggered by DNA breaks. However, a clear understanding of how such breaks arise—including whether there is a dedicated and ES-focused mechanism—is lacking. Here, we synthesize data emerging from recent studies that have proposed a range of mechanisms that could generate these breaks: action of a nuclease or nucleases; repetitive DNA, most notably the 70-bp repeats, providing an intra-ES source of instability; DNA breaks derived from the VSG-adjacent telomere; DNA breaks arising from high transcription levels at the active ES; and DNA lesions arising from replication–transcription conflicts in the ES. We discuss the evidence that underpins these switch-initiation models and consider what features and mechanisms might be shared or might allow the models to be tested further. Evaluation of all these models highlights that we still have much to learn about the earliest acting step in VSG switching, which may have the greatest potential for therapeutic intervention in order to undermine the key reaction used by trypanosomes for their survival and propagation in the mammalian host

A Trypanosoma brucei ORFeome-Based Gain-of-Function Library Identifies Genes That Promote Survival during Melarsoprol Treatment.

ABSTRACT Trypanosoma brucei is an early branching protozoan parasite that causes human and animal African trypanosomiasis. Forward genetics approaches are powerful tools for uncovering novel aspects of trypanosomatid biology, pathogenesis, and therapeutic approaches against trypanosomiasis. Here, we have generated a T. brucei cloned ORFeome consisting of >90% of the targeted 7,245 genes and used it to make an inducible gain-of-function parasite library broadly applicable to large-scale forward genetic screens. We conducted a proof-of-principle genetic screen to identify genes whose expression promotes survival in melarsoprol, a critical drug of last resort. The 57 genes identified as overrepresented in melarsoprol survivor populations included the gene encoding the rate-limiting enzyme for the biosynthesis of an established drug target (trypanothione), validating the tool. In addition, novel genes associated with gene expression, flagellum localization, and mitochondrion localization were identified, and a subset of those genes increased melarsoprol resistance upon overexpression in culture. These findings offer new insights into trypanosomatid basic biology, implications for drug targets, and direct or indirect drug resistance mechanisms. This study generated a T. brucei ORFeome and gain-of-function parasite library, demonstrated the library’s usefulness in forward genetic screening, and identified novel aspects of melarsoprol resistance that will be the subject of future investigations. These powerful genetic tools can be used to broadly advance trypanosomatid research. IMPORTANCE Trypanosomatid parasites threaten the health of more than 1 billion people worldwide. Because their genomes are highly diverged from those of well-established eukaryotes, conservation is not always useful in assigning gene functions. However, it is precisely among the trypanosomatid-specific genes that ideal therapeutic targets might be found. Forward genetics approaches are an effective way to identify novel gene functions. We used an ORFeome approach to clone a large percentage of Trypanosoma brucei genes and generate a gain-of-function parasite library. This library was used in a genetic screen to identify genes that promote resistance to the clinically significant yet highly toxic drug melarsoprol. Hits arising from the screen demonstrated the library’s usefulness in identifying known pathways and uncovered novel aspects of resistance mediated by proteins localized to the flagellum and mitochondrion. The powerful new genetic tools generated herein are expected to promote advances in trypanosomatid biology and therapeutic development in the years to come

A Conserved DNA Repeat Promotes Selection of a Diverse Repertoire of <i>Trypanosoma brucei</i> Surface Antigens from the Genomic Archive

<div><p>African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (<i>VSG</i>) genes and switch from the expression of one <i>VSG</i> to another. Switching <i>VSG</i> expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed <i>VSG</i>, and a donor gene, drawn from an archive of more than 2,000 silent <i>VSG</i>s. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to <i>VSG</i>s within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during <i>VSG</i> switching led to the prediction that <i>VSG</i>-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival <i>VSG</i>s. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between <i>VSG</i> switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival <i>VSGs</i> are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive.</p></div