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

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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

Transcriptional Regulation of the tad Locus in Aggregatibacter actinomycetemcomitans: a Termination Cascade▿

The tad (tight adherence) locus of Aggregatibacter actinomycetemcomitans includes genes for the biogenesis of Flp pili, which are necessary for bacterial adhesion to surfaces, biofilm formation, and pathogenesis. Although studies have elucidated the functions of some of the Tad proteins, little is known about the regulation of the tad locus in A. actinomycetemcomitans. A promoter upstream of the tad locus was previously identified and shown to function in Escherichia coli. Using a specially constructed reporter plasmid, we show here that this promoter (tadp) functions in A. actinomycetemcomitans. To study expression of the pilin gene (flp-1) relative to that of tad secretion complex genes, we used Northern hybridization analysis and a lacZ reporter assay. We identified three terminators, two of which (T1 and T2) can explain flp-1 mRNA abundance, while the third (T3) is at the end of the locus. T1 and T3 have the appearance and behavior of intrinsic terminators, while T2 has a different structure and is inhibited by bicyclomycin, indicating that T2 is probably Rho dependent. To help achieve the appropriate stoichiometry of the Tad proteins, we show that a transcriptional-termination cascade is important to the proper expression of the tad genes. These data indicate a previously unreported mechanism of regulation in A. actinomycetemcomitans and lead to a more complete understanding of its Flp pilus biogenesis

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 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 70-bp repeats on <i>VSG</i> donor selection and switching.

<p>A) Map of BES1 modifications (in comparison to PA) used in analyzed strains illustrate ISceI site introduction with puromycin marker (green), HpaI restriction site location (red) used in Southern blot conformation is indicated along with anticipated sizes following digestion. B) Southern blots of representative clones from each BES1 modified line are shown for HpaI and HpaI/ISceI digestions probed with terminal BES1 <i>VSG</i> (427–2). C) Semi-quantitative RT-PCR of ISCEI enzyme expression for a 5-fold dilution of input RNA under non-inducting (- Dox) and inducing (+ Dox) conditions alongside a tubulin control. D) Heat map showing the number of populations in which clones (n = number of clones analyzed per strain) expressing a specific <i>VSG</i> arose: <i>Lister427 VSG reference number</i> and the predicted genomic locus (BES, MC, or undetermined [UD]) of the <i>VSG</i> are shown together with the number of populations (out of 5 total), in which it arose as depicted by color intensity using the color key (bottom).</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

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

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