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.

Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors.

BACKGROUND: Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6, 9-trisubstituted purines remain unverified. RESULTS: To address this issue, purvalanol B (95. ) and an N6-methylated, CDK-inactive derivative (95M. ) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95. matrix. Casein kinase 1 (CK1) was identified as a principal 95. matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries. CONCLUSIONS: That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds.

Increase in cariogenic bacteria after initial periodontal therapy.

This study examined the hypothesis of an intra-oral shift, during initial periodontal therapy, from a periopathogenic to a cariogenic flora. Seventy-one patients with periodontitis were randomly allocated to one of the following treatment strategies: (1) scaling and root planing, quadrant by quadrant, at two-week intervals (NC); (2) full-mouth scaling and root planing within 24 hrs (FRP); or (3) full-mouth disinfection within 24 hrs, including antiseptics [chlorhexidine (CHX) or amine fluoride/stannous fluoride (F) for 2 mos, or CHX for 2 mos followed by F for 6 mos (CHX+F)]. At baseline and after 2, 4, and 8 mos, bacterial samples were taken from supra- and subgingival plaque, saliva, and tongue. The detection frequencies and relative proportions of Streptococcus mutans increased in the NC and FRP groups, but decreased in the F group. In the CHX group, these species disappeared temporarily, but they disappeared for the entire 8 mos in the CHX+F group. These observations were similar for all sample locations. The periopathogens decreased in all groups. This finding confirms the abovementioned hypothesis and indicates a need for caries prophylactic regimens.

Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre.

Site-specific DNA recombinases from bacteriophage and yeasts have been developed as novel tools for genome engineering both in prokaryotes and eukaryotes. The 38kDa Cre protein efficiently produces both inter- and intramolecular recombination between specific 34bp sites called loxP. We report here the in vivo use of Cre recombinase to manipulate the genome of the protozoan parasite Toxoplasma gondii. Cre catalyzes the precise removal of transgenes from T. gondii genome when flanked by two directly repeated loxP sites. The efficiency of excision has been determined using LacZ as reporter and indicates that it can easily be applied to the removal of undesired sequences such as selectable marker genes and to the determination of gene essentiality. We have also shown that the reversibility of the recombination reaction catalyzed by Cre offers the possibility to target site-specific integration of a loxP-containing vector in a chromosomally placed loxP target in the parasite. In mammalian systems, the Cre recombinase can be regulated by hormone and is used for inducible gene targeting. In T. gondii, fusions between Cre recombinase and the hormone-binding domain of steroids are constitutively active, hampering the utilization of this mode of post-translational regulation as inducible gene expression system.

Molecular cloning of the Toxoplasma gondii sag4 gene encoding an 18 kDa bradyzoite specific surface protein.

An 18 kDa bradyzoite specific surface protein of Toxoplasma gondii (T. gondii) has been purified by affinity chromatography with a specific monoclonal antibody using parasites grown in vitro under conditions inducing the biosynthesis of bradyzoite specific proteins. N-terminal and internal amino acid sequences obtained by microsequencing enabled us to design degenerate oligonucleotides. A fragment of 187 bp was amplified by polymerase chain reaction (PCR). It was used as a probe to clone a 4 kb-Bam HI fragment encompassing the gene encoding the 18 kDa protein. Nucleotide sequence analysis revealed a single open reading frame of 516 nucleotides encoding a 172 amino acid protein. The deduced amino acid sequence matched perfectly the peptides microsequenced from the native protein. The N-terminal hydrophobic region was found to possess the characteristics of a signal peptide of 27 amino acids. The hydrophobic C-terminal part could represent a signal for a glycan-phosphoinositide anchor. The full-length cDNA was also isolated and both the 5' and 3' untranslated regions were determined. Reverse transcriptase-PCR (RT-PCR) using p18-specific primers showed a stage-specific expression of this gene. Comparison of the nucleic acid sequence and the predicted amino acid sequence with databases did not reveal significant homology with known genes or proteins. This gene is proposed to be named sag4, according to the existing T. gondii nomenclature.

Successful reinfection of chronically infected mice by a different Toxoplasma gondii genotype.

It is generally assumed that primary infection by Toxoplasma gondii protects from reinfection. A recent study using a murine model has questioned this dogma using indirect procedures to detect the reinfecting strain. We have reinvestigated this issue using a transfected strain of T. gondii (Prugniaud beta galactosidase: Pru beta gal) which expresses Escherichia coli beta-galactosidase. Detection of enzyme activity on fixed parasites allows a direct distinction between transfected and untransfected strains. We have found that in OF1 mice primary infection with the 76 K strain of T. gondii fully protects mice against tissue cyst production upon reinfection with the Pru beta gal T. gondii strain whereas primary infection with the Pru beta gal T. gondii strain does not impair tissue cyst formation upon reinfection with the Ned strain of T. gondii, which belongs to another T. gondii genotype. These results suggest that the immune protection conferred by one strain of T. gondii can be breached by reinfection with a strain belonging to another genotype; which can have significant consequences in human or veterinary medicine.

