Plasmodium

During the baseline period before the rollout of the intervention commenced, two parasitological prevalence surveys were conducted in a cross section of the study population. Households were randomly selected for inclusion in each prevalence survey to the point where 10 % of the population was included. All members of selected households were informed in advance of the date and time of the survey and were invited to assemble at a public place such as a church or a school near their home for malaria testing. In total, residents of 790 randomly selected households were sampled, covering 1223 houses. The first survey examined 1822 individuals (7.8 % of the total island population) and was carried out during the start of the short rainy season starting from September and finishing in November 2012. A second prevalence survey examined 1810 individuals (7.7 % of the total population) and was conducted from February to April 2013. Individual body temperature was measured by means of a Braun™ IRT 3020 ear thermometer. A drop of blood was obtained through a finger prick and directly tested for antigens of malaria parasites using an SD BIOLINE™ Malaria Ag P.f/Pan (HRP-II/pLDH) Rapid Diagnostic Test (RDT). The SD Bioline RDT kit results distinguish between infection with P. falciparum and other Plasmodium species. However, tests results with more than one positive reading or indicating multiple species of Plasmodium were pooled. If the individual tested positive for malaria antigens, an appropriate dose of Coartem^® (Artemether/lumefantrine) was provided free of charge.

After extraction all RNA samples were tested by qPCR targeting genes encoding 18S rRNA to confirm complete digestion of gDNA in a StepOne Plus® Real-Time PCR system (Applied Biosystems). This was followed by a generic qRT-PCR using the same generic primers and probe but on RNA in a one-tube reaction combining the reverse transcription and amplification reaction. Primers and probes are listed in Table S1, reaction mixes and PCR profiles are listed in Table S2. The RNA-based P. falciparum assay targeted A-type 18S rRNA transcripts expressed in asexual stages [17 (link)], whereas the generic assay targeting conserved regions would amplify all 5 copies of 18S rRNA genes in the genomes of P. falciparum and P. vivax [18 (link)]. All samples positive by the generic Plasmodium sp. assay were further analyzed by species–specific qRT-PCR reactions in a simplex reaction for P. falciparum and P. ovale, and as a duplex reaction for P. vivax and P. malariae. Primers and probes are listed in Table S1. Due to the generally higher parasitaemia in P. falciparum than in P. vivax infections, 18S rRNA transcripts in P. falciparum samples were highly abundant compared to 18S rRNA of for P. vivax. During extensive test evaluation we have observed a low level of aerosol-derived contamination when introducing negative controls, i.e. extraction of water. This low level of air-bourne contamination found in some, but not all negative samples, was corrected for by introducing a cut-off of 10 copies/µl extracted RNA for P. falciparum 18S rRNA qRT-PCR; for P. vivax no cut off was required. The cut off was identified in a plot of all measured transcript copy numbers by the point from which copy numbers rose above a steady baseline. By introducing a cut-off for P. falciparum, 51 previously Pf qRT-PCR positive samples were considered false positive. All except one sample had been P. falciparum negative by qPCR. This finding gave support to the choice of 10 copies/µl extracted RNA as our cut off.
Each plate carried a dilution series of assay-specific control plasmids with the respective template inserted at concentrations of 10⁶,10⁴ and 10² copies of template/reaction in duplicates. For each plate standard curves were generated from these values for quantification of copy numbers in test samples.

For Plasmodium, erythrocytes were lysed with 0.1% saponin for 5 min on ice to release parasites. Parasite pellets were washed twice with ice-cold phosphate-buffered saline (PBS), resuspended in 1× lithium dodecyl sulfate buffer (Life Technologies) in PBS supplemented with 25 μM dithiothreitol (DTT), and boiled for 5 min at 96°C. After separation on Bis-Tris Novex gels (Invitrogen), proteins were transferred to a nitrocellulose membrane and blocked with a blocking buffer containing 0.1% Hammarsten-grade casein (Affymetrix), 0.2× PBS, and 0.01% sodium azide. Next, membranes were incubated with primary antibodies diluted in a wash buffer consisting of 50% blocking buffer, 50% Tris-buffered saline, and 0.25% Tween (TBST) overnight at 4°C or 1 h at room temperature, followed by 3 washes with TBST and 1 h of incubation with the secondary antibodies diluted in the same buffer. Blots were visualized using the LiCor double-color detection system. Blot images were converted to grayscale images for the purpose of this publication and were analyzed using Image Studio Lite software. Primary antibodies and dilutions used in this study were as follows: guinea pig anti-PfAtg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech; 632381), 1:10,000; mouse anti-Plasmodium yoelii BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000. Secondary fluorophore-conjugated antibodies were purchased from Fisher (LiCor) and used at a 1:10,000 dilution.
For T. gondii, protein extracts from 10⁷ freshly egressed tachyzoites were prepared in Laemmli sample buffer, separated by SDS-PAGE, and transferred onto nitrocellulose membrane using the Bio-Rad Mini Trans-Blot system according to the manufacturer’s instructions. Mouse anti-GFP monoclonal antibody (catalog no. 11814460001; Roche; clones 7.1 and 13.1) was used to detect tagged proteins, and mouse anti-SAG1 monoclonal antibody (67 (link)) was used as a loading control.

Primers and gBlocks used in this study are listed in Table S1 in the supplemental material. All constructs for genetic complementation were cloned into pfYC110 using In-Fusion (TaKaRa Bio). Atg8 homologs were cloned without the C-terminal extension following the last glycine residue. To clone pfYC110-GFP-PfAtg8WT, GFP with a Gly-Ala-Gly-Ala linker and AatII site was amplified from pfYC110-ACP_L-GFP, and PfAtg8 was PCR amplified from Plasmodium genomic DNA. The two fragments were inserted into pfYC110, resulting in plasmid pfYC110-GFP-PfAtg8WT. PfAtg8 was removed from that plasmid by AatII-SacII digest and replaced with Atg8 homologs. Human Atg8 homologs LC3A, LC3B, GABARAP, and GABARAPL1 were amplified from cDNA libraries kindly provided by the labs of S. Pfeffer and L. Li; gBlocks (IDT DNA) were used for cloning human LC3C and GABARAPL2; ScAtg8G was amplified from yeast S288C genomic DNA. To clone the Plasmodium-yeast loop hybrids, parts of Atg8 from the start codon to the loop or from the loop to the stop codon were amplified from the plasmids carrying PfAtg8 or ScAtg8 and the desired loop sequence was added on the primer; the N- and C-terminal parts of the hybrid were then fused by overlap extension PCR; the N-terminal GFP tag was amplified with a Gly-Ala-Gly-Ala linker and PvuI site downstream of it; both fragments were simultaneously inserted into pfYC110 via AvrII-SacII restriction sites. The remaining hybrid constructs as well as CpAtg8 were purchased as gBlocks (IDT DNA) and inserted into pfYC110 via AvrII-SacII sites. Accession numbers for the aligned sequences are as follows: hLC3A isoform 1, NP_115903; hLC3B, NP_073729; hLC3C, NP_001004343; hGABARAP, NP_009209; hGABARAPL1 isoform 1, NP_113600; hGABARAPL2, NP_009216; PfAtg8, PF3D7_1019900; TgAtg8, TGME49_254120; CpAtg8, CPATCC_0009830.