Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii.

The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

Molecular characterization of the glutathione peroxidase gene of the human malaria parasite Plasmodium falciparum.

In this paper we report the isolation and the characterization of a gene encoding the antioxidant enzyme glutathione peroxidase from the human malaria parasite Plasmodium falciparum. This gene contains two introns of 208 and 168 bp and is present in a single copy on chromosome 13. The open reading frame encodes a protein with a predicted length of 205 amino acids, which possesses a potential cleavage site between residues 21 and 22 after a hydrophobic region with the characteristics of a signal sequence. Therefore, the mature protein is predicted to be 184 residues long with a molecular mass of 21404 Da. In comparison with other known glutathione peroxidases many amino acid residues implicated in catalysis are conserved in the malarial enzyme. Phylogenetic analysis indicates that the deduced protein sequence is more closely related to plant glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase. A 1.5-kb transcript was identified in asynchronous erythrocytic stages.

The MIC1 microneme protein of Toxoplasma gondii contains a duplicated receptor-like domain and binds to host cell surface.

The cDNA encoding the Toxoplasma gondii microneme protein MIC1 and the corresponding gene have been cloned and sequenced. The MIC1 gene contains three introns. The cDNA encodes a 456 amino acid (aa) sequence, with a typical signal sequence and no other trans-membrane domain. The protein contains a tandemly duplicated domain with conservation of cysteines and presents distant homology with the Plasmodium sp. microneme protein TRAP-SSP2. The MIC1 protein from tachyzoite lysates and a PMAL recombinant expressing the N-terminal duplicated domain of the protein bound to the surface of putative host cells, suggesting a possible involvement of MIC1 in host cell binding/recognition.

Characterization of iron-dependent endogenous superoxide dismutase of Plasmodium falciparum.

Two main superoxide dismutase activities at isoelectric points (pI) 6.2 and 6.8 and two minor at pI 5.6 and 6.4 were found in crude extracts of Plasmodium falciparum. These activities were cyanide-resistant and hydrogen peroxide-sensitive and represented 20-30% of the total SOD activity found in the crude extract. A fragment of 424 bp, amplified from genomic DNA from P. falciparum, was cloned and sequenced. The deduced amino acid sequence identified this fragment as a coding region of an SOD gene. A cDNA corresponding to SOD was then isolated from a P. falciparum cDNA library and sequenced. The deduced amino acid sequence of SOD (197 aa) was compared with 32 known Feor Mn-SODs by the 'DARWIN' system. This analysis showed that the parasitic enzyme was related to typical Fe-SODs. The SOD subunit was purified and the N-terminal sequence, determined up to 29 residues, corresponded to that of cDNA isolated. The iron-dependent SOD activity found in Plasmodium falciparum represents the first level of the antioxidant defence system of the parasite. It is also the first SOD characterized in the parasitic Apicomplexa phylum whose sequence can be compared to equivalent iron-dependent enzymes known in other protozoa and bacteria.

Apical organelles and host-cell invasion by Apicomplexa.

Host-cell invasion by apicomplexan parasites involves the successive exocytosis of three different secretory organelles; namely micronemes, rhoptries and dense granules. The findings of recent studies have extended the structural homologies of each set of organelles between most members of the phylum and suggest shared functions of each set. Micronemes are apparently used for host-cell recognition, binding, and possibly motility; rhoptries for parasitophorous vacuole formation; and dense granules for remodeling the vacuole into a metabolically active compartment. In addition, gene cloning and sequencing have demonstrated conserved domains, which are likely to serve similar functions in the invasion process. This is especially true for microneme proteins containing thrombospondin-like domains, which are likely to be involved in binding to sulphated glycoconjugates. One such protein was recently shown to be required for the motility of Plasmodium sporozoites. These molecules have been shown to be shed on the parasite and/or cell surfaces during the invasion process in Plasmodium, Toxoplasma and Eimeria. For rhoptries and dense granules, the association between exocytosed proteins and the parasitophorous vacuole membrane had been analyzed extensively in Toxoplasma, as these proteins are likely to play a crucial role in metabolic interactions between the parasites and their host cells. The development of parasite transformation by gene transfection has provided powerful tools to analyze the fate and function(s) of the corresponding proteins.

Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence.

In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10/10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7/13 displayed the calf profile, and 6/13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates.

Polymorphism of the thymidylate synthase gene of Pneumocystis carinii from different host species.

Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400-bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat-derived P. carinii was hybridized with the amplified products from rat- and mouse-derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplified from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host-species specific. A specific probe which recognized all human P. carinii isolates was defined.

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite.

Assay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein-protein or protein-carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii.