Transmission Stages Dominate Trypanosome Within-Host Dynamics during Chronic Infections

Summary Sleeping sickness is characterized by waves of the extracellular parasite Trypanosoma brucei in host blood, with infections continuing for months or years until inevitable host death. These waves reflect the dynamic conflict between the outgrowth of a succession of parasite antigenic variants and their control by the host immune system. Although a contributor to these dynamics is the density-dependent differentiation from proliferative “slender forms” to transmissible “stumpy forms,” an absence of markers discriminating stumpy forms has prevented accurate parameterization of this component. Here, we exploit the stumpy-specific PAD1 marker, which functionally defines transmission competence, to quantitatively monitor stumpy formation during chronic infections. This allows reconstruction of the temporal events early in infection. Mathematical modeling of these data describes the parameters controlling trypanosome within-host dynamics and provides strong support for a quorum-sensing-like mechanism. Our data reveal the dominance of transmission stages throughout infection, a consequence being austere use of the parasite's antigen repertoire.

For the purposes of production of a standard curve, the concentration of parasites in the blood was required to be known. In this case, slender or stumpy parasites were harvested and purified from a mouse infection. The cells were counted and a known number of cells were centrifuged at 800g for 10 minutes to pellet the cells. The parasites were then resuspended in a known volume of blood such that the final concentration of parasites per ml blood could be calculated. This blood was divided into 10µl aliquots added to 30µl lysis mix (see above) before being stored at -80°C. These standard curve samples were processed in the same manner as experimental samples.

RNA extraction from whole blood
RNA was extracted using an ABI Prism 6100 Nucleic Acid PrepStation according to manufacturer's instructions. The platform takes 96-well RNA purification trays (ABI 4305673) to which the sample and appropriate washes are applied, and pulls the flowthrough into the waste collection by applying a vacuum across the wells. A splashguard (ABI 4311758) was used in order to prevent contamination between wells. All RNA extractions were carried out using the 'RNA Blood-DNA' programme. For this, wells were pre-wet with 40µl of RNA Purification Wash Solution 1 (ABI 4305891) and then 40µl of lysate was added to each well. An 80% vacuum was applied to the wells for 3 minutes. Wells were washed with 650µl of RNA Purification Wash Solution 1 and the wash was removed by applying an 80% vacuum to the wells for 3 minutes. Wells were then washed with 650µl of RNA Purification Wash Solution 2 (ABI 4305890) and the wash was removed by applying an 80% vacuum to the wells for 3 minutes. Taking care to apply directly to the bottom of the well, 50µl of AbsoluteRNA Wash Solution (ABI 4305545) was added to the wells and incubated for 15 minutes. The AbsoluteRNA Wash Solution contains a DNase which should remove genomic DNA from the sample. Due to the labile nature of DNase this was defrosted on ice with care taken not to destroy the DNase mechanically. Without removing the AbsoluteRNA Wash Solution, 400µl of RNA Purification Wash Solution 2 was added and this was incubated for 5 minutes before removal by applying an 80% vacuum to the wells for 3 minutes. The well was washed twice more with RNA Purification Wash Solution 2, firstly with 650µl and then 400µl, each time removing the wash by applying an 80% vacuum to the wells for 3 minutes. A 90% pre-elution vacuum was applied for 5 minutes to remove any remaining wash solution before moving the purification try from the waste collection position to the sample collection position. To elute the sample, 100µl of Nucleic Acid Purification Elution Solution was added to each well and a 20% vacuum was applied to the wells for 2 minutes. Samples were eluted into a MicroAmp Optical 96-well Reaction Plate (ABI 4306737), sealed with an adhesive film (ABI 4311971) and stored at -80°C until use.

Treatment of RNA with TURBO DNase
An additional DNase treatment was added after RNA extraction using an Ambion TURBO DNA-free kit (Applied Biosystems AM1907) according to manufacturers' instructions. A typical 50µl reaction is shown below.
DNase reaction: 5µl 10X TURBO DNase Buffer 44µl RNA 1µl TURBO DNase The reaction was mixed gently and incubated at 37°C for 30 minutes before deactivating the DNase with 0.1 volume of DNase Inactivation Reagent (in this case, 5µl). The reaction was mixed well and incubated for 5 minutes with occasional mixing to disperse the DNase Inactivation Reagent. The sample was centrifuged at 4000g for 1.5 minutes and the RNA was transferred to a fresh tube. The RNA was stored at -80°C until use and care is taken to avoid repeated freeze thaw cycles. cDNA production cDNA was produced using the ABI High-Capacity cDNA Reverse Transcription kit (ABI 4368813) according to manufacturer's instructions. A typical 20µl reaction is shown below. The reaction was carried out in 0.2ml PCR tubes (Axygen 321-10-051) in a Thermo Electron Corporation PCR SPRINT thermal cycler. Controls which replace the reverse transcriptase with dH 2 O were used to ensure that there was not any contamination from DNA in the sample. The cDNA was stored at -20°C until required.

Quantitative RT-PCR
Quantitative RT-PCR (qRT-PCR) was carried out on an ABI StepOnePlus RT-PCR machine to amplify either PAD1 or TbZFP3 mRNA. Reactions were carried out in 25µl volumes, as detailed below. A melt curve was added to the end of each qRT-PCR to ensure that there was only one amplification product. For TbZFP3, a standard curve was for absolute quantification of parasite number. For PAD1 the ΔΔCT method of data analysis was used for relative quantification, using TbZFP3 as an internal control. The data was analysed using the ABI StepOne software version 2.