2A and the Auxin-Based Degron System Facilitate Control of Protein Levels in <i>Plasmodium falciparum</i>

<div><p>Analysis of gene function in <i>Plasmodium falciparum,</i> the most important human malaria parasite, is restricted by the lack of robust and simple reverse genetic tools. Approaches to manipulate protein levels post-translationally are powerful tools to study <i>protein-off</i> effects especially in the haploid malaria parasite where genetic knockouts of essential genes are lethal. We investigated if the auxin-inducible degron system is functional in <i>P. falciparum</i> and found that degron-tagged yellow fluorescent protein levels were efficiently reduced upon addition of auxin which otherwise had no effect on parasite viability. The genetic components required in this conditional approach were co-expressed in <i>P. falciparum</i> by applying the small peptide 2A. 2A is a self-processing peptide from Foot-And-Mouth Disease virus that allows the whole conditional system to be accommodated on a single plasmid vector and ensures stoichiometric expression levels.</p></div

2A mediated co-expression of AID-system functional components in <i>P. falciparum.</i>

<p>The AID system was realized in <i>P. falciparum</i> by transfecting the plasmid pCHD_T2AY resulting in D10_T2AY parasites. A) Scheme of T2AY polycistronic mRNA encoding c-Myc tagged TIR1 and the reporter eYFP N-terminally tagged with AID degron. B) Immunoblots of D10_T2AY protein lysates (without auxin added) to control for expression and correct cleavage of AID system essential components, namely TIR1 and AID-eYFP. Blots with anti-c-Myc antibodies and anti-GFP antibodies were performed separately applying the same parasite protein samples: 1: D10_T2AY, 2: D10_ACP-GFP, D3: D10. D10 and D10_ACP-GFP and are control parasites unresponsive to anti-c-Myc but the latter reactive with anti-GFP antibodies.</p

Cleavage activity of 2A in <i>P. falciparum.</i>

<p>Cleavage activity of 2A in <i>P. falciparum</i> was assessed by transgenic parasites (3D7_R2Y2B) expressing A) an open reading frame encoding for Ds<u>R</u>ed, e<u>Y</u>FP and <u>b</u>lasticidin-S deaminase (BSD). <u>2</u>A and <i>P. falciparum</i> codon usage adapted version thereof (Pf<u>2</u>A) was placed between the genes. Co-translational cleavage leads to separated proteins C-terminally tagged with 2A and downstream sequences start with a 2A derived proline. B) Fluorescent microscopy showed DsRed and eYFP expressing transgenic parasites (schizont stage) grown under blasticidin selection. Hoechst 33342 staining indicated intraerythrocytic parasites C) Western blot analysis with anti-GFP antibody showed fully cleaved eYFP (30 kDa) and a small proportion of uncleaved polyprotein (73 kDa) of 3D7_R2Y2B parasites. Controls for anti-GFP antibody binding pattern are HeLa cells expressing eYFP (26 kDa), D10_ACP-GFP (32 kDa) and untransfected D10 parasites only. Samples are from two different blots probed with anti-GFP antibody.</p

Auxin reduces AID-tagged eYFP levels in <i>P. falciparum.</i>

<p>D10_T2AY parasites were treated with auxin and eYFP levels were measured by flow cytometry at the respective time points. A) Histogram overlay of eYFP+ parasites of one representative experiment in which D10_T2AY parasites were treated with 0.5 mM IAA for 0.5 h, 2 h and 5 h, respectively, assessed by flow cytometry. B) D10_T2AY treated with 0.5 mM IAA and eYFP median fluorescent intensities (MFI) measured at the respective time point presented as % relative MFI compared to the untreated control. The maximum observed fluorescence intensity of the control was set to 100%. C) D10_T2AY parasites were pretreated with 0.5 µM epoxomicin (1 h) before adding 0.5 mM IAA for 2 h or 5 h, respectively. MFI signals were assessed with flow cytometry. B) and C) represent the MFI and the respective standard deviation bars based on five independently performed experiments. Difference between 0.5 h and 2 h are statistically significant (paired t-test, p<0.005).</p

Surrogate markers for extravascular hemolysis and disease severity on admission.

<p>MM: mild malaria, SMA: severe malarial anemia, relative spleen volume: calculated as spleen volume at recruitment/spleen volume after reconvalescence (measured by sonography), Spleen (cm) represents subcoastal spleen enlargement, sMODS: simplified multi organ dysfunction score, results are in median (interquartile ranges).</p>a<p>Kruskal-Wallis rank sum test.</p

Clinical data of anemic and non-anemic patients on admission.

<p>MM: mild malaria, SMA: severe malarial anemia, Hb: hemoglobin, Hct: hematocrit, RBCs: red blood cells, MCV: mean corpuscular volume, results are in median (interquartile ranges) and count data (#).</p>a<p>parameter used for group definition - no p-value calculated, ^bKruskal-Wallis rank sum test, ^cχ²-test.</p

Phagocytosis rate in severe malarial anemia (SMA) and mild malaria (MM) patients: The plot shows individual values of relative phagocytosis of autologous samples (patients monocytes phagocytosing autologous RBCs) and positive control samples (patients monocytes phagocytosing anti-D IgG opsonized control RBCs).

<p>Thick lines represent median values. Phagocytosis rates were investigated on admission and after reconvalescence on day 56. In A) phagocytosis rates are shown for SMA patients. Phagocytosis rates of autologous samples at day 0 are as high as positive control. On day 56, autologous sample is significantly lower than positive control (p<0.05, ○ SMA - non-transfused, • SMA - transfused). In B), phagocytosis rates of MM patients are illustrated. Phagocytosis rates of autologous samples are significantly lower on day 0 as well as on day 56 (p<0.05, Δ MM).</p

Erythrocyte surface makers on admission.

<p>MM: mild malaria, SMA: severe malarial anemia, %: percentage of fluorescence positive cells, MFI: mean fluorescence intensity, IgG: immunoglobulin G, results are in median (interquartile ranges).</p>a<p>Kruskal-Wallis rank sum test.</p

Markers of intravascular hemolysis on admission.

<p>MM: mild malaria, SMA: severe malarial anemia, LDH: lactate dehydrogenase, α-HBDH: alpha hydroxybutyrate dehydrogenase, results are in median (interquartile ranges).</p>a<p>Kruskal-Wallis rank sum test.</p

Study-flow chart.

<p>Study-flow chart.</p