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Andrew Hemphill - One of the best experts on this subject based on the ideXlab platform.
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neospora caninum protein disulfide isomerase is involved in tachyzoite Host Cell interaction
International Journal for Parasitology, 2005Co-Authors: Arunasalam Naguleswaran, Nathalie Vonlaufen, Ferial Alaeddine, Christophe Guionaud, Sabrina Sonda, Paul Jenoe, Meike Mevissen, Andrew HemphillAbstract:We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces Host Cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-Host Cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite Cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to Host Cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced Host Cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-Host Cell interaction.
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neospora caninum and toxoplasma gondii a novel adhesion invasion assay reveals distinct differences in tachyzoite Host Cell interactions
Experimental Parasitology, 2003Co-Authors: Arunasalam Naguleswaran, Norbert Muller, Andrew HemphillAbstract:This paper describes an adhesion/invasion assay, based on combined pyrrolidine dithiocarbamate (PDTC) and antibody treatment of parasites followed by quantitative real-time PCR. This PDTC-PCR assay can be used to comparatively assess the participation of Host Cell- and parasite-associated components during Host Cell adhesion and entry by Neospora caninum and Toxoplasma gondii tachyzoites, respectively, and is potentially applicable to any other apicomplexan parasite. The assay allows to determine the parasite invasion rate in relation to the overall number of parasites which interact with Host Cells in any given experiment, and thus represents a significant improvement to conventional microscopic assays in terms of accuracy and reproducibility. Using this assay it was possible to show that adhesion and invasion of N. caninum tachyzoites are two distinct and separated events, in that N. caninum tachyzoites preferentially utilise Host Cell surface chondroitin sulphates for adhesion, but not for the Host Cell invasion process. Application of the PDTC-PCR assay also demonstrated that N. caninum and T. gondii tachyzoites differ largely with regard to the functional involvement of proteases in adhesion and invasion of Host Cells. Thus, although phylogenetically closely related, N. caninum and T. gondii are biologically quite different and exhibit distinct dissimilarities with regard to Host Cell interactions.
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identification of a neospora caninum microneme protein ncmic1 which interacts with sulfated Host Cell surface glycosaminoglycans
Infection and Immunity, 2002Co-Authors: Nadine Keller, Nathalie Vonlaufen, Arunasalam Naguleswaran, Angela Cannas, Camilla Bjorkman, M Bienz, Wolfgang Bohne, Andrew HemphillAbstract:The invasive stages of apicomplexan parasites enter their Host Cells through mechanisms which are largely conserved throughout the phylum. Host Cell invasion is divided into two distinct events, namely, adhesion onto the Host Cell surface and the actual Host Cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the Host Cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero Cells, as well as to Vero Cell monolayers. Removal of glycosaminoglycans from the Host Cell surface and modulation of Host Cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the Host Cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.
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neospora caninum microneme protein ncmic3 secretion subCellular localization and functional involvement in Host Cell interaction
Infection and Immunity, 2001Co-Authors: Arunasalam Naguleswaran, Nathalie Vonlaufen, Angela Cannas, Nadine Keller, Gereon Schares, Franz Josef Conraths, Camilla Bjorkman, Andrew HemphillAbstract:In apicomplexan parasites, Host Cell adhesion and subsequent invasion involve the sequential release of molecules originating from secretory organelles named micronemes, rhoptries, and dense granules. Microneme proteins have been shown to be released at the onset of the initial contact between the parasite and the Host Cell and thus mediate and establish the physical interaction between the parasite and the Host Cell surface. This interaction most likely involves adhesive domains found within the polypeptide sequences of most microneme proteins identified to date. NcMIC3 is a microneme-associated protein found in Neospora caninum tachyzoites and bradyzoites, and a large portion of this protein is comprised of a stretch of four consecutive epidermal growth factor (EGF)-like domains. We determined the subCellular localization of NcMIC3 prior to and following Host Cell invasion and found that NcMIC3 was secreted onto the tachyzoite surface immediately following Host Cell lysis in a temperature-dependent manner. Surface-exposed NcMIC3 could be detected up to 2 to 3 h following Host Cell invasion, and at later time points the distribution of the protein was again restricted to the micronemes. In vitro secretion assays using purified tachyzoites showed that following secretion onto the surface, NcMIC3 was largely translocated towards the posterior end of the parasite, employing a mechanism which requires a functional actin microfilament system. Following this, the protein remained bound to the parasite surface, since it could not be detected in a soluble form in respective culture supernatants. Secretion of NcMIC3 onto the surface resulted in an outward exposure of the EGF-like domains and coincided with an increased capacity of N. caninum tachyzoites to adhere to Vero Cell monolayers in vitro, a capacity which could be inhibited by addition of antibodies directed against the EGF-like domains. NcMIC3 is a prominent component of Triton X-100 lysates of tachyzoites, and cosedimentation assays employing prefixed Vero Cells showed that the protein binds to the Vero Cell surface. In addition, the EGF-like domains, expressed as recombinant proteins in Escherichia coli, also interacted with the Vero Cell surface, while binding of NcSRS2 and NcSAG1, the major immunodominant surface antigens, was not as efficient. Our data are indicative of a functional role of NcMIC3 in Host Cell infection.
