Tracking the In Vivo Dynamics of Antigenic Variation in the African Trypanosome

Trypanosoma brucei, a causative agent of African sleeping sickness in humans and nagana in animals, constantly changes its dense variant surface glycoprotein (VSG) coat to avoid elimination by the immune system of its mammalian host, using an extensive repertoire of dedicated genes. Although this process, referred to as antigenic variation, is the major mechanism of pathogenesis for T. brucei, the dynamics of VSG expression in T. brucei during an infection are poorly understood. In this thesis, I describe the development of VSG-seq, a method for quantitatively examining the diversity of expressed VSGs in any population of trypanosomes. Using VSG-seq, I monitored VSG expression dynamics in vivo during both acute and chronic mouse infections. My experiments revealed unexpected diversity within parasite populations, and the expression of as much as one-third of the functional genomic VSG repertoire after only one month of infection. In addition to suggesting that the host-pathogen interaction in T. brucei infection is substantially more dynamic and nuanced than previously expected, this observed diversity highlighted the importance of the mechanisms by which T. brucei diversifies its genome-encoded VSG repertoire. During infection, the parasite can form mosaic VSGs, novel variants that arise through recombination events within the parasite genome during infection. Though these novel variants had been identified previously, little was known about the mechanisms by which they form. VSG-seq facilitated the identification of mosaic VSGs during the infection, which allowed me to track their formation over time. My results provide the first temporal data on the formation of these variants and suggest that mosaic VSGs likely form at sites of VSG transcription. VSG-seq, which is based on the de novo assembly of VSGs, obviates the requirement for a reference genome for the analysis of expressed VSG populations. This allows the method to be used for the high-resolution study of VSG expression in any strain of T. brucei, whether in the lab or in the field. To this end, I have applied VSG-seq to samples grown in vitro, parasites isolated from natural infections, and extravascular parasites occupying various tissues in vivo. These extensions of the method reveal new aspects of T. brucei biology and demonstrate the potential of high-throughput approaches for studying antigenic variation, both in trypanosomes and in any pathogen that uses antigenic variation as a means of immune evasion

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

A dynamic T cell–limited checkpoint regulates affinity-dependent B cell entry into the germinal center

Entry into the germinal center requires antigen-bearing B cells to compete for cognate T cell help at the T–B border

A model of the VSG–antibody interaction.

<p>A hypothetical model of immunoglobulin M (IgM) antibody (teal) binding to VSG (pink and blue). The precise arrangement of VSG on the cell membrane (gray) is unknown, but the packing of VSG on the cell membrane is known to be extremely dense. It is unknown whether IgM binds VSG in this particular configuration, but the dense packing of VSG may nevertheless affect the accessibility of antibody to the C-terminus (pink), as illustrated here. The multimeric IgM is also likely to interact with more than one VSG on the cell surface, resulting in the depicted “staple” conformation. The figure was produced by combining (1) the pentameric C-alpha model of IgM (PDB ID 2RCJ), (2) modeling the remainder of the chains using the FG-MD Server [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005784#ppat.1005784.ref008" target="_blank">8</a>] over (3) a planar array of manually positioned VSG N-terminal and C-terminal domains based on the crystal structures of VSG221 (PDB ID 1VSG, 1XU6).</p

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

Effects of engineered 70-bp repeat sequences on <i>VSG</i> switching and donor selection.

<p>A) BES1 maps of cell lines bearing repeat deletions (Δ70) and introductions, shown as pink boxes, or in reverse (pink arrow). B) Observed switching frequencies of doxycycline induced strains bearing alternative repeat regions: wild-type repeats (70.II, ★), no repeats (Δ70, ●), monomeric motif (Monomer, ◆), dimeric motif (Dimer, ▴), and reverse dimeric motif (Dimer_Rv). Difference in switching between Δ70 and Dimer motif introduction is statistically significant (asterisk over bracket pval = 0.027). C) Flow-cytometry analysis of switched cells (427–2 negative population) and cell cycle (DNA content frequency histogram measured by PI fluorescence) at 48 hours following doxycycline induction. D) VSG-seq analysis of MACS-isolated switchers from three biological replicates of Δ70, Dimer, & Dimer_Rv is shown in the form of a heat diagram where the color intensity reflects the proportion of each <i>VSG</i> RNA in the population. The <i>Lister427</i> VSG number and its predicted genomic location are shown on the right of the heat diagram where var indicates that the assembled <i>VSG</i> sequence had minor sequence variations from the most similar 427 reference <i>VSG</i>.</p

Conservation and genomic distribution of the 70-bp repeat sequence.

<p>A) Map of <i>T</i>. <i>brucei Lister 427</i> BES1 with promoter (bent arrow), terminal ESAG (ESAG1), <i>VSG</i> (blue), <i>VSG</i> pseudogene (pink), telomere (black circle), and 70-bp repeat regions (yellow) illustrated (70.I contains 3 repeats and 70.II contains 39 repeats). B) The identified 70-bp consensus sequence from shown as a logo created from the 42 repeats found in BES1 (weblogo.berkely.edu). C) Graph of the number of 70b-bp repeats (determined by e-value>40, identities> 70%, and length>40bp) enumerated per chromosome from the genomes of <i>T</i>. <i>brucei brucei TREU927</i> (blue), <i>T</i>. <i>brucei brucei Lister427</i> (green), <i>T</i>. <i>evansi STIB805</i> (orange), <i>T</i>. <i>brucei gambiense DAL972</i> (red), <i>T</i>. <i>congolense IL3000</i> (yellow), and <i>T</i>. <i>vivax Y486</i> (light blue).</p

BES1 repeat content following GC with BES7.

<p>Field-inversion gel electrophoresis and Southern blot analysis of <i>VSG427-3</i> switched clones arising from A) BES1-70.II-ISceI or B) BES1-Δ70-ISceI GC with silent BES7 donor digested BglII digest or HindIII, respectively. BES7 probe (red bar) proximal to <i>427–3</i> results in the formation of a new band whose size approximates the region of BES7 transferred during gene conversion. Diagrams to the right of Southern blots show the predicted composition of the newly formed band.</p

Growth and <i>VSG</i> switching in 70-bp repeat analysis lines.

<p>A) Growth of 70-bp repeat-analysis strains is shown over 5 days of consistent cell passage with (dashed lines) and without (solid lines) doxycycline induction for the ISCEI-bearing parental line (black ●), 70.II-ISceI (blue ■), and Δ70-ISceI (red ▲), as well as Δ70-No ISceI (green ◆). B) DNA content frequency histogram measured by PI fluorescence used to estimate the percentage of cells in G₁ and G₂/M at 24 & 48 hours following doxycycline induction for ISceI bearing strains (red) in comparison with parental strain (blue). C) Switching frequencies of 70-bp repeat analysis strains (PA = ♦, 70.II = ●, 70.I = ▲, Δ70 = ■) during growth with (filled symbol) or without (hollow symbol) doxycycline induction (three days) is normalized to the number of population doublings (***<0.0001, F-test) as determined from growth data in panel A. D) Flow-cytometry analysis of proportion of cells without <i>VSG427-2</i> (x-axis) and staining with propidium iodide (y-axis), as a measure of <i>VSG</i> switching and cell death, respectively, shown as a zebra plot with frequencies of each population per quadrant shown.</p