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Michael J Linke - One of the best experts on this subject based on the ideXlab platform.
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gene expression of pneumocystis murina after treatment with anidulafungin results in strong signals for sexual reproduction cell wall integrity and cell cycle arrest indicating a requirement for ascus formation for proliferation
Antimicrobial Agents and Chemotherapy, 2018Co-Authors: Melanie T Cushion, Michael J Linke, Alan Ashbaugh, Keeley Hendrix, Nikeya Tisdale, Steven G Sayson, Aleksey PorolloAbstract:The echinocandins are a class of antifungal agents that target β-1,3-d-glucan (BG) biosynthesis. In the ascigerous Pneumocystis species, treatment with these drugs depletes the ascus life cycle stage, which contains BG, but large numbers of forms which do not express BG remain in the infected lungs. In the present study, the gene expression profiles of Pneumocystis murina were compared between infected, untreated mice and mice treated with anidulafungin for 2 weeks to understand the metabolism of the persisting forms. Almost 80 genes were significantly up- or downregulated. Like other fungi exposed to echinocandins, genes associated with sexual replication, cell wall integrity, cell cycle arrest, and stress comprised the strongest upregulated signals in P. murina from the treated mice. The upregulation of the P. murina β-1,3-d-glucan endohydrolase and endo-1,3-glucanase was notable and may explain the disappearance of the existing asci in the lungs of treated mice since both enzymes can degrade BG. The biochemical measurement of BG in the lungs of treated mice and fluorescence microscopy with an anti-BG antibody supported the loss of BG. Downregulated signals included genes involved in cell replication, genome stability, and ribosomal biogenesis and function and the Pneumocystis-specific genes encoding the Major Surface Glycoproteins (Msg). These studies suggest that P. murina attempted to undergo sexual replication in response to a stressed environment and was halted in any type of proliferative cycle, likely due to a lack of BG. Asci appear to be a required part of the life cycle stage of Pneumocystis, and BG may be needed to facilitate progression through the life cycle via sexual replication.
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Biofilm Formation by Pneumocystis spp.
Eukaryotic Cell, 2008Co-Authors: Melanie T Cushion, Margaret S. Collins, Michael J LinkeAbstract:Pneumocystis spp. can cause a lethal pneumonia in hosts with debilitated immune systems. The manner in which these fungal infections spread throughout the lung, the life cycles of the organisms, and their strategies used for survival within the mammalian host are largely unknown, due in part to the lack of a continuous cultivation method. Biofilm formation is one strategy used by microbes for protection against environmental assaults, for communication and differentiation, and as foci for dissemination. We posited that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system comprised of insert wells suspended in multiwell plates containing supplemented RPMI 1640 medium supported biofilm formation by P. carinii (from rat) and P. murina (from mouse).Dramatic morphological changes accompanied the transition to a biofilm. Cyst and trophic forms became highly refractile and produced branching formations that anastomosed into large macroscopic clusters that spread across the insert. Confocal microscopy revealed stacking of viable organisms enmeshed in concanavalin A-staining extracellular matrix. Biofilms matured over a 3-week time period and could be passaged. These passaged organisms were able to cause infection in immunosuppressed rodents. Biofilm formation was inhibited by farnesol, a quorum-sensing molecule in Candida spp., suggesting that a similar communication system may be operational in the Pneumocystis biofilms. Intense staining with a monoclonal antibody to the Major Surface Glycoproteins and an increase in (1,3)-β-d-glucan content suggest that these components contributed to the refractile properties. Identification of this biofilm process provides a tractable in vitro system that should fundamentally advance the study of Pneumocystis.
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Characterization of Rat CD4 T Cell Clones Specific for the Major Surface Glycoprotein of Pneumocystis carinii
The Journal of eukaryotic microbiology, 1997Co-Authors: Sue A. Theus, Michael J Linke, Ryan P. Andrews, Peter D. WalzerAbstract:Pneumocystis carinii are coated by abundant heterogenous Major Surface Glycoproteins (MSGs), which facilitate interaction with the host. We have produced MSG-specific T-cell clones from the spleens of P. carinii-exposed Lewis rats and analyzed five for antigen specificity to native MSG and a recombinant form of MSG, cell Surface markers, and cytokine profiles. All five of the clones were CD4+. All of the clones proliferated specifically to both the native MSG and the recombinant MSG only in the presence of antigen presenting cells demonstrating that the response is antigen/driven rather than mitogen/driven. All five of the clones secreted IL-2 and IFN-gamma, although in differing amounts, implicating a Th1 response. Only one of the clones produced any detectable IL-4. This is the first report of T cell clones responsive to a specific antigen of P. carinii, MSG. We conclude that the T cell clones will be helpful in mapping protective epitopes present in MSG and in functional studies of MSG.
Peter Bütikofer - One of the best experts on this subject based on the ideXlab platform.
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Major Surface Glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse.
PloS one, 2009Co-Authors: Erik Vassella, Christina Kunz Renggli, Peter Bütikofer, Michael Oberle, Simon Urwyler, Erwin Studer, Andrew Hemphill, Cristina Fragoso, Reto Brun, Isabel RoditiAbstract:Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant Surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.
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Trypanosoma congolense Procyclins: Unmasking Cryptic Major Surface Glycoproteins in Procyclic Forms
Eukaryotic cell, 2006Co-Authors: Silvia Utz, Isabel Roditi, Christina Kunz Renggli, Igor C. Almeida, Alvaro Acosta-serrano, Peter BütikoferAbstract:In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant Surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these Surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the Surface coat of cells that are negative for both PRS and GARP.
Isabel Roditi - One of the best experts on this subject based on the ideXlab platform.
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Major Surface Glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse.
