Article Figures & Data
Figures
- Fig 1
Py-MIF transcript is expressed in liver-stage (LS) and blood-stage (BS) parasites. The expression of Py-mif was determined by RT-PCR on RNA derived from P. yoelii 17XNL. RNA was isolated from salivary gland sporozoites, mixed blood-stage parasites, or mouse livers infected with 1 × 106 sporozoites (isolated at 24 or 44 hpi). Genomic DNA was removed by DNase treatment, and cDNA was generated by reverse transcriptase single-strand DNA synthesis. Either Py-mif or 18S rRNA was amplified from cDNAs via 35 cycles of PCR. Non-RT controls (−RT) were included to confirm that mif amplification was due to cDNA and not to residual genomic DNA.
- Fig 2
Localization of Py-MIF during in vitro liver-stage development. (A) P. yoelii sporozoites were isolated from salivary glands of Anopheles stephensi mosquitoes and stained with polyclonal mouse antibody to Py-MIF (green) and CSP (red). (B and C) HepG2-CD81 cells were infected with P. yoelii salivary gland sporozoites and fixed at 24 (B) or 48 (C) hpi. Cells were stained with a mouse anti-Py-MIF antibody (green), and a polyclonal rabbit anti-UIS4 antibody (red) was used to localize the parasitophorous vacuole membrane. DAPI (blue) was used to stain the nuclei of both parasites and host hepatocytes.
- Fig 3
Localization of Py-MIF in liver-stage parasite mouse infections. One million P. yoelii Δmif sporozoites were injected intravenously into BALB/cJ mice to study liver-stage development in vivo. Liver sections were stained at 2 (A), 16 (B), 30 (C), 44 (D), and 52 (E) hpi with a polyclonal mouse anti-Py-MIF antibody (green). (A to D) A polyclonal rabbit anti-UIS4 antibody (red) was used to localize the parasitophorous vacuole membrane of liver-stage parasites. (E) Cells were stained with a polyclonal rabbit antibody against Py-falstatin (red), a protein that is released into the host cell during late liver-stage development. DAPI was used to stain the nuclei of both parasites and host hepatocytes.
- Fig 4
Generation of P. yoelii Δmif parasites. (A) Py-mif was replaced with a selectable drug resistance marker (TgDHFR, the dhfr gene from Toxoplasma gondii [positive selection marker]; and AMP, ampicillin resistance gene for bacterial selection) and a cassette encoding red fluorescent protein by double-crossover homologous recombination using the 5′UTR and 3′UTR regions at the Py-mif locus. The resulting mif-deficient parasite population was fluorescent red. (B) The mixed population of wild-type and transgenic parasites was cloned by serial limiting dilution into SW mice. The genotyping gel shows successful integration on the 3′ (3′ test) and 5′ (5′ test) flanks, as well as the loss of the Py-mif open reading frame, in two independent clones (PyΔmif1 and PyΔmif2). (C) RT-PCR using primers amplifying either mif or 18S rRNA from WT or P. yoelii Δmif1 blood-stage parasites. RT-PCR was performed on cDNA derived from 1 μg of parasite RNA.
- Fig 5
mif-deficient parasites develop normally during mosquito or blood stages. (A) Blood-stage growth curve. One million control or P. yoelii Δmif1 blood-stage parasites were injected into BALB/c mice, and parasitemia in the blood was measured daily until all parasites were cleared. (B) P. yoelii Δmif parasites form oocysts. Midguts from mosquitoes infected with the P. yoelii Δmif1 strain were collected at 10 days p.i. and viewed by fluorescence microscopy. P. yoelii Δmif oocysts are fluorescent red. (C) P. yoelii Δmif strain generates salivary gland sporozoites. P. yoelii Δmif1 sporozoites were isolated from mosquito salivary glands and stained with a mouse polyclonal antibody against CSP (green). Parasite DNA was stained with DAPI (blue).
- Fig 6
Livers from mice infected with the P. yoelii Δmif strain contain less parasite biomass than those infected with WT P. yoelii. BALB/cJ mice were infected with 1 × 106 WT (Py) or mif-deficient clone 1 (PyΔmif1) P. yoelii sporozoites. Livers were harvested at 44 hpi, and the relative amounts of parasite biomass were determined by QPCR for 18S rRNA and normalized to the wild type. Data for a representative sample of two repetitions are shown (n = 5 mice per group). *, P < 0.05.
- Fig 7
Py-mif-deficient liver-stage parasites exhibit a growth defect. (A and B) Livers from mice infected with 1 × 106 P. yoelii WT or Δmif parasites were analyzed at 44 hpi. (A) Quantification of the number of liver-stage parasites per cm2 of liver (for WT, 25 ± 5.60; for P. yoelii Δmif strain, 17.67 ± 8.31; P = 0.09). The data are combinations for 2 experimental replicates. ns, not significant. (B) Liver sections were stained with a rabbit polyclonal antibody against UIS4 (red) to mark the parasitophorous vacuole membrane in infected hepatocytes. DNA is stained with DAPI (blue). Images of a representative sample are shown. (C) Diameters of RFP-expressing P. yoelii control (n = 18) and P. yoelii Δmif (n = 21) parasites were measured using the ruler tool in the Metamorph software package. Three or four diameters were measured for each parasite and then averaged. ***, P < 0.001.
Tables
- Table 1
Deletion of Py-mif does not affect sporozoite development in Anopheles mosquitoes
Genotype No. of sporozoites per mosquitoa Oocystsb Salivary glandsc RFP-expressing WT 65,800 ± 26,543 17,275 ± 7,864 Δmif1 81,500 ± 37,772 20,383 ± 9,442 Δmif2 64,083 ± 18,304 15,016 ± 7,311 ↵a Values are means ± standard deviations.
↵b Mosquito midguts were removed at 10 days p.i., and oocyst sporozoites were quantified (combination of 3 independent experiments, with >30 mosquitoes per experiment).
↵c Salivary gland sporozoites were dissected at 14 days p.i. and quantified (combination of 4 independent experiments, with >30 mosquitoes per experiment).
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Fig. S1. Py-mif expression in sporozoites.
Fig. S2. Localization of Py-MIF-myc in vitro.
Fig. S3. Confirmation of PyΔmif.Table S1. Description of primers utilized.
Supplemental methods.
PDF file, 185K.
- Supplemental file 1 -
Fig. S1. Py-mif expression in sporozoites.
