Cerebral malaria claims more than 1 million lives per year. We report that heme oxygenase-1 (HO-1, encoded by Hmox1) prevents the development of experimental cerebral malaria (ECM). BALB/c mice infected with Plasmodium berghei ANKA upregulated HO-1 expression and activity and did not develop ECM. Deletion of Hmox1 and inhibition of HO activity increased ECM incidence to 83% and 78%, respectively. HO-1 upregulation was lower in infected C57BL/6 compared to BALB/c mice, and all infected C57BL/6 mice developed ECM (100% incidence). Pharmacological induction of HO-1 and exposure to the end-product of HO-1 activity, carbon monoxide (CO), reduced ECM incidence in C57BL/6 mice to 10% and 0%, respectively. Whereas neither HO-1 nor CO affected parasitemia, both prevented blood-brain barrier (BBB) disruption, brain microvasculature congestion and neuroinflammation, including CD8(+) T-cell brain sequestration. These effects were mediated by the binding of CO to hemoglobin, preventing hemoglobin oxidation and the generation of free heme, a molecule that triggers ECM pathogenesis.
An obligatory step of malaria parasite infection is Plasmodium sporozoite invasion of host hepatocytes, and host lipoprotein clearance pathways have been linked to Plasmodium liver infection. By using RNA interference to screen lipoprotein-related host factors, we show here that the class B, type I scavenger receptor (SR-BI) is the strongest regulator of Plasmodium infection among these factors. Inhibition of SR-BI function reduced P. berghei infection in Huh7 cells, and overexpression of SR-BI led to increased infection. In vivo silencing of liver SR-BI expression in mice and inhibition of SR-BI activity in human primary hepatocytes reduced infection by P. berghei and by P. falciparum, respectively. Heterozygous SR-BI(+/-) mice displayed reduced P. berghei infection rates correlating with liver SR-BI expression levels. Additional analyses revealed that SR-BI plays a dual role in Plasmodium infection, affecting both sporozoite invasion and intracellular parasite development, and may therefore constitute a good target for malaria prophylaxis.
SummaryThe study of the liver stage of malaria has been hampered by limitations in the experimental approaches required to effectively dissect and quantify hepatocyte infection by Plasmodium. Here, we report on the use of flow cytometry, in conjunction with GFP-expressing Plasmodium sporozoites, to assess the various steps that constitute a successful malaria liver infection: cell traversal, hepatocyte invasion and intrahepatocyte parasite development. We show that this rapid, efficient and inexpensive method can be used to overcome current limitations in the independent quantification of those steps, facilitating routine or large-scale studies of host-pathogen molecular interactions.
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, PKCς (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 PKCς-targeting siRNAs. Additional cell-based analyses using a pseudo-substrate inhibitor of PKCς added further RNAi-independent support, indicating a role for host PKCς 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.
Shigella, the Gram-negative enteroinvasive bacterium that causes shigellosis, relies on its type III secretion system (TTSS) and injected effectors to modulate host cell functions. However, consequences of the interaction between Shigella and lymphocytes have not been investigated. We show that Shigella invades activated human CD4(+) T lymphocytes. Invasion requires a functional TTSS and results in inhibition of chemokine-induced T cell migration, an effect mediated by the TTSS effector IpgD, a phosphoinositide 4-phosphatase. Remarkably, IpgD injection into bystander T cells can occur in the absence of cell invasion. Upon IpgD-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), the pool of PIP(2) at the plasma membrane is reduced, leading to dephosphorylation of the ERM proteins and their inability to relocalize at one T cell pole upon chemokine stimulus, likely affecting the formation of the polarized edge required for cell migration. These results reveal a bacterial TTSS effector-mediated strategy to impair T cell function.
Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level.
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