Figure 1.
Localization of PbROM1 protein in merozoites and sporozoites.
PbROM1 protein expression was assayed by indirect immunofluorescence (IFA). (A) IFA of fully segmented schizonts and free merozoites, double labeled with anti-PbROM1 (green) and anti-AMA1 (red) antibodies. (B) IFA of midgut and salivary gland sporozoites double labeled with anti-PbROM1 (green) and anti-CSP (red) antibodies. Little or no PbROM1 protein can be detected in midgut sporozoites while the protein is distributed in a punctuate pattern throughout salivary gland sporozoites. DAPI is shown in blue in the merged panels. The dotted line separates the fields of two separate images. PbROM1, P. berghei rhomboid 1, AMA1, apical membrane antigen 1, CSP, circumsporozoite protein. (Scale bars, 3 µm).
Figure 2.
Immuno-electron microscopic localization of PbROM1 in salivary gland sporozoites.
(A,B) Immunogold labeling of merozoites. PbROM1 is detected in the apical end (arrows) of merozoites within secretory organelles, predominantly within micronemes (Mi). The insert in panel B shows a microneme from another merozoite labeled with gold particles. (C,D) Immunogold labeling with anti-PbROM1 antibody of P. berghei-infected mosquito salivary gland sporozoite cryosections. The protein is detected on the parasite plasma membrane (PM) as well as on the membrane of micronemes (Mi) (see text for distribution statistics). The typical folded posterior end seen in sporozoites is marked with asterisk. DG: dense granule, SD: salivary duct, N: Nucleus, R: Rhoptries. (Scale bars, 250 nm).
Figure 3.
(A) Schematic representation of the targeting strategy. The wild-type PbROM1 genomic locus (WT) was targeted with an NdeI-linearized plasmid (pROM1) containing the 5′ and 3′ truncations of the PbROM1 open reading frame and the TgDHFR positive selection marker. Upon a single crossover event, the region of homology is duplicated, resulting in two truncated, nonexpressed PbROM1 copies in the integrated locus [PbROM1(−)]. The homologous regions in the disruption plasmid are shaded gray. Arrowheads indicate primer pairs used to confirm gene disruption. Hatched areas represent the region of the ORF that is outside the homologous region. (B) Integration-specific PCR analysis. Genomic DNA was prepared from wild-type P. berghei and drug resistant parasite clones and PCR was performed using the primer pairs indicated in panel A. The presence of the 1.2 kb integration-specific PCR product (P3/P2) but not the 1.7 kb WT locus-specific PCR product (P1/P2) in the PbROM1(−) lanes confirm gene disruption. Note that WT lanes show the presence of the wild-type locus (P1/P2) as expected but not the integration locus (P3/P2). (C) RT-PCR confirmation of PbROM1 disruption. Salivary gland sporozoites from PbROM1(−)-infected mosquitoes did not express PbROM1, as expected. PbCS was used as a positive control and can be seen expressed in both WT and PbROM1(−) sporozoites.
Figure 4.
PbROM1 is required for efficient infection of mosquito midgut and mouse hepatocytes.
(A) PbROM1 is not required for ookinete formation. Individual midguts from mosquitoes fed either on WT- or ROM1(−)-infected mice were analyzed for ookinete numbers. ROM1(−) parasites differentiated into ookinetes as efficiently as wild-type parasites (P>0.05, unpaired t test). (B) PbROM1(−) ookinetes are impaired in the ability to form oocysts. In six out of seven experiments, mosquitoes fed on mice infected with ROM1(−) parasites formed significantly fewer oocysts compared to four out of five experiments using mice infected with WT parasites. Experiments labeled ROM1(−)4, ROM1(−)5 and ROM1(−)6 were performed using an independent clone. Oocysts were counted on day 15 after blood feeding (*: P<0.05, ANOVA, Tukey's multiple comparison test). (C) PbROM1 is not required for efficient invasion of mosquito salivary glands. Sporozoites were isolated from midguts and salivary glands of mosquitoes (day 25–26) infected with WT and ROM1(−) parasites and counted on a hemocytometer. To estimate the efficiency of sporozoite infection of salivary glands, total midgut sporozoites were normalized for prevalence (mosquito infectivity) and mean oocysts per mosquito (day 15) for each experiment (P >0.05, unpaired t test). Bars show mean±SEM. (D) ROM1 is required for efficient infection of the mouse liver. The same number (1000) of WT or ROM1(−) salivary gland sporozoites were injected intravenously into mice and the efficiency of liver infection was measured 36 h later by quantitative PCR of P. berghei 18S rRNA normalized using mouse GAPDH (P<0.05, unpaired t test). Parasite load in livers of mice infected with mutant sporozoites was 68% lower than in livers infected by wild-type sporozoites.
Table 1.
Prepatent period of blood infection is longer for mice infected with PbROM1(−) sporozoites
Figure 5.
PbTRAP is not a substrate for PbROM1.
(A) PbTRAP is cleaved by a serine protease. Total cell lysate from 3×104 salivary gland sporozoites was loaded in each lane of a 4–25% denaturing SDS-PAGE. Western blot was performed using an anti-PbTRAP-repeat rabbit polyclonal antibody. The TRAP fragment recognized by the anti-repeat antibody but not by antibody against the cytoplasmic tail is indicated with an asterisk. Ctrl, control; E, empty lane. (B) PbTRAP processing in PbROM1(−) sporozoites. Experiments with 3×104 sporozoites/lane were conducted as described for experiments in panel A with wild-type (WT) and mutant parasites. (C) Gliding motility of PbROM1(−) sporozoites. PbROM1(−) salivary gland sporozoites were placed in a 2-well chamber slide coated with BSA and incubated at 37°C for 30 min. After fixation with paraformaldehyde CSP trails were detected with an anti-CSP antibody (3D11) and a rhodamine-conjugated anti-mouse secondary antibody. *, sporozoite at the leading end of the trail.
Figure 6.
PbROM1(−) blood-stage parasites grow slower. Mice infected with the mutant parasite survive longer and become protected from a WT parasite challenge.
Swiss Webster mice were injected intravenously with 1,000 wild-type (WT) or PbROM1(−) salivary gland sporozoites (Spz) (A) or intraperitonealy with 105 infected RBCs (iRBC) (independent clone (B)) and parasitemia was measured daily and expressed as mean of two consecutive days. Three to four mice were used for each group and parasitemia is expressed as mean±SEM. The dotted line represents the point beyond which parasitemia in WT and ROM1(−) differed significantly (P<0.05, repeated measures ANOVA). (C) Survival of mice infected with either wild-type (WT) or PbROM1(−) sporozoites. Swiss Webster mice were injected intravenously with the indicated number of either WT or PbROM1(−) sporozoites and animal survival was monitored daily. PbROM1(−) infected mice survive significantly longer than WT infected mice (P<0.0001). Numbers in parenthesis indicate the number of mice assayed. (D) Mice that clear PbROM1(−) infection are protected from WT parasite challenge. Mice (M) from three experiments (E) such as the ones described in panel A and B that had survived PbROM1(−) infection and cleared the parasites were re-infected by either a) intravenous (i.v) injection of 104 wild-type sporozoites (E1), b) intra-peritoneal injection of 106 infected RBCs (E2), or c) intra-venous injection of 106 infected RBCs (E3) at least 30 days after the clearance of the original PbROM1(−) infection. Seven out of 14 mice developed very mild parasitemia (0.004%–2.6%) that was subsequently completely cleared. These mice were also protected from a subsequent 2nd and a 3rd WT parasite challenge (Table 2).
Table 2.
PbROM1(−) parasites generate protective immunity