One-stage full-mouth disinfection. Long-term microbiological results analyzed by checkerboard DNA-DNA hybridization.

BACKGROUND: Recent studies reported significant, additional clinical and microbiological improvements when severe adult periodontitis was treated via the one-stage full-mouth (OSFM) disinfection approach, instead of a standard treatment scheme with staged instrumentation per quadrant. The OSFM disinfection involves dealing with the remaining oropharyngeal niches such as tonsils, saliva, tongue, and mucosa. The OSFM disinfection procedure involves scaling and root planing of all pockets within 24 hours in combination with chlorhexidine application to all oropharyngeal niches (chairside and at home for 2 months). This study aimed to compare the microbiological shifts with the OSFM approach versus standard therapy. METHODS: Nineteen patients with advanced chronic periodontitis (AP) and 12 patients with early-onset periodontitis (EOP) were randomly assigned to the test and control groups. The control group (9 AP patients, 6 EOP patients) was scaled and root planed, per quadrant, with 2-week intervals. The test group (10 AP patients and 6 EOP patients) underwent OSFM disinfection treatment. At baseline and after 2, 4, and 8 months, pooled subgingival plaque samples were taken from single- and multi-rooted teeth. The presence and levels of 30 subgingival taxa were determined using whole genomic DNA probes and checkerboard DNA-DNA hybridization. RESULTS: Both treatments resulted in important reductions of the pathogenic species up to 8 months after therapy, both for their detection level and frequency. The OSFM disinfection resulted in an additional improvement, especially in the AP group. P. gingivalis and B. forsythus were reduced below detection level. The number of beneficial species remained nearly unchanged. CONCLUSIONS: The OSFM disinfection results in supplementary reductions of periodontal pathogens even after 8 months in the treatment of patients with advanced or early-onset periodontitis.

[Full mouth one stage disinfection of the oropharynx. A new treatment strategy for periodontitis]. / De éénfasige volledige orofaryngeale desinfectie. Een nieuwe behandelingsstrategie bij parodontitis.

Most periodontal pathogens colonise different niches such as the oral mucosa, tongue, saliva, periodontal pockets, all hard intra-oral surfaces and the tonsils. Since intra-oral translocation of these pathogens between these niches is possible, a 'one stage' disinfection of the whole mouth in one or two consecutive days as treatment strategy for periodontal infections must be considered. A one stage full-mouth disinfection is achieved by a scaling and root planing of all periodontal pockets within 24 hours, in combination with a chlorhexidine application in all niches (subgingival irrigation, mouth wash, brushing of the tongue, and spray for the pharynx). As such, reinfection by bacteria of recently instrumented sites is inhibited. This approach results in significant additional clinical and microbiological results compared to the classical approach (scaling and rootplaning per quadrant with several days or weeks between each session).

The importance of reverse genetics in determining gene function in apicomplexan parasites.

The phylum Apixomplexa includes obligate intracellular parasites that are of enormous medical and veterinary significance, as they are responsible for a wide variety of diseases including malaria, toxoplasmosis, coccidiosis, cryptosporidiosis, theileriosis and babesiosis. The EST sequencing projects in Toxoplasma gondii and the Plasmodium falciparum genome sequencing project have greatly accelerated gene discovery, revealing for example novel coding sequences restricted to the Apicomplexa. However, easy acquisition of sequence is almost useless if the function of any given gene cannot be tested. The establishment of transfection systems in Toxoplasma gondii, Neospora and in several Plasmodium species has provided us with the reverse genetics methods appropriate to the functional analysis of genes. Over the past few years, the discovery of novel genes coupled to the ability to introduce or modify genes has already contributed to a better understanding of cell biology and pathogenesis of these obligate intracellular parasites. Some insights into the complex processes of parasite invasion, differentiation, regulation of gene expression and protein trafficking are emerging although identification of the exact functional roles for many molecules is still awaiting more investigation. This review summarizes progress in this area. It also emphasises the tight link and synergy between Toxoplasma and malaria research. The use of reverse genetics does not guarantee the answer to gene function, so we can learn from both failed and successful experiments about how better and more efficiently to use 'genomics' to accelerate discoveries relevant to the understanding of parasitism by Apicomplexa.

Toxoplasma gondii: kinetics of bradyzoite-tachyzoite interconversion in vitro.

