Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to ...control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3' untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The SHERP genes are found as a tandem pair within the differentially regulated LmcDNA16 locus of Leishmania major. The SHERP gene product (small hydrophilic endoplasmic reticulum-associated protein) ...is unusual in its small size (6.2 kDa), its acidic pI (4.6) and its exclusive, high-level expression ( approximately 100000 copies per cell) in infective non-replicative parasite stages. No homologues have been found to date. Secondary-structure predictions suggest that SHERP contains an amphiphilic alpha-helix that is presumably involved in protein-protein interactions. SHERP has been localized to the endoplasmic reticulum as well as to the outer mitochondrial membrane in both wild-type and over-expressing parasites. Given the absence of an N-terminal signal sequence, transmembrane-spanning domains or detectable post-translational modifications, it is likely that this hydrophilic molecule is a peripheral membrane protein on the cytosolic face of intracellular membranes. This weak membrane association has been confirmed in cell-fractionation assays, in which SHERP redistributes from the cytoplasmic to the membrane fraction after in vivo cross-linking. SHERP does not appear to be involved in rearrangements of the cytoskeleton or conservation of organelle morphology during parasite differentiation. The role of this novel protein, presumed to be part of a protein complex, in infective parasites that are nutrient-deficient and pre-adapted for intracellular survival in the mammalian host is under investigation.
Plasmodium falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport. However, PfRab5B is unique in lacking a C-terminal geranyl-geranylation motif, while having N-terminal ...palmitoylation and myristoylation motifs. We show that the N-terminal glycine is required for PfRab5B myristoylation in vitro and when an N-terminal PfRab5B fragment possessing both acylation motifs is fused to GFP and expressed in transgenic P. falciparum parasites, the chimeric PfRab5B protein localizes to the plasma membrane. Upon substitution of the modified glycine by alanine the staining becomes diffuse and GFP is found in soluble subcellular fractions. Immuno-electron microscopy shows endogenous PfRab5B decorating the parasite's plasma and food vacuole membranes. Using reverse genetics rab5b couldn't be deleted from the haploid genome of asexual blood stage P. berghei parasites. The failure of PbRab5A or PbRab5C to complement for loss of PbRab5B function indicates non-overlapping roles for the three Plasmodium Rab5s, with PfRab5B involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane. We discuss similarities between Plasmodium Rab5B and Arabidopsis thaliana ARA6, a similarly unusual Rab5-like GTPase of plants.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The LmcDNA16 locus of Leishmania major contains three highly related genes HASPA1, HASPA2 and HASPB, encoding hydrophilic, acylated surface proteins and a tandem pair of unrelated sequences, SHERP1 ...and SHERP2, coding for a small, hydrophilic protein that localizes to the endoplasmic reticulum and outer mitochondrial membrane. Differential regulation of these genes results in expression of a subset of the HASP proteins and SHERP only in infective stage parasites. To assess the contribution of these molecules to parasite virulence, the diploid LmcDNA16 gene locus has been removed by targeted gene deletion. Homozygous null mutants have precise deletions of both alleles and exhibit no HASP or SHERP expression. They are at least as virulent as wild‐type parasites in macrophage invasion and intracellular survival assays, both in vitro and in vivo. Conversely, null mutants engineered to overexpress the entire LmcDNA16 gene locus are unable to survive within the intramacrophage environment despite their differentiation into infective metacyclic parasites. Both null and overexpressing null parasites show increased sensitivity to complement‐mediated lysis, suggesting perturbation of their surface architecture. Avirulence in overexpressing parasites correlates with selective depletion of a specific lipid species, decreased expression of the major surface glycoprotein GP63, but no significant downregulation of the glycoconjugate lipophosphoglycan.
Regulation of gene expression in trypanosomatid parasites is predominantly post-transcriptional. Primary transcripts are trans-spliced and polyadenylated to generate mature mRNAs and transcript ...stability is a major factor controlling stage-specific gene expression. Degenerate PCR has been used to clone the gene encoding the Leishmania homologue of poly(A)-binding protein (Lm PAB1), as an approach to the identification of trans-acting factors involved in this atypical mode of eukaryotic gene expression. lmpab1 is a single copy gene encoding a 63 kDa protein which shares major structural features but only 35-40% amino acid identity with other PAB1 sequences, including those of other trypanosomatids. Lm PAB1 is expressed at constant levels during parasite differentiation and is phosphorylated in vivo. It is localised predominantly in the cytoplasm but inhibition of transcription with actinomycin D also reveals diffuse localisation in the nucleus. Lm PAB1 binds poly(A) with high specificity and affinity but fails to complement a null mutation in Saccharomyces cerevisiae. These properties are indicative of functional divergence in vivo.
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