Journal of the Australian Ceramic Society

Additional file 1. Quality control of parasite samples used for lipidomic analysis. A. Giemsa stain of cells before magnet purification. Scale bar, 5 Âľm. B. Western blot analysis of magnet purified parasites. An antiserum against Pfs16 was used as a marker for gametocytes and an antiserum against PfHSP70 was used as a control for the amount of protein loaded. Troph, trophozoite; I-V, Gametocyte stages I-V

MOESM4 of Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum

Additional file 4. Content (nmol / 109 cells, Mean Âą SD) and percentage (in parentheses) of the lipid classes and major lipid species of the blood-stage P. falciparum infected red blood cells. Parasites were enriched with higher than 95% parasitemia by magnet purification and global lipidomics of whole-cell extracts were analysed by mass spectrometry. uRBC, uninfected red blood cell; I-V, gametocytes stage I to V; n.d., not detected

Generation of <i>ipis2-</i>[GFP-Luc;mCherry] and <i>ipis3</i>-[GFP-Luc;mCherry] was achieved through double homologous recombination and confirmed by PCR.

(A) Homology regions (labeled as the 5’ and 3’ genomic regions) flanking the gene of interest were cloned into the GOMO GFP-Luciferase vector. The resulting parasites lack either IPIS2 or IPIS3, express both mCherry and GFP-luciferase, and contain a DHFR-γFCU drug resistance cassette. (B) Successful integration of the construct was verified by PCR amplification of the products indicated in grey in panel A. Produ cts were sequenced to confirm the genomic editing. (C) Excision of the mCherry and drug resistance expression cassette from the recycled lines was confirmed by the presence of product using the mCherry excision (mCherry exc) and absence of the 3’ integration (3’) product. (TIF)</p

<i>ipis2-</i> and <i>ipis3-</i> contribute to blood-stage growth efficiency during <i>P</i>. <i>berghe</i>i infection.

A) The appearance of ipis3- parasites in the blood following sporozoite injection is delayed in comparison to wild-type. C57BL/6 mice were infected with 1,000 sporozoites of either ipis2-, ipis3- or wild-type parasites, and blood-stage infections were monitored starting from day 3 post-infection with Giemsa-stained blood smears. Statistics: Log-rank (Mantel-cox) test; n = 5 (ipis2-), n = 10 (ipis3-). B) The growth of ipis2- and ipis3- parasites in the liver does not differ significantly from wild-type P. berghei. The relative parasite burden in the liver was measured by qPCR of RNA extracted from livers forty-two hours after intravenous injection of either ipis2-, ipis3-, or wild-type sporozoites. Levels of Pb18s rRNA were normalized to levels of mouse GAPDH RNA in each infected liver. Statistics: unpaired two-tailed t-test; n = 5; ns: not significant. C) The ipis2- and ipis3- lines do not grow as efficiently as wild type in the blood of co-infected animals. Mice were infected with equal amounts of wild-type P. berghei-infected erythrocytes and ipis2-[mCherry; PyrS] or ipis3-[mCherry; PyrS] infected erythrocytes. Parasitemia was measured by flow cytometry between days 4–7 after infection. * p ipis2- line, a statistically significant increase in mCherry signal was detected; however the extent was negligible. * p <0.05, unpaired two-tailed t-test.</p

Mass-spec results of immunoprecipitation experiments.

(XLSX)</p

Phenotypes of <i>ipis2</i>- and <i>ipis3</i>- in mosquito and blood infection stages.

(A) Midguts of mosquitoes were harvested 14 days after an infectious blood meal and microscopy was used to determine the presence or absence of oocysts. (B) The number of sporozoites per mosquito were calculated at least 17 days following infected blood meal. (C) Liver stage ipis2- and ipis3- parasites grow similarly to wild-type parasites in vitro. HepG2 cells infected with either wild-type, ibis2-[GFP-Luc;mCherry], or ibis3-[GFP-Luc;mCherry] sporozoites were fixed 48 hours after infection, and the parasites were stained with antibodies against PbHSP70. Exo-erythrocytic forms were imaged with the fluorescence microscope, and their size was determined using FIJI. ns, not significant, unpaired t-test. (D) Parasitemia in the blood of mice following infection with 1000 sporozoites was quantified by microscopy from Giemsa-stained blood smears. *, P ipis2- and ipis3- are less pathogenic in mice than wild-type P. berghei. Survival of mice after infection with 1000 sporozoite. n = 5 (ipis2-), n = 10 (ipis3-). ns, not significant **, P (TIF)</p

Primers used in this study.

(PDF)</p

PBANKA_0524300 (IPIS2), PBANKA_0623100 (IPIS3), PBANKA_1400700 and putative <i>P</i>. <i>vivax</i> orthologs localize to discrete structures in the cytoplasm of infected erythrocytes.

A) PBANKA_0524300-mCherry, PBANKA_0623100-mCherry, and PBANKA_1400700-mCherry colocalize with the IBIS marker IBIS1-GFP in the erythrocyte cytoplasm. Infected cells were fixed and labeled with anti-GFP and anti-mCherry antibodies to amplify the fluorescent signal. Scale bars, 5 μm. B) Erythrocytes infected with P. berghei expressing the HA-tagged P. vivax proteins PvTRAg8 or PvTRAg2, which are putative orthologs of IPIS2 and IPIS3, respectively, were fixed and labeled with anti-HA antibodies. Scale bars, 5 μm.</p

Synthesized sequences of <i>PvTRAg8</i> and <i>PvTRAg2</i>.

(PDF)</p

mCherry tagged PBANKA_0524300 (IPIS2), PBANKA_0623100 (IPIS3) colocalize with IBIS1-GFP at the liver-stage parasitophorous vacuole.

A) Protein features of candidate proteins that coprecipitated with IBIS1. B) Hepatoma cells were infected with sporozoites after the cross-fertilization of IBIS1-GFP and either PBANKA_0524300-mCherry, PBANKA_0623100-mCherry or PBANKA_1400700-mCherry transgenic parasite lines. Cells were fixed at indicated time points and the mCherry and GFP signals were amplified by using anti-RFP and anti-GFP antibodies. Scale bars, 10 μm.</p