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1.
Front Cell Infect Microbiol ; 11: 704662, 2021.
Article En | MEDLINE | ID: mdl-34268141

Hepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target.


Aquaporins , Sporozoites , Animals , Hepatocytes/metabolism , Humans , Plasmodium falciparum , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Sporozoites/metabolism , Tetraspanin 28/metabolism
2.
PLoS One ; 6(3): e18162, 2011 Mar 31.
Article En | MEDLINE | ID: mdl-21483865

BACKGROUND: Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites. METHODOLOGY: P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured. RESULTS: The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine. CONCLUSION: Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium-throughput in vitro screening assay for novel hypnozoiticidal drugs.


Hepatocytes/parasitology , Plasmodium/physiology , Sporozoites/physiology , Animals , Antimalarials/pharmacology , Cells, Cultured , Humans , Macaca fascicularis , Plasmodium/drug effects , Sporozoites/drug effects
3.
Cell Host Microbe ; 4(3): 283-92, 2008 Sep 11.
Article En | MEDLINE | ID: mdl-18779054

Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as specific SR-BI-blocking antibodies, we demonstrate that SR-BI significantly boosts hepatocyte permissiveness to P. falciparum, P. yoelii, and P. berghei entry and promotes parasite development. We show that SR-BI, but not the low-density lipoprotein receptor, acts as a major cholesterol provider that enhances Plasmodium infection. SR-BI regulates the organization of CD81 at the plasma membrane, mediating an arrangement that is highly permissive to penetration by sporozoites. Concomitantly, SR-BI upregulates the expression of the liver fatty-acid carrier L-FABP, a protein implicated in Plasmodium liver-stage maturation. These findings establish the mechanistic basis of the CD81-dependent Plasmodium sporozoite invasion pathway.


Hepatocytes/metabolism , Hepatocytes/parasitology , Host-Parasite Interactions , Malaria/metabolism , Malaria/parasitology , Plasmodium/physiology , Scavenger Receptors, Class B/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cells, Cultured , Cholesterol/metabolism , Female , Humans , Liver Diseases/metabolism , Liver Diseases/parasitology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Scavenger Receptors, Class B/genetics , Schizonts/physiology , Sporozoites/physiology , Tetraspanin 28
4.
Antivir Ther ; 13(5): 643-54, 2008.
Article En | MEDLINE | ID: mdl-18771048

BACKGROUND: Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89 (the large and small subunits of the HCMV terminase, respectively) their mechanism of action is not yet fully understood. We aimed here to better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives. METHODS: The HCMV pUL56 protein was studied by sequence analysis of the HCMV UL56 gene and herpesvirus counterparts combined with primary structure analysis of the corresponding amino acid sequences. RESULTS: The UL56 sequence analysis of 45 HCMV strains and counterparts among herpesviruses allowed the identification of 12 conserved regions. Moreover, comparison with the product of gene 49 (gp49) of bacteriophage T4 suggested that the pUL56 zinc finger is localized close to the dimerization site of pUL56, providing a spatial organization of the catalytic site that allows recognition and cleavage of DNA. CONCLUSIONS: This study provides a basis to investigate the mechanism of concatemeric DNA cleavage and a biochemical basis for DNA packaging inhibition by benzimidazole derivatives.


Benzimidazoles/pharmacology , Cytomegalovirus/drug effects , Cytomegalovirus/metabolism , Ribonucleosides/pharmacology , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolism , Amino Acid Sequence , Benzimidazoles/chemistry , Cells, Cultured , Cytomegalovirus/genetics , Cytomegalovirus/growth & development , DNA, Viral/metabolism , Dimerization , Fibroblasts , Humans , Models, Molecular , Molecular Sequence Data , Ribonucleosides/chemistry , Sequence Analysis, DNA , Viral Structural Proteins/genetics , Virus Assembly/drug effects
5.
J Biol Chem ; 279(52): 54518-28, 2004 Dec 24.
Article En | MEDLINE | ID: mdl-15475565

Neurofibrillary tangles (NFTs) are classic lesions of Alzheimer's disease. NFTs are bundles of abnormally phosphorylated tau, the paired helical filaments. The initiating mechanisms of NFTs and their role in neuronal loss are still unknown. Accumulating evidence supports a role for the activation of proteolytic enzymes, caspases, in neuronal death observed in brains of patients with Alzheimer's disease. Alterations in tau phosphorylation and tau cleavage by caspases have been previously reported in neuronal apoptosis. However, the links between the alterations in tau phosphorylation and its proteolytic cleavage have not yet been documented. Here, we show that, during staurosporine-induced neuronal apoptosis, tau first undergoes transient hyperphosphorylation, which is followed by dephosphorylation and cleavage. This cleavage generated a 10-kDa fragment in addition to the 17- and 50-kDa tau fragments previously reported. Prior tau dephosphorylation by a glycogen synthase kinase-3beta inhibitor, lithium, enhanced tau cleavage and sensitized neurons to staurosporine-induced apoptosis. Caspase inhibition prevented tau cleavage without reversing changes in tau phosphorylation linked to apoptosis. Furthermore, the microtubule depolymerizing agent, colchicine, induced tau dephosphorylation and caspase-independent tau cleavage and degradation. Both phenomena were blocked by inhibiting protein phosphatase 2A (PP2A) by okadaic acid. These experiments indicate that tau dephosphorylation precedes and is required for its cleavage and degradation. We propose that the absence of cleavage and degradation of hyperphosphorylated tau (due to PP2A inhibition) may lead to its accumulation in degenerating neurons. This mechanism may contribute to the aggregation of hyperphosphorylated tau into paired helical filaments in Alzheimer's disease where reduced PP2A activity has been reported.


Apoptosis , Neurons/physiology , tau Proteins/metabolism , Alzheimer Disease , Animals , Apoptosis/drug effects , Caspase Inhibitors , Cells, Cultured , Cerebral Cortex/chemistry , Cerebral Cortex/cytology , Colchicine/pharmacology , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta , Lithium/pharmacology , Neurons/chemistry , Okadaic Acid/pharmacology , Peptide Fragments/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation , Protein Phosphatase 2 , Rats , Rats, Wistar , Staurosporine/pharmacology , tau Proteins/analysis , tau Proteins/chemistry
6.
J Biol Chem ; 279(25): 25978-85, 2004 Jun 18.
Article En | MEDLINE | ID: mdl-15069072

To rescue stalled ribosomes, eubacteria employ a molecule, transfer messenger RNA (tmRNA), which functions both as a tRNA and as an mRNA. With the help of small protein B (SmpB), tmRNA restarts protein synthesis and adds by the trans-translation mechanism a peptide tag to the stalled protein to target it for destruction by cellular proteases. Here, the cellular location and expression of endogenous SmpB were monitored in vivo. We report that SmpB is associated with 70S ribosomes and not in the soluble fraction, independently of the presence of tmRNA. In vitro, SmpB that is pre-bound to a stalled ribosome can trigger initiation of trans-translation. Our results demonstrate the existence of a novel pathway for the entry of tmRNA to the ribosome and for the trans-transfer of a nascent peptide chain from peptidyl-tRNA to charged tmRNA.


Protein Biosynthesis , RNA, Bacterial/chemistry , RNA-Binding Proteins/physiology , Ribosomes/chemistry , Blotting, Northern , Cell Division , Centrifugation, Density Gradient , Escherichia coli/metabolism , Genetic Complementation Test , Immunoblotting , Mutation , Protein Binding , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribosomes/metabolism , Subcellular Fractions , Sucrose/pharmacology , Time Factors
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