Kinome-wide RNAi screen implicates at least 5 host hepatocyte kinases in Plasmodium sporozoite infection.
Plasmodium sporozoites, the causative agent of malaria, are injected into their vertebrate host through the bite of an infected Anopheles mosquito, homing to the liver where they invade hepatocytes to proliferate and develop into merozoites that, upon reaching the bloodstream, give rise to the clinical phase of infection. To investigate how host cell signal transduction pathways affect hepatocyte infection, we used RNAi to systematically test the entire kinome and associated genes in human Huh7 hepatoma cells for their potential roles during infection by P. berghei sporozoites. The three-phase screen covered 727 genes, which were tested with a total of 2,307 individual siRNAs using an automated microscopy assay to quantify infection rates and qRT-PCR to assess silencing levels. Five protein kinases thereby emerged as top hits, all of which caused significant reductions in infection when silenced by RNAi. Follow-up validation experiments on one of these hits, PKCsigma (PKCzeta), confirmed the physiological relevance of our findings by reproducing the inhibitory effect on P. berghei infection in adult mice treated systemically with liposome-formulated PKCsigma-targeting siRNAs. Additional cell-based analyses using a pseudo-substrate inhibitor of PKCsigma added further RNAi-independent support, indicating a role for host PKCsigma on the invasion of hepatocytes by sporozoites. This study represents the first comprehensive, functional genomics-driven identification of novel host factors involved in Plasmodium sporozoite infection.
Prudêncio
Miguel
M
Unidade de Malária, Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa, Portugal.
Rodrigues
Cristina D
CD
Hannus
Michael
M
Martin
Cécilie
C
Real
Eliana
E
Gonçalves
Lígia A
LA
Carret
Céline
C
Dorkin
Robert
R
Röhl
Ingo
I
Jahn-Hoffmann
Kerstin
K
Luty
Adrian J F
AJ
Sauerwein
Robert
R
Echeverri
Christophe J
CJ
Mota
Maria M
MM
D000818:Animals; D002460:Cell Line; D020868:Gene Silencing; D015894:Genome, Human; D022781:Hepatocytes / Q000201:enzymology / Q000469:parasitology; D006801:Humans; D008288:Malaria; D051379:Mice; D008810:Mice, Inbred C57BL; D010770:Phosphotransferases / Q000235:genetics*; D010962:Plasmodium berghei / Q000472:pathogenicity*; D011493:Protein Kinase C; D034741:RNA, Small Interfering / Q000494:pharmacology*; D015398:Signal Transduction; D034101:Sporozoites; D000099221:Protein Kinase C zeta
D010770:Phosphotransferases; D011493:Protein Kinase C; D034741:RNA, Small Interfering; D000099221:Protein Kinase C zeta