Multiple forms of the serine protease<i> Alp</i> of <i>Aspergillus fumigatus</i>

Kunert, J.; Kopeček, Petr

doi:10.1046/j.1439-0507.2000.00586.x

Cited by 29 publications

(17 citation statements)

References 42 publications

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“…A fumigatus is known to secrete a variety of lytic enzymes including proteases and phospholipases. 27,28 It is possible that these secreted enzymes are most active when they achieve high concentrations in the endocytic vacuole of the endothelial cell. As mentioned previously, it is also likely that the hyphal cell wall itself is toxic to endothelial cells.…”

Section: Discussionmentioning

confidence: 99%

Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity

Lópes-Bezerra

Filler

2004

Blood

114

112

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Invasive aspergillosis causes significant mortality among patients with hematologic malignancies. This infection is characterized by vascular invasion and thrombosis. To study the pathogenesis of invasive aspergillosis, we investigated the interactions of Aspergillus fumigatus conidia and hyphae with endothelial cells in vitro. We found that both forms of the organism induced endothelial cell microfilament rearrangement and subsequent endocytosis. Conidia were endocytosed 2-fold more avidly than hyphae, and endocytosis was independent of fungal viability. Endocytosed conidia and hyphae caused progressive endothelial cell injury after 4 hours of infection. Live conidia induced more endothelial cell injury than did live hyphae. However, endothelial cell injury caused by conidia was dependent on fungal viability, whereas injury caused by hyphae was not, indicating that conidia and hyphae injure endothelial cells by different mechanisms. Neither live nor killed conidia increased tissue factor activity of endothelial cells. In contrast, both live and killed hyphae stimulated significant endothelial cell tissue factor activity, as well as the expression of tissue factor antigen on the endothelial cell surface. These results suggest that angioinvasion and thrombosis caused by A fumigatus hyphae in vivo may be due in part to endothelial cell invasion, induction of injury, and stimulation of tissue factor activity. ( IntroductionInvasive aspergillosis has become one of the leading causes of death among bone marrow transplantation and leukemia patients. Even with current therapy, the mortality rate of invasive aspergillosis is 80% to 90% in immunocompromised patients. 1 Aspergillus fumigatus is responsible for 90% of invasive Aspergillus infections. 1 This ubiquitous mould releases numerous conidia into the atmosphere, which are small enough (2 to 3 m in diameter) to reach the pulmonary alveoli after they are inhaled. 2 Once the conidia reach the alveoli, they swell and germinate, producing hyphae that invade the pulmonary parenchyma. These hyphae have a marked tropism for blood vessels. A key finding in invasive aspergillosis is angioinvasion, which subsequently leads to thrombosis and tissue infarction. [3][4][5] In immunocompromised hosts, invasion of the pulmonary vasculature can result in widespread hematogenous dissemination to organs such the brain, kidneys, heart, and eyes. 1,3 During angioinvasion, A fumigatus hyphae interact with vascular endothelium. We hypothesize that this interaction plays an important role in the pathogenesis of invasive aspergillosis. Once the hyphae have entered the bloodstream, they must adhere to and penetrate the endothelial cell lining of the blood vessels to invade the deep tissues of the target organs.In addition, the prominent thrombosis at sites of A fumigatus angioinvasion suggests that the organism stimulates endothelial cells to become prothrombotic. A major mechanism by which endothelial cells can promote intravascular thrombus formation is by expressing tissue factor, also k...

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Section: Discussionmentioning

confidence: 99%

Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity

Lópes-Bezerra

Filler

2004

Blood

114

112

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“…Other fungi do secrete serine proteinase and/or metalloproteinase activity (Abbas et al, 1989;Ahman et al, 2002;Chou et al, 2002;Jaton-Ogay et al, 1994;Kunert & Kopecek, 2000;Monod et al, 1991Monod et al, , 1993Moutaouakil et al, 1993;van den Hombergh et al, 1994;Zhu et al, 1990). Second, a public B. cinerea EST database contains a number of sequences that presumably encode a non-KEX-like serine proteinase and a carboxypeptidase, respectively.…”

Section: B Cinerea Cultures Exclusively Contain Secreted Ap Activitymentioning

confidence: 99%

An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features

et al. 2004

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Botrytis cinerea, an important fungal plant pathogen, secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity could be detected. Proteinase activity was higher in culture medium containing BSA or wheat germ extract, as compared to minimal medium. A proportion of the enzyme activity remained in the extracellular glucan sheath. AP was also the only type of proteinase activity in fluid obtained from B. cinerea-infected tissue of apple, pepper, tomato and zucchini. Five B. cinerea genes encoding an AP were cloned and denoted Bcap1–5. Features of the encoded proteins are discussed. BcAP1, especially, has novel characteristics. A phylogenetic analysis was performed comprising sequences originating from different kingdoms. BcAP1 and BcAP5 did not cluster in a bootstrap-supported clade. BcAP2 clusters with vacuolar APs. BcAP3 and BcAP4 cluster with secreted APs in a clade that also contains glycosylphosphatidylinositol-anchored proteinases from Saccharomyces cerevisiae and Candida albicans. All five Bcap genes are expressed in liquid cultures. Transcript levels of Bcap1, Bcap2, Bcap3 and Bcap4 are subject to glucose and peptone repression. Transcripts from all five Bcap genes were detected in infected plant tissue, indicating that at least part of the AP activity in planta originates from the pathogen.

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“…To date, a number of secreted proteases have been identiWed in A. fumigatus including a serine protease (ALP1) [7,8], a metalloprotease (MEP) 1 [9] and an aspartic protease (PEP1) [10]. It appears that these enzymes alone may not play a signiWcant role in the invasive characteristics of A. fumigatus following animal studies with single alp1, mep and pep1 mutants and double alp1 and mep mutants [4,11,12].…”

mentioning

confidence: 99%

Isolation, activity and immunological characterisation of a secreted aspartic protease, CtsD, from Aspergillus fumigatus

Vickers

Reeves

Kavanagh

et al. 2007

Protein Expression and Purification

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Aspergillus fumigatus is an opportunistic fungal pathogen that infects immunocompromised patients. A putative aspartic protease gene (ctsD; 1425 bp; intron-free) was identiWed and cloned. CtsD is evolutionarily distinct from all previously identiWed A. fumigatus aspartic proteases. Recombinant CtsD was expressed in inclusion bodies in Escherichia coli (0.2 mg/g cells) and subjected to extensive proteolysis in the baculovirus expression system. Activation studies performed on puriWed, refolded, recombinant CtsD resulted in protease activation with a pH opt 4.0 and speciWc activity D 10 U/mg. Pepstatin A also inhibited recombinant CtsD activity by up to 72% thereby conWrming classiWcation as an aspartic protease. Native CtsD was also immunologically identiWed in culture supernatants and puriWed from fungal cultures using pepstatin-agarose aYnity chromatography (7.8 g CtsD/g mycelia). In A. fumigatus, semi-quantitative RT-PCR analysis revealed expression of ctsD in minimal and proteinaceous media only. Expression of ctsD was absent under nutrient-rich conditions. Expression of ctsD was also detected, in vivo, in the Galleria mellonella virulence model following A. fumigatus infection.

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Multiple forms of the serine protease Alp of Aspergillus fumigatus

Cited by 29 publications

References 42 publications

Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity

Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity

An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features

Isolation, activity and immunological characterisation of a secreted aspartic protease, CtsD, from Aspergillus fumigatus

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