Stage-specific monoclonal antibodies have been used to investigate the bradyzoite-tachyzoite interconversion of Toxoplasma gondii in vitro. The differentiation of bradyzoites isolated from brain cysts (strains 76K and BQNC2) and grown in culture proceeded through intermediate stages which expressed both the specific markers of bradyzoites (P36, P34, P21, and P18) and a specific marker of tachyzoites (SAG1-P30). Differentiation started before parasite division, but large vacuoles containing intermediate stages were also found, suggesting that these were able to multiply. Intermediate stages were also observed during the differentiation of peritoneal tachyzoites (strain Prugniaud) into bradyzoites in vitro. Triggering of bradyzoite protein synthesis is not a single event since one of the four bradyzoite-specific proteins (P21) always appeared later than the others during bradyzoite differentiation. During interconversion, heterogeneous vacuoles containing parasites expressing different levels of bradyzoite or tachyzoite proteins were observed. This observation together with the fact that differentiation is not synchronous within a culture or within a host cell suggest a complex triggering process.

Developmental differentiation between tachyzoites and bradyzoites of Toxoplasma gondii.

An important event in the pathogenesis of toxoplasmosis is the interconversion between the bradyzoite and the tachyzoite stage of Toxoplasma gondii within the intermediate host. The factors that influence either cyst formation (bradyzoites) or reactivation (tachyzoites) are unknown. Uwe Gross, Wolfgang Bohne, Martine Soête and Jean François Dubremetz here describe current knowledge about the mechanisms that might lead to the induction of stage differentiation of this protozoan parasite.

Experimental induction of bradyzoite-specific antigen expression and cyst formation by the RH strain of Toxoplasma gondii in vitro.

The differentiation of Toxoplasma gondii tachyzoites into bradyzoites has been studied experimentally in vitro using the virulent RH strain. The differentiation was monitored by immunofluorescence detection of stage-specific proteins by monoclonal antibodies and by electron microscopy. The expression of bradyzoite-specific proteins has been induced by modifying the culture conditions in any of the following three ways: increasing the pH of the culture medium (pH 8), shifting temperature from 37 to 43 degrees C, or performing a sodium arsenite treatment. Interferon-gamma, described as involved in the control of toxoplasmosis in vivo, was inefficient to trigger bradyzoite proteins expression in HFF host cells in vitro. The pH increase and heat treatment, but not the sodium arsenite, induced the formation of cysts whose fine structure was similar to that of cysts found in the brain of mice infected by avirulent strains. Our results therefore show that the tachyzoite-bradyzoite switch is not directly dependent on an immunomodulator, but is likely to arise from an alteration of the environment of the host cell-parasite complex.

Serine protease inhibitors block invasion of host cells by Toxoplasma gondii.

We investigated the effect of protease inhibitors on the asexual development of the protozoan parasite Toxoplasma gondii. Among the inhibitors tested only two irreversible serine protease inhibitors, 3,4-dichloroisocoumarin and 4-(2-aminoethyl)-benzenesulfonyl fluoride, clearly prevented invasion of the host cells by specifically affecting parasite targets in a dose-dependent manner, with 50% inhibitory concentrations between 1 and 5 and 50 and 100 microM, respectively. Neither compound significantly affected parasite morphology, basic metabolism, or gliding motility within the range of the experimental conditions in which inhibition of invasion was demonstrated. No partial invasion was observed, meaning that inhibition occurred at an early stage of the interaction. These results suggest that at least one serine protease of the parasite is involved in the invasive process of T. gondii.

Characterization of bradyzoite-specific antigens of Toxoplasma gondii.

Monoclonal antibodies that react specifically with bradyzoite antigens of Toxoplasma gondii were selected by differential immunofluorescence among hybridomas produced against these organisms. These antigens were further characterized by immunofluorescence on living bradyzoites and Western immunoblotting. Four pellicular antigens (36, 34, 21, and 18 kDa) were identified; three of these are exposed on the surface of the organism and accessible to either antibodies, trypsin cleavage, or both of these surface-probing procedures. These antigens were found on recent human isolates of T. gondii, as well as on laboratory strain bradyzoites obtained from either brain cysts or in vitro-grown parasites.

The intra-oral translocation of periodontopathogens jeopardises the outcome of periodontal therapy. A review of the literature.

