Parasite motility is critical for virulence of African trypanosomes

Abstract

African trypanosomes, Trypanosoma brucei spp., are lethal pathogens that cause substantial human suffering and limit economic development in some of the world's most impoverished regions. The name Trypanosoma ("auger cell") derives from the parasite's distinctive motility, which is driven by a single flagellum. However, despite decades of study, a requirement for trypanosome motility in mammalian host infection has not been established. LC1 is a conserved dynein subunit required for flagellar motility. Prior studies with a conditional RNAi-based LC1 mutant, RNAi-K/R, revealed that parasites with defective motility could infect mice. However, RNAi-K/R retained residual expression of wild-type LC1 and residual motility, thus precluding definitive interpretation. To overcome these limitations, here we generate constitutive mutants in which both LC1 alleles are replaced with mutant versions. These double knock-in mutants show reduced motility compared to RNAi-K/R and are viable in culture, but are unable to maintain bloodstream infection in mice. The virulence defect is independent of infection route but dependent on an intact host immune system. By comparing different mutants, we also reveal a critical dependence on the LC1 N-terminus for motility and virulence. Our findings demonstrate that trypanosome motility is critical for establishment and maintenance of bloodstream infection, implicating dynein-dependent flagellar motility as a potential drug target.

Figure 1

Constitutive LC1 double knock-in (DKI) motility mutant. (a) Schematic of strategy for replacing both LC1 alleles with an LC1 mutant transgene. Asterisks represent the K203A and R210A substitutions in LC1. Gray arrows show position of primers used for PCR in panel b. (b) PCR amplification of the LC1 locus from parental cells (WT), single knock-in cells (SKI) and double knock-in cells (DKI). Knock-in constructs are larger than WT owing to presence of the drug resistance marker and intergenic region. Primer positions are shown in panel a. Right side shows sequence of codons 203 and 210 of the LC1 gene in parental cells (WT) and the double knock-in (DKI). (c) Mean squared displacement (MSD) was determined for the indicated cell lines as described in Methods. Bold lines represent the mean MSD of two biological replicates from two independent experiments (>70 cells tracked per line per experiment for a total of >175 cells tracked per cell line). Dashed lines show the standard error of the mean between two independent replicates. Inset shows growth curve of the indicated cell lines in culture. Error bars show standard deviation of three replicates. For WT and DKI, standard deviation is smaller than the size of the symbols.

Figure 2

Virulence defect of LC1 DKI motility mutants. (a) Survival curves for mice infected intraperitoneally with the indicated cell lines. Graphs show data from two independent experiments, RNAi-K/R vs DKI vs SKI (inoculum = 100 parasites, n = 4 mice each) and WT vs DKI-62 (inoculum = 1000 parasites, n = 3 mice each). Data are representative of the phenotypes observed for WT, RNAi-K/R and DKI cell lines across multiple independent experiments (Figs 2c, 3c, and data not shown). DKI and DKI-62 are independently derived LC1 K203A/R210A double knock-in mutants. (b) Parasitemia of mice used for panel a. Parasitemia in blood was measured beginning four days post infection. Detection limit is ∼1e5 cells/ml. (c) Survival curves for mice infected intraperitoneally with the indicated cell lines. Addback is a DKI mutant line into which a WT copy of LC1 has been introduced at an ectopic locus, as described in Methods. (d) Immunofluorescence microscopy of detergent-extracted cytoskeletons. HA-tagged LC1 (green), PFR (red), DAPI (blue). The white arrows and arrowheads mark the proximal ends of LC1 and PFR staining, respectively. RNAi-K/R cells were examined 72 h after Tet-induction^,. Images were collected under identical conditions and processed identically.

Figure 3

The LC1 N-terminus is important for motility. (a) Drawing depicts the flagellar axoneme of T. brucei and model for LC1 function with C-terminus (C) and N-terminus (N) contributing to interaction with the outer dynein gamma heavy chain (γ-HC) and microtubule doublet, respectively. Based on^,. (b) Mean squared displacement (MSD) was determined for the indicated cell lines as described in Methods. Bold lines represent the average MSD across seven biological replicates from three independent experiments (>400 cells tracked per cell line per replicate for a total of >4200 cells tracked per cell line). Dashed lines represent the standard error of the mean between seven replicates. Inset shows the HA-tag and point mutant status of LC1 in each cell line. Asterisks represent K203A and R210A substitutions in LC1. (c) Survival curves for mice infected with the indicated cell lines. The HA-tag and point mutant status are as indicated in the panel b inset.

Figure 4

The LC1 DKI motility mutant is defective in establishing and maintaining bloodstream infection. (a) Survival curves for mice infected intravenously with the indicated cell lines. Parasitemias are shown in Supplementary Fig. S3. (b) Time course of VSG-Ig clearance after shift to 37 °C. Error bars show standard error of the mean of three replicates. (c) Survival curves for control mice and mice lacking B cells (B cell−) that were infected intraperitoneally with the indicated trypanosome cell lines (WT or DKI). Data are from two independent experiments (n = 3 mice per experiment). Parasitemias from one of the experiments are shown in Supplementary Fig. S4.