Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii☆

Pszenny, Viviana; Angel, Sérgio O.; Duschak, Vilma Gladys; Paulino, Margot; Ledesma, Bibiana; Yabo, Miriam I.; Guarnera, Eduardo; Ruiz, Andrés M.; Bontempi, Esteban J.

doi:10.1016/s0166-6851(00)00202-4

Cited by 46 publications

(35 citation statements)

References 29 publications

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“…In addition to oomycetes, they are mainly found in animal species and in apicomplexans such as T. gondii, a parasite that transits through the mammalian digestive tract (15)(16)(17)(18)(19)). An interesting analogy can be made between plant apoplasts and mammalian digestive tracts.…”

Section: Discussionmentioning

confidence: 99%

“…Some of these parasites secrete inhibitors that target host proteases and may aid in survival and colonization of the host. For instance, the apicomplexan obligate parasite Toxoplasma gondii secretes TgPI-1 and TgPI-2, four-domain serine protease inhibitors of the Kazal family (15)(16)(17)(18)(19), and the intestinal hookworm Ancylostoma ceylanicum secretes an 8-kDa broad spectrum serine protease inhibitor of the Kunitz family (14). In plants, the apoplast (intercellular fluid) forms a protease-rich environment that is colonized by many pathogens, including P. infestans and the fungus Cladosporium fulvum.…”

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confidence: 99%

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A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the Tomato Pathogenesis-related Protease P69B

Tian

Huitema

Cunha

et al. 2004

Journal of Biological Chemistry

292

268

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The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic lifestyle and consequently are thought to have developed alternative mechanisms of pathogenicity. The oomycete Phytophthora infestans causes late blight, a ravaging disease of potato and tomato. Little is known about processes associated with P. infestans pathogenesis, particularly the suppression of host defense responses. We describe and functionally characterize an extracellular protease inhibitor, EPI1, from P. infestans. EPI1 contains two domains with significant similarity to the Kazal family of serine protease inhibitors. Database searches suggested that Kazal-like proteins are mainly restricted to animals and apicomplexan parasites but appear to be widespread and diverse in the oomycetes. Recombinant EPI1 specifically inhibited subtilisin A among major serine proteases and inhibited and interacted with the pathogenesis-related P69B subtilisin-like serine protease of tomato in intercellular fluids. The epi1 and P69B genes were coordinately expressed and up-regulated during infection of tomato by P. infestans. Inhibition of tomato proteases by EPI1 could form a novel type of defense-counterdefense mechanism between plants and microbial pathogens. In addition, this study points to a common virulence strategy between the oomycete plant pathogen P. infestans and several mammalian parasites, such as the apicomplexan Toxoplasma gondii.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the Tomato Pathogenesis-related Protease P69B

Tian

Huitema

Cunha

et al. 2004

Journal of Biological Chemistry

292

268

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“…A reduction in the degradative capacity of lysosomal contents may therefore be important to sustain parasite growth. Of interest, several serpin class protease inhibitors are secreted by the parasite into the PV lumen (64,70). The absence of a marked increase in the levels of enzymes directly involved in amino acid biosynthesis suggests that, like the situation observed for lipids, the changes instituted in other aspects of intermediary metabolism satisfy the needs for parasite growth.…”

Section: Vol 76 2008 Modulation Of the Host Cell Proteome By T Gonmentioning

confidence: 99%

Modulation of the Host Cell Proteome by the Intracellular Apicomplexan ParasiteToxoplasma gondii

et al. 2008

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To investigate how intracellular parasites manipulate their host cell environment at the molecular level, we undertook a quantitative proteomic study of cells following infection with the apicomplexan parasite Toxoplasma gondii. Using conventional two-dimensional electrophoresis, difference gel electrophoresis (DIGE), and mass spectrometry, we identified host proteins that were consistently modulated in expression following infection. We detected modification of protein expression in key metabolic pathways, including glycolysis, lipid and sterol metabolism, mitosis, apoptosis, and structural-protein expression, suggestive of global reprogramming of cell metabolism by the parasite. Many of the differentially expressed proteins had not been previously implicated in the response to the parasite, while others provide important corroborative protein evidence for previously proposed hypotheses of pathogen-cell interactions. Significantly, over one-third of all modulated proteins were mitochondrial, and this was further investigated by DIGE analysis of a mitochondrion-enriched preparation from infected cells. Comparison of our proteomic data with previous transcriptional studies suggested that a complex relationship exits between transcription and protein expression that may be partly explained by posttranslational modifications of proteins and revealed the importance of investigating protein changes when interpreting transcriptional data. To investigate this further, we used phosphatase treatment and DIGE to demonstrate changes in the phosphorylation states of several key proteins following infection. Overall, our findings indicate that the host cell proteome responds in a dramatic way to T. gondii invasion, in terms of both protein expression changes and protein modifications, and reveal a complex and intimate molecular relationship between host and parasite.The obligate intracellular protozoan Toxoplasma gondii has evolved an intimate relationship with its host that extends to the cellular and molecular levels (82, 83). The absolute requirement for invasion of an appropriate host cell in the replication of the parasite suggests that modification of host functions is central to pathogenesis. Intuitively, this subversion of the cell must be a complex process, since host cells are not inherently programmed to provide an environment conducive to pathogens. Host cells have evolved primary lines of defense as countermeasures to pathogen invasion, establishment, and replication. These include elaborate systems, such as the phagolysosomal fusion, reactive oxygen and nitrogen intermediates, the sequestration of nutrients, and even cell suicide (apoptosis), as defenses to limit pathogen growth. The implementation of these systems and the ability of successful pathogens to mitigate their effects are ultimately mediated by changes in both the levels and activities of key proteins. While some of these changes may be transcriptionally regulated and thus potentially revealed by microarray analysis, the true effectors are proteins,...

