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No full textFunction of region I and II adhesive motifs of Plasmodium falciparum circumsporozoite protein in sporozoite motility and infectivity
Full text linkThe circumsporozoite protein of Plasmodium falciparum contains two conserved motifs (regions I and II) that have been proposed to interact with mosquito and vertebrate host molecules in the process of sporozoite invasion of salivary glands and hepatocytes, respectively. To study the function of this protein we have replaced the endogenous circumsporozoite protein gene of Plasmodium berghei with that of P. falciparum and with versions lacking either region I or region II. We show here that P. falciparum circumsporozoite protein functions in rodent parasite and that P. berghei sporozoites carrying the P. falciparum CS gene develop normally, are motile, invade mosquito salivary glands, and infect the vertebrate host. Region I-deficient sporozoites showed no impairment of motility or infectivity in either vector or vertebrate host. Disruption of region II abolished sporozoite motility and dramatically impaired their ability to invade mosquito salivary glands and infect the vertebrate host. These data shed new light on the role of the CS protein in sporozoite motility and infectivity
Analysis of a malaria sporozoite protein family required for gliding motility and cell invasion: Response
No full textWe were surprised to read such strong objections to the experimental strategy that we developed to investigate the functional significance of the amino acid residues shared by the thrombospondin-related adhesive proteins (TRAP) found in Plasmodium berghei (PbTRAP) and Plasmodium falciparum (PfTRAP)1. Not only have complementation experiments, using heterologous host systems, been widely used to elucidate gene function in a variety of organisms, but Drs Nussenzweig and Menard themselves have used such an approach to assess the function of conserved residues in the TRAP cytoplasmic tail: ‘cytoplasmic domains of this family (TRAP) are interchangeable indicating that they perform a similar function’2. What Drs Nussenzweig and Menard omit to say is that MIC2, the homologue of PbTRAP in Toxoplasma gondii, is less than 30% homologous to PbTRAP in the cytoplasmic tail. We wonder what makes it appropriate for them to use an experimental procedure that they regard as daring and unorthodox when used by us. As far as the vertebrate host range of P. berghei is concerned, we are aware that this parasite species does not infect humans. However, in vitro, P. berghei sporozoites are perfectly capable of invading human hepatocytes, where they can complete their development; this provided the rationale behind the study of P. falciparum molecules, that were implicated in the recognition and invasion process, in P. berghei
Structure-function analysis of malaria proteins by gene targeting--a response
Full text linkWe have generated transgenic Plasmodium berghei parasites in which the endogenous gene encoding thrombospondin-related anonymous protein (TRAP) was replaced by either wild-type (WT) P. falciparum TRAP (PfTRAP) or by mutated versions of PfTRAP carrying amino acids substitutions or deletions in the A domain or in the thrombospondin-related motif1. This strategy was employed in a structure–function analysis, which aimed to circumvent limitations due to the availability of only one selectable marker to transform P. berghei. We did not attempt to modify the endogenous gene encoding TRAP by using an insertion-targeting approach because we were (and still are) convinced that genetic reversion is a limitation in structure–function studies. A reversion frequency of 1% should not be underestimated. If the revertant parasites were 50–100% more efficient than the mutated ones in infecting target cells or in gliding motility, their presence would confound the phenotypic analysis of the mutated parasites
THROMBOSPONDIN-RELATED ADHESIVE PROTEIN (TRAP) OF PLASMODIUM BERGHEI AND PARASITE MOTILITY
Full text linkThrombospondin-related adhesive protein (TRAP) plays a part in malaria sporozoite recognition and entry into host hepatocytes. [1] and [2] Little is known about how this molecule, mainly localised in the parasite micronemes, contributes to sporozoite invasion.3 The presence of conserved adhesive motifs within the aminoacid sequence of TRAP suggested that this molecule could interact directly with ligands on the surface of host cells.4 We observed that air-dried Plasmodium berghei sporozoites, while being processed for immuno-fluorescence, leave a trail of TRAP immunoreactive material on the microscope slide, suggesting that TRAP was released during parasite movement. We looked for TRAP after inducing sporozoites to glide on microscope slides at 37°C. Trails of TRAP immunoreactive material were observed behind most sporozoites (figure). Gliding sporozoites occasionally detached from microscope slides leaving an immunoreactive footprint on