Development of Pyb9 was like WT parasites in the blood (as they develop a 0.5-2% parasitemia after 8 days from an infection initiated by a single parasite) and in mosquito-stage development (normal production of oocysts and sporozoites). Liver-stage development was analysed in BALB/c mice after inoculation of 104 or 2x105 sporozoites by in vivo imaging and analysis of subsequent blood-stage infections. In vivo imaging of mice infected with 2x105 Pyb9 sporozoites showed faint luminescent signals in the liver at 3hpi that were significantly higher than background values of uninfected mice. At 40hpi none of the infected mice showed luminescence signals above background. A blood-stage infection was only detected in 1 out of 8 mice infected with 2x105 sporozoites with a long prepatent period of 10 days. These results indicate that Pyb9 parasites, like P. berghei b9, are severely compromised in liver stage development.

BALB/c mice injected with 10,000 Pyb9 sporozoites all developed a patent blood-stage infection. Dramatic reduction in the number of Pyb9 exoerythrocytic forms (EEFs) in vitro cultured HepG2 cells. Evidence presented that Pyb9 were not able to form productive vacuoles in hepatocytes.

Expression of RON4 in merozoites and sporozoites

Expression of RON4 in merozoites and sporozoites

Normal numbers of motile salivary gland sporozoites are formed. While 30 wild-type sporozoites were sufficient to establish infection in all rats, even 1 10 B9(-) sporozoites were insufficient to establish infection in all rats.

Normal sporozoite production. Sporozoites showed normal gliding motility and WT-levels of hepatocyte invasion. When Swiss or BALB/c mice were infected by intravenous inoculation of either 1 or 5x104 Pbb9 sporozoites none of the mice developed blood-stage infections. When C57BL6 mice were infected with a high dose of 5x104 Pbb9 sporozoites, 10-20% developed a blood-stage infection with a 3-4 days prolonged prepatent period. Immunofluorescence analyses show that Pbb9 parasites arrest early after invasion of hepatocytes. Pbb9 sporozoites exhibit normal hepatocyte invasion, but at 24hpi most intra-cellular parasites had disappeared and only a few small parasites could be observed with a size that was similar to 5-10 hpi liver stages. Analysis of Pbb9 parasites in the liver, using real-time in vivo imaging, confirmed the early growth-arrest observed in cultured hepatocytes.

Normal sporozoite production. Sporozoites showed normal gliding motility and WT-levels of hepatocyte invasion. When Swiss or BALB/c mice were infected by intravenous inoculation of either 1 or 5x104 Pbb9 sporozoites none of the mice developed blood-stage infections. When C57BL6 mice were infected with a high dose of 5x104 Pbb9 sporozoites, 10-20% developed a blood-stage infection with a 3-4 days prolonged prepatent period. Immunofluorescence analyses show that Pbb9 parasites arrest early after invasion of hepatocytes. Pbb9 sporozoites exhibit normal hepatocyte invasion, but at 24hpi most intra-cellular parasites had disappeared and only a few small parasites could be observed with a size that was similar to 5-10 hpi liver stages. Analysis of Pbb9 parasites in the liver, using real-time in vivo imaging, confirmed the early growth-arrest observed in cultured hepatocytes.

Normal sporozoite production. Sporozoites showed normal gliding motility and WT-levels of hepatocyte invasion. When Swiss or BALB/c mice were infected by intravenous inoculation of either 1 or 5x104 Pbb9 sporozoites none of the mice developed blood-stage infections. When C57BL6 mice were infected with a high dose of 5x104 Pbb9 sporozoites, 10-20% developed a blood-stage infection with a 3-4 days prolonged prepatent period. Immunofluorescence analyses show that Pbb9 parasites arrest early after invasion of hepatocytes. Pbb9 sporozoites exhibit normal hepatocyte invasion, but at 24hpi most intra-cellular parasites had disappeared and only a few small parasites could be observed with a size that was similar to 5-10 hpi liver stages. Analysis of Pbb9 parasites in the liver, using real-time in vivo imaging, confirmed the early growth-arrest observed in cultured hepatocytes.

C57BL/6 mice injected with 10,000 Pbb9 did not develop a patent blood infection. Dramatic reduction in the number of Pbb9 exoerythrocytic forms (EEFs) in vitro cultured HepG2 cells. Evidence presented that Pbb9 were not able to form productive vacuoles in hepatocytes.A cell wound-repair assay indicated that the cell traversal activity of Pbb9 sporozoites is not different to wild-type sporozoites, in both HepG2 and HepG2/CD81 cells.

In vitro in HepG2 cells RON4-mCherry signal was lost in a vast majority of intracellular wild-type PbGFP/RON4-mCherry sporozoites, reflecting rhoptry discharge during productive invasion. In sharp contrast, RON4-mCherry was detected in all examined Pbb9/RON4-mCherry intracellular sporozoites, indicating that sporozoites lacking B9 invade cells without secreting their rhoptries, i.e. through traversal mode only.

B9(-) parasites formed abnormal liver stages (LS) and displayed increased cell traversal activity. The number of B9(-) LS parasites formed in HepG2 cells was 6% that of wild-type parasites at 24 and 48 h post-inoculation (hpi) of sporozoites. The average diameter of the B9(-) LS parasites decreased to approximately 80 % that of wild-type LSs at 48 hpi. There was a decreased number of sporozoites entering the hepatocytes by productive invasion and reciprocally, an increased number of sporozoites that continued migration through the hepatocytes. Evidence is presented that B9(-) LS parasites develop in the nucleus of hepatocytes.