The in vivo dynamics of antigenic variation in Trypanosoma brucei

Abstract

Trypanosoma brucei, a causative agent of African Sleeping Sickness, 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. However, the dynamics of VSG expression in T. brucei during an infection are poorly understood. We have developed a method, based on de novo assembly of VSGs, for quantitatively examining the diversity of expressed VSGs in any population of trypanosomes and monitored VSG population dynamics in vivo. Our experiments revealed unexpected diversity within parasite populations and a mechanism for diversifying the genome-encoded VSG repertoire. The interaction between T. brucei and its host is substantially more dynamic and nuanced than previously expected.

Fig. 1. VSG-seq for assembly of VSGs and quantification VSG expression in a population of African trypanosomes

(A) Efficiency of VSG assembly (mean +/− SD). Control libraries made from a mixture of cell lines expressing different VSGs in known proportions were sequenced, and sequencing reads were assembled using Trinity(13). Control mixtures were made from either one million or ten million cells. (B) Quantification of VSG expression in control libraries (mean +/− SD). The black bar (“Expected”) represents the proportion of cells expressing that VSG in the control mixture, and the gray bars represent quantification for each library using VSG-seq.

Fig. 2. Complex dynamics throughout T. brucei infection

(A) Dynamics of VSG expression during early infection (d6-30). Each colored line represents an individual VSG's presence in the population, while the black line represents total parasitemia. Only variants present at greater than 0.1% of the population at that time point are shown. When parasitemia could not be measured by hemacytometer (<10⁶/ml), parasitemia is artificially set at 10⁵/ml to allow for visualization of the population. Note that, because there are so many VSGs expressed during infection, colors are difficult to distinguish; overall, variants do not reappear later in the same infection. A smooth curve connects points where expression or parasitemia was measured; these curves are for visualization and do not imply the actual kinetics of variant expression between points. This figure is representative of four infection experiments (Mouse 2 is shown). (B) Dynamics of VSG expression during late infection (d96-105) for Mouse 3. (C) Number of VSGs present at each time point. Any variants quantified as greater than 0.01% of the population are included.

Fig. 3. Variant emergence during infection

(A) Minor variants present at each time point (mean +/− SD). A minor variant is arbitrarily defined as any VSG that never exceeds 1% of the population during the course of infection in a single mouse. Major variants are any variant that exceeds 1% of the population at some point during infection. (B) Venn diagram comparing the fates of VSGs appearing in all four infections. (C) Intersection of sets of VSGs expressed during early infection (d6-d30). The total number of VSGs is listed in parentheses below the mouse number. (D) Venn diagrams showing intersection of VSGs expressed early in infection (VSGs from Mouse 1, 2, or 4 vs VSGs from Mouse 3, d7-30) and intersection of VSGs expressed early in infection with VSGs expressed late in infection (VSGs from Mouse 1, 2, or 4 vs VSGs from Mouse 3, d96-105).

Fig. 4. Mosaic VSGs can be identified throughout infection

(A) Shows transient expression of a mosaic VSG in the population, and PCR confirmation of the mosaic is shown below. The black line represents total parasitemia at each day post infection, and the green line represents the number of parasites expressing the mosaic VSG. “n.q.” (not quantifiable) indicates that the VSG is detectable within the population, but not quantifiable. “n.d.” indicates that the VSG is not detectable within the population. Below the graph are products from PCR of gDNA at each time point, using either primers specific for the mosaic VSG or the control gene, ura3. This VSG could not be amplified when first detected by VSG-seq, likely because of low cell numbers in the DNA sample (probably less than 10 cells). (B) Mosaic from late infection with PCR confirmation of the mosaic shown below.