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“…Besides oomycetes, Ser protease inhibitors of the Kazal family are widely distributed in animals and apicomplexans. They are thought to play important roles in maintenance of normal cellular and physiological processes of animals (Magert et al, 2002;Kreutzmann et al, 2004) and pathogenesis of mammalian parasitic apicomplexans (Pszenny et al, 2000(Pszenny et al, , 2002Morris et al, 2004). As a result of exhaustive biochemical structure function analyses of the third domain of turkey ovomucoid protein conducted by the late Michael Laskowski Jr. and collaborators Lu et al, 1997Lu et al, , 2001, much is known about the relationship between domain sequence and inhibition specificity in Kazal protein-Ser protease interactions.…”

mentioning

confidence: 99%

“…1A). We used ClustalX (Thompson et al, 1997) to generate a multiple alignment of Kazal domains from P. infestans EPI1, EPI10, the signal crayfish Pacifastacus leniusculus Kazal inhibitor PAPI-1 (Johansson et al, 1994), and the apicomplexan Toxoplasma gondii TgPI1 (Pszenny et al, 2000;Fig. 1B).…”

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confidence: 99%

A Second Kazal-Like Protease Inhibitor from Phytophthora infestans Inhibits and Interacts with the Apoplastic Pathogenesis-Related Protease P69B of Tomato

2005

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The plant apoplast forms a protease-rich environment in which proteases are integral components of the plant defense response. Plant pathogenic oomycetes, such as the potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) pathogen Phytophthora infestans, secrete a diverse family of serine protease inhibitors of the Kazal family. Among these, the two-domain EPI1 protein was shown to inhibit and interact with the pathogenesis-related protein P69B subtilase of tomato and was implicated in counter-defense. Here, we describe and functionally characterize a second extracellular protease inhibitor, EPI10, from P. infestans. EPI10 contains three Kazal-like domains, one of which was predicted to be an efficient inhibitor of subtilisin A by an additivity-based sequence to reactivity algorithm (Laskowski algorithm). The epi10 gene was up-regulated during infection of tomato, suggesting a potential role during pathogenesis. Recombinant EPI10 specifically inhibited subtilisin A among the major serine proteases, and inhibited and interacted with P69B subtilase of tomato. The finding that P. infestans evolved two distinct and structurally divergent protease inhibitors to target the same plant protease suggests that inhibition of P69B could be an important infection mechanism for this pathogen.The plant apoplast forms a protease-rich environment in which proteases are integral components of the plant defense response (Tornero et al., 1997;Jorda et al., 1999;van Loon and van Strien, 1999;Kruger et al., 2002;Xia et al., 2004). For example, the subtilisin-like Ser protease P69B, an apoplastic pathogenesis-related (PR) protein of tomato (Lycopersicon esculentum), has long been tied to plant defense (Tornero et al., 1997;Zhao et al., 2003;Tian et al., 2004). P69B is induced by multiple plant pathogens, including the oomycete Phytophthora infestans, citrus exocortis viroid, and the bacterium Pseudomonas syringae (Tornero et al., 1997;Zhao et al., 2003;Tian et al., 2004), and appears to account for a significant portion of the increase in total endoprotease activity observed in defense-activated tomato (Tornero et al., 1997;Zhao et al., 2003;Tian et al., 2004). Rcr3, an apoplastic papain-like Cys protease from tomato, is required for specific resistance to the plant pathogenic fungus Cladosporium fulvum (Kruger et al., 2002). In Arabidopsis (Arabidopsis thaliana), the extracellular aspartic protease CDR1 functions in disease resistance signaling as a positive regulator of cell death (Xia et al., 2004). These findings suggest that suppression of plant protease-mediated host defenses could be one of the diverse strategies that plant pathogens have evolved to survive in the plant intercellular space and colonize plant tissue. Indeed, our laboratory recently reported that plant pathogenic oomycetes secrete a diverse family of Kazal-like Ser protease inhibitors with at least 35 members identified from P. infestans, Phytophthora sojae, Phytophthora ramorum, Phytophthora brassicae, and the downy mildew Plasmopara halstedii (Tian et ...

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Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii☆

Cited by 46 publications

References 29 publications

A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the Tomato Pathogenesis-related Protease P69B

A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the Tomato Pathogenesis-related Protease P69B

Modulation of the Host Cell Proteome by the Intracellular Apicomplexan ParasiteToxoplasma gondii

A Second Kazal-Like Protease Inhibitor from Phytophthora infestans Inhibits and Interacts with the Apoplastic Pathogenesis-Related Protease P69B of Tomato

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