Jorge E Galan - One of the best experts on this subject based on the ideXlab platform.
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visualization of the type iii secretion mediated salmonella Host Cell interface using cryo electron tomography
eLife, 2018Co-Authors: Donghyun Park, Maria Laratejero, Neal M Waxham, Wenwei Li, Bo Hu, Jorge E GalanAbstract:: Many important gram-negative bacterial pathogens use highly sophisticated type III protein secretion systems (T3SSs) to establish complex Host-pathogen interactions. Bacterial-Host Cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target Cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and Host Cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella-Host Cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the Host Cell membrane, and the establishment of an intimate association between the bacteria and the target Cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.
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visualization of the type iii secretion mediated salmonella Host Cell interface using cryo electron tomography
bioRxiv, 2018Co-Authors: Jun Liu, Donghyun Park, Maria Laratejero, Jorge E Galan, Neal M WaxhamAbstract:Many important gram-negative bacterial pathogens use highly sophisticated type III secretion systems (T3SSs) to establish complex Host-pathogen interactions. Bacterial-Host Cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target Cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and Host Cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella-Host Cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the Host Cell membrane, and the establishment of an intimate association between the bacteria and the target Cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.
Jan Potempa - One of the best experts on this subject based on the ideXlab platform.
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intraCellular staphylococcus aureus employs the cysteine protease staphopain a to induce Host Cell death in epithelial Cells
PLOS Pathogens, 2021Co-Authors: Kathrin Stelzner, Jan Potempa, Aziza Boyny, Tobias Hertlein, Aneta Sroka, Adriana Moldovan, Kerstin Paprotka, David Kessie, Helene MehlingAbstract:Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by Host Cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial Cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce Host Cell death. Here, we identified a staphylococcal cysteine protease to induce Cell death after translocation of intraCellular S. aureus into the Host Cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of Host Cell death and prolonged intraCellular replication of S. aureus in epithelial Cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intraCellular killing of the Host Cell even in the absence of detectable intraCellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic Cells, where intraCellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial Host Cell and thus contributes to tissue destruction and dissemination of infection.
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intraCellular staphylococcus aureus employs the cysteine protease staphopain a to induce Host Cell death in epithelial Cells
bioRxiv, 2020Co-Authors: Kathrin Stelzner, Jan Potempa, Tobias Hertlein, Aneta Sroka, Adriana Moldovan, Kerstin Paprotka, David Kessie, Helene Mehling, Knut OhlsenAbstract:Abstract Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. IntraCellularity is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial Cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce Host Cell death. Here, we identified a staphylococcal cysteine protease to induce Cell death by intraCellular S. aureus after translocation into the Host Cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of Host Cell death and prolonged intraCellular replication of S. aureus in epithelial Cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intraCellular killing of the Host Cell even in the absence of detectable intraCellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. Our study suggests that staphopain A is utilized by S. aureus to mediate escape from the Host Cell and thus contributes to tissue destruction and dissemination of infection. Author Summary Staphylococcus aureus is a well-known antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium asymptomatically colonizes the upper respiratory tract and skin of about one third of the human population and takes advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus is not regarded as a professional intraCellular bacterium, it can be internalized by human Cells and subsequently exit the Host Cells by induction of Cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intraCellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to Host Cell death mediated by intraCellular S. aureus. Staphopain A induced killing of the Host Cell after translocation of the pathogen into the Cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intraCellular role for the bacterial protease staphopain A.
Sylvie Kieffer - One of the best experts on this subject based on the ideXlab platform.
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Host Cell subversion by toxoplasma gra16 an exported dense granule protein that targets the Host Cell nucleus and alters gene expression
Cell Host & Microbe, 2013Co-Authors: Alexandre Bougdour, Eric Durandau, Mariepierre Brenierpinchart, Philippe Ortet, Mohamed Barakat, Sylvie KiefferAbstract:After invading Host Cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the Host Cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the Host Cell nucleus, where it positively modulates genes involved in Cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two Host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the Cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these Host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate Host function.
Arunasalam Naguleswaran - One of the best experts on this subject based on the ideXlab platform.
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neospora caninum protein disulfide isomerase is involved in tachyzoite Host Cell interaction
International Journal for Parasitology, 2005Co-Authors: Arunasalam Naguleswaran, Nathalie Vonlaufen, Ferial Alaeddine, Christophe Guionaud, Sabrina Sonda, Paul Jenoe, Meike Mevissen, Andrew HemphillAbstract:We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces Host Cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-Host Cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite Cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to Host Cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced Host Cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-Host Cell interaction.
