ABSTRACT
The experiments described in this paper were designed to try and isolate a recombinant DNA clone encoding a Trypanosoma cruzi homologue of the Trypanosoma brucei glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) gene. Despite the ready biochemical detection of phospholipase C activities that hydrolyse GPI-anchors of cell surface proteins in T. cruzi, it did not prove possible to isolate any recombinant DNA clones using the T. brucei gpi-plc gene as a probe. On determining the DNA sequence to the 5' side of the gpi-plc gene it was found to be adjacent to a gene that encodes a 100 kDa heat shock protein (HSP100). To investigate whether this linkage between the hspl00 and gpi-plc genes was conserved in T. cruzi, a probe derived from the T. brucei hsp100 gene was used to isolate T. cruzi genomic clones. These were partially sequenced and shown to contain an hsp100 gene. Restriction enzyme fragments located to the 3' side of the T. cruzi hsp100 gene were then sequenced and found to contain a gene that encodes a polypeptide (TcPLC1) that has 46% amino acid sequence identity with the T. brucei GPI-PLC including most of the key residues involved in inositol binding and the catalytic histidine. A recombinant form of TcPLC1 was produced and shown to possess phospholipase C activity towards a GPI-substrate. Thus, the hsp100 and gpi-plc genes are adjacent in T. brucei and this linkage is conserved in T. cruzi. This observation has been used to facilitate the isolation of a clone encoding a T. cruzi phospholipase C gene.
Subject(s)
Genetic Linkage , Heat-Shock Proteins/genetics , Trypanosoma brucei brucei/genetics , Trypanosoma cruzi/genetics , Type C Phospholipases/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA Probes , Electrophoresis, Polyacrylamide Gel , Fluorometry , Genes, Protozoan , Glycosylphosphatidylinositol Diacylglycerol-Lyase , Molecular Sequence Data , Phosphatidylinositol Diacylglycerol-Lyase , Polymerase Chain Reaction , Recombinant Proteins/analysis , Sequence Homology, Amino Acid , Substrate Specificity , Trypanosoma brucei brucei/enzymology , Trypanosoma cruzi/enzymologyABSTRACT
The purpose of this review is to consider recent results obtained concerning the properties and function of the glycosylphosphatidylinositol-phospholipase C (GPI-PLC) in Trypanosoma brucei. A mutagenesis study that provides evidence that the GPI-PLC is more closely related to bacterial PI-PLCs than previously realised is described. The variant specific glycoprotein (VSG), which dominates the surface of the mammalian stages of the trypanosome, is almost certainly the major substrate of the GPI-PLC. The hydrolysis of the GPI-anchor of the VSG under stress conditions and hypotonic lysis is well established. To investigate whether this hydrolysis of the GPI-anchor plays any role during the life cycle a GPI-PLC null mutant has been made. The phenotype indicates that the gene is non-essential, but its absence alters the course of infection in mice.
Subject(s)
Trypanosoma brucei brucei/enzymology , Trypanosomiasis, African/parasitology , Type C Phospholipases/metabolism , Amino Acid Sequence , Animals , Gene Deletion , Glycosylphosphatidylinositol Diacylglycerol-Lyase , Glycosylphosphatidylinositols/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Phosphatidylinositol Diacylglycerol-Lyase , Substrate Specificity , Trypanosoma brucei brucei/genetics , Trypanosomiasis, African/immunology , Type C Phospholipases/chemistry , Type C Phospholipases/genetics , Variant Surface Glycoproteins, Trypanosoma/metabolismABSTRACT
In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.
Subject(s)
Glycosylphosphatidylinositols/physiology , Parasitemia/enzymology , Trypanosoma brucei brucei/enzymology , Trypanosomiasis, African/enzymology , Type C Phospholipases/physiology , Animals , Disease Models, Animal , Glycosylphosphatidylinositols/genetics , Mice , Mice, Inbred Strains , Mutagenesis, Insertional , Parasitemia/genetics , Parasitemia/parasitology , Phenotype , Sequence Deletion , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/growth & development , Trypanosomiasis, African/genetics , Trypanosomiasis, African/parasitology , Type C Phospholipases/biosynthesis , Type C Phospholipases/geneticsABSTRACT
The glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei is particularly effective in hydrolysing the GPI-anchors of some proteins. The enzyme is inhibited by Zn2+ and p-chloromercurylphenylsulphonic acid, both of which can act as sulphydryl reagents, suggesting that a cysteine residue may be important in catalysis. Single cysteine to serine mutants have been produced for all eight cysteines in GPI-PLC; all the mutants were fully active in vitro and were still susceptible to p-chloromercurylphenylsulphonic acid inhibition. In contrast, a single histidine 34 to glutamine mutation totally inactivated GPI-PLC. The histidine was chosen after a sequence alignment with the Bacillus cereus phosphatidylinositol phospholipase C (PI-PLC) suggested a conservation of active site residues, including histidine 34 which is central to the proposed reaction mechanism (Heinz D.W., Ryan M., Bullock T.L., Griffith O.H. EMBO J 1995;14:3855-3863). The results suggest that the GPI-PLC and bacterial PI-PLCs have conserved active sites and that the inhibition of GPI-PLC by sulphydryl reagents can occur through more than one residue.
Subject(s)
Trypanosoma brucei brucei/enzymology , Type C Phospholipases/metabolism , 4-Chloromercuribenzenesulfonate/pharmacology , Amino Acid Sequence , Animals , Bacillus cereus/enzymology , Bacillus cereus/genetics , Binding Sites , Cysteine/chemistry , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Glycosylphosphatidylinositol Diacylglycerol-Lyase , Histidine/chemistry , Listeria monocytogenes/enzymology , Listeria monocytogenes/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Recombinant Proteins/metabolism , Sequence Alignment , Sulfhydryl Reagents/pharmacology , Trypanosoma brucei brucei/genetics , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/chemistry , Type C Phospholipases/geneticsABSTRACT
The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG). However the evidence that it is involved in VSG metabolism in the living trypanosome is circumstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotonic lysis. Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease. To try to determine the role of the GPI-PLC, a homozygous null mutant T. brucei has been constructed. The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes. The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype. The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be.
Subject(s)
Glycosylphosphatidylinositols/metabolism , Phosphoric Diester Hydrolases/physiology , Trypanosoma brucei brucei/enzymology , Variant Surface Glycoproteins, Trypanosoma/metabolism , Animals , Gene Deletion , Glycosylphosphatidylinositol Diacylglycerol-Lyase , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoric Diester Hydrolases/genetics , Restriction Mapping , Trypanosoma brucei brucei/geneticsABSTRACT
The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG). However the evidence that it is involved in VSG metabolism in the living trypanosome is circunstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotomic lysis. Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease. To try to determine the role of the GPI-PLC, a homozygous mull mutant T. brucei has been constructed. The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes. The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype. The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be