BACKGROUND: Although periodontitis has a multi-factorial aetiology, the success of its therapy mainly focuses on the eradication/reduction of the exogenous/endogenous periodontopathogens. Most of the species colonise several niches within the oral cavity (e.g. the mucosae, the tongue, the saliva, the periodontal pockets and all intra-oral hard surfaces) and even in the oro-pharyngeal area (e.g., the sinus and the tonsils). METHODS: This review article discusses the intra-oral transmission of periodontopathogens between these niches and analyses clinical studies that support the idea and importance of such an intra-oral translocation. RESULTS AND CONCLUSIONS: Based on the literature, the oro-pharyngeal area should indeed be considered as a microbiological entity. Because untreated pockets jeopardise the healing of recently instrumented sites, the treatment of periodontitis should involve "a one stage approach" of all pathologic pockets (1-stage full-mouth disinfection) or should at least consider the use of antiseptics during the intervals between consecutive instrumentations, in order to prevent a microbial translocation of periodontopathogens during the healing period. For the same reason, regeneration procedures or the local application of antibiotics should be postponed until a maximal improvement has been obtained in the remaining dentition. This more global approach offers significant additional clinical and microbiological benefits.

Stage conversion of Toxoplasma gondii in mouse brain during infection and immunodepression.

The kinetics and pattern of expression of bradyzoite-specific proteins were studied in mouse brain during infection with Toxoplasma gondii. Parasites found in the brain 6 days after ingestion of cysts were expressing only tachyzoite-specific proteins (anti-SAG1 antibodies being used as a marker). Bradyzoite-specific protein (Pb36) expression was first found after 9 days in vacuoles containing mixed parasites simultaneously expressing SAG1 and Pb36 or cysts containing parasites expressing only the bradyzoite marker. Reactivation of toxoplasmosis was studied in mouse brain using corticosteroids for immunosuppression. Parasites expressing SAG1 were first found 6 days after the beginning of treatment, but a very heterogeneous pattern was found throughout the study. We simultaneously found vacuoles containing parasites expressing only SAG1 or containing intermediate stages or cysts containing parasites expressing only bradyzoite proteins. A striking observation was the multiplication of cysts in foci, suggesting that the immune suppression triggered the release of parasites from preexisting cysts but that the factors inducing bradyzoite development remained fully effective in driving parasites into this pathway.

[Structure and biology of Toxoplasma gondii bradyzoites]. / Structure et biologie des bradyzoïtes de Toxoplasma gondii.

Tachyzoite-bradyzoite interconversion is one characteristic feature of Toxoplasma gondii. Although highly similar in structure, tachyzoite and bradyzoite differ by the relative amount of certain organelles and by specific surface or cytoplasmic molecules. Differences in structure and contents also exist between parasitophorous vacuoles and cysts. Using stage specific markers, it was shown the quickness of stage switching in vivo as well as in vitro, together with the occurrence of intermediate stages. Regulatory mechanisms of interconversion remain unknown. However, stress or inhibition of the mitochondrial metabolism of the parasite trigger bradyzoite formation.

Toxoplasma gondii: appearance of specific markers during the development of tissue cysts in vitro.

Cultures were initiated in Madin-Darby bovine kidney (MDBK) cells from ME49 strain bradyzoites. Specific antibody staining showed that two populations of parasites exist, one being a predominant population of tachyzoites that were positive for the tachyzoite-specific marker SAG1 and negative for the bradyzoite-specific marker P36. All of these parasites expressed the dense granule molecule GRA5, which in larger clusters was seen faintly in the membrane of the parasitophorous vacuole. No rosette formation or monolayer destruction was observed. Also seen was a sub-population of bradyzoites that were positive for P36 and negative for SAG1. Approximately 90% of these parasites expressed the matrix molecule P29. These parasites were also positive for the dense granule molecule GRA5, which was highly concentrated in the wall of the cyst. These bradyzoite clusters contained fewer parasites and were smaller in diameter than those expressing tachyzoite markers.

[Effects of three types of respiratory assistance (continuous positive pressure, intermittent positive pressure, classical respiratory physiotherapy) on PaO2 and PaCO2 in patients undergoing digestive tract surgery]. / Effets de trois méthodes d'assistance respiratoire sur la PaO2 et la PaCO2 chez l'opéré digestif (pression positive continue, pression positive intermittente, kinésithérapie respiratoire classique)

Two hundred and seventeen patients undergoing digestive tract surgery, either randomly or non-randomly allocated in two separate groups, and presenting postoperative hypoxemia, were studied during intensive care : 77 were ventilated by continuous positive pressure (CPP), 70 by intermittent positive pressure (IPP), and 70 were treated by classical respiratory physiotherapy. Results showed that CPP was the method of choice for ventilatory assistance following digestive tract surgery: hematosis was improved in a prolonged manner without modification of CO2 levels. The other procedures had a beneficial effect on PaO2 but at the price of a hyperventilation. Patients receiving IPP developed hypocapnea and hypoxemia five minutes after the end of the treatment. Physiopathological bases for these differences in gasometric behaviour in patients undergoing digestive tract surgery are analyzed as a function of the characteristics of CPP on the one hand, and IPP and respiratory physiotherapy on the other.