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neospora caninum and toxoplasma gondii a novel adhesion invasion assay reveals distinct differences in tachyzoite Host Cell interactions
Experimental Parasitology, 2003Co-Authors: Arunasalam Naguleswaran, Norbert Muller, Andrew HemphillAbstract:This paper describes an adhesion/invasion assay, based on combined pyrrolidine dithiocarbamate (PDTC) and antibody treatment of parasites followed by quantitative real-time PCR. This PDTC-PCR assay can be used to comparatively assess the participation of Host Cell- and parasite-associated components during Host Cell adhesion and entry by Neospora caninum and Toxoplasma gondii tachyzoites, respectively, and is potentially applicable to any other apicomplexan parasite. The assay allows to determine the parasite invasion rate in relation to the overall number of parasites which interact with Host Cells in any given experiment, and thus represents a significant improvement to conventional microscopic assays in terms of accuracy and reproducibility. Using this assay it was possible to show that adhesion and invasion of N. caninum tachyzoites are two distinct and separated events, in that N. caninum tachyzoites preferentially utilise Host Cell surface chondroitin sulphates for adhesion, but not for the Host Cell invasion process. Application of the PDTC-PCR assay also demonstrated that N. caninum and T. gondii tachyzoites differ largely with regard to the functional involvement of proteases in adhesion and invasion of Host Cells. Thus, although phylogenetically closely related, N. caninum and T. gondii are biologically quite different and exhibit distinct dissimilarities with regard to Host Cell interactions.
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identification of a neospora caninum microneme protein ncmic1 which interacts with sulfated Host Cell surface glycosaminoglycans
Infection and Immunity, 2002Co-Authors: Nadine Keller, Nathalie Vonlaufen, Arunasalam Naguleswaran, Angela Cannas, Camilla Bjorkman, M Bienz, Wolfgang Bohne, Andrew HemphillAbstract:The invasive stages of apicomplexan parasites enter their Host Cells through mechanisms which are largely conserved throughout the phylum. Host Cell invasion is divided into two distinct events, namely, adhesion onto the Host Cell surface and the actual Host Cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the Host Cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero Cells, as well as to Vero Cell monolayers. Removal of glycosaminoglycans from the Host Cell surface and modulation of Host Cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the Host Cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.
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neospora caninum microneme protein ncmic3 secretion subCellular localization and functional involvement in Host Cell interaction
Infection and Immunity, 2001Co-Authors: Arunasalam Naguleswaran, Nathalie Vonlaufen, Angela Cannas, Nadine Keller, Gereon Schares, Franz Josef Conraths, Camilla Bjorkman, Andrew HemphillAbstract:In apicomplexan parasites, Host Cell adhesion and subsequent invasion involve the sequential release of molecules originating from secretory organelles named micronemes, rhoptries, and dense granules. Microneme proteins have been shown to be released at the onset of the initial contact between the parasite and the Host Cell and thus mediate and establish the physical interaction between the parasite and the Host Cell surface. This interaction most likely involves adhesive domains found within the polypeptide sequences of most microneme proteins identified to date. NcMIC3 is a microneme-associated protein found in Neospora caninum tachyzoites and bradyzoites, and a large portion of this protein is comprised of a stretch of four consecutive epidermal growth factor (EGF)-like domains. We determined the subCellular localization of NcMIC3 prior to and following Host Cell invasion and found that NcMIC3 was secreted onto the tachyzoite surface immediately following Host Cell lysis in a temperature-dependent manner. Surface-exposed NcMIC3 could be detected up to 2 to 3 h following Host Cell invasion, and at later time points the distribution of the protein was again restricted to the micronemes. In vitro secretion assays using purified tachyzoites showed that following secretion onto the surface, NcMIC3 was largely translocated towards the posterior end of the parasite, employing a mechanism which requires a functional actin microfilament system. Following this, the protein remained bound to the parasite surface, since it could not be detected in a soluble form in respective culture supernatants. Secretion of NcMIC3 onto the surface resulted in an outward exposure of the EGF-like domains and coincided with an increased capacity of N. caninum tachyzoites to adhere to Vero Cell monolayers in vitro, a capacity which could be inhibited by addition of antibodies directed against the EGF-like domains. NcMIC3 is a prominent component of Triton X-100 lysates of tachyzoites, and cosedimentation assays employing prefixed Vero Cells showed that the protein binds to the Vero Cell surface. In addition, the EGF-like domains, expressed as recombinant proteins in Escherichia coli, also interacted with the Vero Cell surface, while binding of NcSRS2 and NcSAG1, the major immunodominant surface antigens, was not as efficient. Our data are indicative of a functional role of NcMIC3 in Host Cell infection.