the glass that reveals a uniform distribution of TRAP along the surface of the parasite body (figure arrow). These findings show that in motile sporozoites TRAP is translocated to the parasite surface and is continuously shed behind in a similar fashion to that described for the circumsporozoite (CS) protein. We have investigated TRAP function during sporozoite movement with freshly dissected P berghei salivary-gland sporozoites and a polyclonal mouse serum previously shown to specifically recognise TRAP. Examination by direct microscopy of live sporozoites revealed that parasites incubated with TRAP antiserum at 37°C showed a reduction in gliding motility. We carried out a quantitative analysis of this inhibitory effect by immunofluorescence with a biotin-labelled monoclonal antibody directed against the CS protein. We identified motile sporozoites by the presence of an immunoreactive trail of CS protein. This experiment showed that the TRAP antiserum inhibited, in a dose-dependent manner, sporozoite gliding. Sporozoites incubated with TRAP antiserum diluted 1/50 and 1/100 slowed CS-protein gliding trails by 19·5% and 41%, respectively. These percentages were significantly lower than those observed by incubating sporozoites with a control mouse serum raised against the recombinant protein 85A from Mycobacterium tuberculosis. TRAP binding to host ligands would allow sporozoites to interact with biological surfaces thus providing molecular anchors for parasite gliding. These results also shed new light on the mode of action of TRAP antibodies in blocking sporozoite invasion of hepatocytes. TRAP antibodies would primarily impair sporozoite motility and, as a consequence of this, the ability of parasites to invade host cells. The demonstration that TRAP is implicated in sporozoite motility contributes to the understanding of the molecular mechanisms that lead to parasite infection of host cells, and provides the rationale for eliciting TRAP antibodies to block sporozoite invasion of hepatocytes
Temporal and spatial distribution of Toxoplasma gondii differentiation into Bradyzoites and tissue cyst formation in vivo
Full text linkDuring Toxoplasma gondii infection, a fraction of the multiplying parasites, the tachyzoites, converts into bradyzoites, a dormant stage, which form tissue cysts localized mainly in brain, heart, and skeletal muscles that persist for several years after infection. At this stage the parasite is protected from the immune system, and it is believed to be inaccessible to drugs. While the long persistence of tissue cysts does not represent a medical problem for healthy individuals, this condition represents a major risk for patients with a compromised immune system, who can develop recrudescent life-threatening T. gondii infections. We have investigated for the first time the dynamics and the kinetics of tachyzoite-to-bradyzoite interconversion and cyst formation in vivo by using stage-specific bioluminescent parasites in a mouse model. Our findings provide a new framework for understanding the process of bradyzoite differentiation in vivo. We have also demonstrated that complex molecules such as d-luciferin have access to tissue cysts and are metabolically processed, thus providing a rationale for developing drugs that attack the parasite at this developmental stage
EARLY REGULATION OF IFN-GAMMA PRODUCTION IN POLARIZED T-HELPER CELL RESPONSES IN CANDIDA-ALBICANS INFECTION
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Addition of allochthonous fungi to a historically contaminated soil affects both remediation efficiency and bacterial diversity.
No full textBotryosphaeria rhodina DABAC P82 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow and to degrade aromatic hydrocarbons in an aged contaminated soil. To evaluate the impact of indigenous microflora on the overall process, incubations were performed on both fumigated and nonfumigated soils. Fungal colonization by B. rhodina was unexpectedly lower in the fumigated than in the nonfumigated soil while the growth of P. pulmonarius showed an opposite response. Degradation performances and detoxification by both fungi in the nonfumigated soil were markedly higher than those observed in the fumigated one. Heterotrophic bacterial counts in nonfumigated soil augmented with either B. rhodina or P. pulmonarius were significantly higher than those of the corresponding incubation control (6.7 ± 0.3 × 108 and 8.35 ± 0.6 × 108, respectively, vs 9.2 ± 0.3 × 107). Bacterial communities of both incubation controls and fungal-augmented soil were compared by numerical analysis of denaturing gradient gel electrophoresis profiles of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA (rRNA) genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Besides increasing overall diversity, fungal augmentation led to considerable qualitative differences with respect to the pristine soil. © 2007 Springer-Verlag
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