PloS one, 2009Co-Authors: Erik Vassella, Christina Kunz Renggli, Peter Bütikofer, Michael Oberle, Simon Urwyler, Erwin Studer, Andrew Hemphill, Cristina Fragoso, Reto Brun, Isabel RoditiAbstract:Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant Surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.
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Trypanosoma congolense Procyclins: Unmasking Cryptic Major Surface Glycoproteins in Procyclic Forms
Eukaryotic cell, 2006Co-Authors: Silvia Utz, Isabel Roditi, Christina Kunz Renggli, Igor C. Almeida, Alvaro Acosta-serrano, Peter BütikoferAbstract:In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant Surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these Surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the Surface coat of cells that are negative for both PRS and GARP.
Pamela J. Langer - One of the best experts on this subject based on the ideXlab platform.
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molecular karyotype and chromosomal localization of genes encoding two Major Surface Glycoproteins gp63 and gp46 m2 hsp70 and β tubulin in cloned strains of several leishmania species
Molecular and Biochemical Parasitology, 1991Co-Authors: Theodor Hanekamp, Pamela J. LangerAbstract:Abstract The molecular karyotypes of several Leishmania isolates ( Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis, Leishmania donovani, Leishmania Major, Leishmania aethiopica, Leishmania tropica, Leishmania enriettii ) have been analyzed by clamped homogeneous electric field (CHEF) gel electrophoresis. The chromosomal localization of genes encoding 2 Major Surface Glycoproteins, gp63 and gp46/M2, heat shock protein 70 (hsp70), and β-tubulin was determined for cloned isolates of 8 of these Leishmania species. The chromosome size class assignment of hsp70 genes was most conserved in that all species contained a single hybridizing DNA band of approximately 1200 kb. The β-tubulin gene probe hybridized predominantly to large (1600–1750 kb) chromosome-size DNA and to 1–5 additional bands, the number of which depended on the species. The number and size of DNA bands hybridizing to gp63 or gp46/M2 gene probes were not uniformly conserved among species. In contrast to previous reports of gp63 genes being located on a single chromosome, using various CHEF gel conditions we observed a Leishmania Major gp63 gene probe hybridizing to at least 2 chromosomal DNA bands in the New World species and in L. tropica . Gp46/M2 genes were located on 1 band in L. donovani, L. Major , and L. aethiopica or 2 bands in L. tropica and L. amazonensis , but surprisingly, do not hybridize to any chromosomal DNA of species in the L. braziliensis complex or in L. enriettii . Whenever both genes were present in a species, gp63 and gp46/M2 genes were located on different chromosomal DNA bands.
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Molecular karyotype and chromosomal localization of genes encoding two Major Surface Glycoproteins, gp63 and gp46/M2, hsp70, and β-tubulin in cloned strains of several Leishmania species
Molecular and biochemical parasitology, 1991Co-Authors: Theodor Hanekamp, Pamela J. LangerAbstract:Abstract The molecular karyotypes of several Leishmania isolates ( Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis, Leishmania donovani, Leishmania Major, Leishmania aethiopica, Leishmania tropica, Leishmania enriettii ) have been analyzed by clamped homogeneous electric field (CHEF) gel electrophoresis. The chromosomal localization of genes encoding 2 Major Surface Glycoproteins, gp63 and gp46/M2, heat shock protein 70 (hsp70), and β-tubulin was determined for cloned isolates of 8 of these Leishmania species. The chromosome size class assignment of hsp70 genes was most conserved in that all species contained a single hybridizing DNA band of approximately 1200 kb. The β-tubulin gene probe hybridized predominantly to large (1600–1750 kb) chromosome-size DNA and to 1–5 additional bands, the number of which depended on the species. The number and size of DNA bands hybridizing to gp63 or gp46/M2 gene probes were not uniformly conserved among species. In contrast to previous reports of gp63 genes being located on a single chromosome, using various CHEF gel conditions we observed a Leishmania Major gp63 gene probe hybridizing to at least 2 chromosomal DNA bands in the New World species and in L. tropica . Gp46/M2 genes were located on 1 band in L. donovani, L. Major , and L. aethiopica or 2 bands in L. tropica and L. amazonensis , but surprisingly, do not hybridize to any chromosomal DNA of species in the L. braziliensis complex or in L. enriettii . Whenever both genes were present in a species, gp63 and gp46/M2 genes were located on different chromosomal DNA bands.
Michael A. J. Ferguson - One of the best experts on this subject based on the ideXlab platform.
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Partial structure of glutamic acid and alanine-rich protein, a Major Surface glycoprotein of the insect stages of Trypanosoma congolense.
The Journal of biological chemistry, 2002Co-Authors: Lynn M. Thomson, Douglas J. Lamont, Angela Mehlert, J. David Barry, Michael A. J. FergusonAbstract:The tsetse fly transmitted salivarian trypanosome, Trypanosoma congolense of the subgenus Nanomonas, is the most significant of the trypanosomes with respect to the pathology of livestock in sub-Saharan Africa. Unlike the related trypanosome Trypanosoma brucei of the subgenus Trypanozoon, the Major Surface molecules of the insect stages of T. congolense are poorly characterized. Here, we describe the purification and structural characterization of the glutamic acid and alanine-rich protein, one of the Major Surface Glycoproteins of T. congolense procyclic and epimastigote forms. The glycoprotein is a glycosylphosphatidylinositol-anchored molecule with a galactosylated glycosylphosphatidylinositol anchor containing an sn-1-stearoyl-2-l-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol phospholipid moiety. The 21.6-kDa polypeptide component carries two large mannose- and galactose-containing oligosaccharides linked to threonine residues via phosphodiester linkages. Mass spectrometric analyses of tryptic digests suggest that several or all of the closely related glutamic acid and alanine-rich protein genes are expressed simultaneously in a T. congolense population growing in vitro.
