Inducible chitinolytic system of Aspergillus fumigatus

Escott, Gemma M.; Hearn, Veronica M.; Adams, David J.

doi:10.1099/00221287-144-6-1575

Cited by 42 publications

(24 citation statements)

References 29 publications

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“…Unfortunately, all attempts to inhibit the exponential growth of either yeasts or filamentous fungi (including P. chrysogenum) by allosamidin have failed (Dickinson et al, 1989;Escott et al, 1998;Gooday et al, 1992;Pócsi et al, 2000;Sándor et al, 1998). Although chitinases are always found in growing fungal mycelia together with chitin synthase (Gooday et al, 1986;Hearn et al, 1997;Rast et al, 1991) it is possible that they are protected from the inhibitory effect of allosamidin (Gooday, 1995;Gooday et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…The pseudotrisaccharide allosamidin, which is a wellknown potent inhibitor of numerous fungal chitinases (Cabib et al, 1992;Dickinson et al, 1989;Escott et al, 1998;Gooday et al, 1992;Hodge et al, 1996;Sándor et al, 1998), also inhibited the outgrowth of surviving hyphal fragments in aging P. chrysogenum cultures relieved of carbon starvation by the addition of an extra dose of glucose (Pócsi et al, 2000). To the best of our knowledge, this is the first indication of a possible antifungal effect of allosamidin on a filamentous growth form.…”

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confidence: 99%

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Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin.

Sámi

Pusztahelyi

Emri

et al. 2001

J. Gen. Appl. Microbiol.

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IntroductionThe autolysis taking place in submerged carbon-depleted cultures of Penicillium chrysogenum is influenced by numerous intrinsic (vacuolation, age-related hydrolase production, aging) and extrinsic (power input, shearing forces, nutrient and O 2 limitations, penicillin side-chain precursors) factors (Harvey et al., 1998;McIntyre et al., 1999McIntyre et al., , 2000Nielsen and Krabben, 1995;Paul et al., 1994;Paul and Thomas, 1996;Pócsi et al., 2000; Pusztahelyi et al., 1997a, b;White et al., 1999). In some aspects, the autolysis of P. chrysogenum is reminiscent of the apoptosis of higher eucaryotes under these conditions, e.g., it is an en- In carbon-depleted cultures of Penicillium chrysogenum, age-related chitinases were shown to play a crucial role in both autolysis and fragmentation as indicated by in vivo enzyme inhibition experiments using allosamidin. This pseudotrisaccharide even hindered significantly the outgrowth of new hyphal tips from the surviving yeastlike fragments after glucose supplementation. The antifungal effect of allosamidin on autolyzing P. chrysogenum mycelia was fungistatic rather than fungicidal. In growing hyphae, membrane-bound microsomal chitinase zymogen(s) were detected, which may be indicative of some compartmentalization of these hydrolases. Later, during autolysis, no zymogenic chitinase was detected in any enzyme fraction studied, including microsomes. These observations may explain the different sensitivity of growing and autolyzing mycelia to allosamidin. Chitinases taking part in the age-related fragmentation of hyphae and the outgrowth of surviving hyphal fragments seem to be potent targets for future antifungal drug research. Autolysis and aging of

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin.

Sámi

Pusztahelyi

Emri

et al. 2001

J. Gen. Appl. Microbiol.

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“…Considering the high similarity of Pc22g01100 to chitinase ChiB1 from A. fumigatus (Escott et al, 1998), its gene was named PcchiB1.…”

Section: Sequence Analysis Of Gene Pc22g01100mentioning

confidence: 99%

PcchiB1, encoding a class V chitinase, is affected by PcVelA and PcLaeA, and is responsible for cell wall integrity in Penicillium chrysogenum

et al. 2011

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Penicillin production in Penicillium chrysogenum is controlled by PcVelA and PcLaeA, two components of the regulatory velvet-like complex. Comparative microarray analysis with mutants lacking PcVelA or PcLaeA revealed a set of 62 common genes affected by the loss of both components. A downregulated gene in both knockout strains is PcchiB1, potentially encoding a class V chitinase. Under nutrient-depleted conditions, transcript levels of PcchiB1 are strongly upregulated, and the gene product contributes to more than 50 % of extracellular chitinase activity. Functional characterization by generating PcchiB1-disruption strains revealed that PcChiB1 is responsible for cell wall integrity and pellet formation in P. chrysogenum. Further, fluorescence microscopy with a DsRed-labelled chitinase suggests a cell wall association of the protein. An unexpected phenotype occurred when knockout strains were grown on media containing N-acetylglucosamine as the sole C and N source, where, in contrast to the recipient, a penicillin producer strain, the mutants and an ancestral strain show distinct mycelial growth. We discuss the relevance of this class V chitinase for morphology in an industrially important fungus. INTRODUCTIONPenicillium chrysogenum is the main industrial producer of the b-lactam antibiotic penicillin. In this filamentous ascomycete, developmental processes and secondary metabolism are tightly linked and controlled by diverse conserved regulators. Among these are PcVelA and PcLaeA, two global regulators, which have recently been shown to control penicillin biosynthesis as well as hyphal and conidiophore morphogenesis (Hoff et al., 2010a). Homologues of both proteins have been intensively studied in the model fungus Aspergillus nidulans (Calvo, 2008;Bok & Keller, 2004;Sarikaya Bayram et al., 2010). For example, it has been shown that both proteins, together with VelB, form the core of a regulatory heterotrimeric complex, which we refer to as the velvet complex (Bayram et al., 2008a). The general regulatory function of these proteins is highly conserved in diverse filamentous fungi (Calvo, 2008), and it has been shown that velvet-like complexes exist in other ascomycetes (Hoff et al., 2010a;Wiemann et al., 2010).Despite its importance for development and secondary metabolism, so far only a few high-throughput transcriptional analyses have been conducted with strains lacking components of the velvet complex. Cary et al. (2007) performed a microarray analysis with a DveA mutant of Aspergillus flavus to analyse the expression levels of 5002 genes. That study identified 136 differentially regulated genes in the disruption strain compared with the control strain, some of these representing known aflatoxin biosynthetic genes. In Aspergillus fumigatus, about 20-40 % of all secondary metabolite biosynthesis genes are negatively affected by the loss of laeA, as detected by whole-genome microarray analysis (Perrin et al., 2007).Using a PcvelA knockout strain, our previous microarray study to understand the expression profi...

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“…To obtain conidia, A. fumigatus was grown on Sabouraud's agar plates for 3 d at 37 uC. Spore suspensions were prepared as described by Escott et al (1998). For induction of extracellular chitinase by chitin, Vogel's N medium (Vogel, 1964) containing sucrose (2 %, w/v) (50 ml) was inoculated with A. fumigatus conidia (1610 6 spores ml 21 ) and incubated at 37 uC at 200 r.p.m.…”

Section: Methodsmentioning

confidence: 99%

“…Chitinase activity was detected in polyacrylamide gels containing chitin as described by Escott et al (1998).…”

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confidence: 99%

Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product

et al. 2003

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The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the b-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc 4 , GlcNAc 5 and GlcNAc 6 indicated that the predominant reaction involved hydrolysis of GlcNAc 2 from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

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Inducible chitinolytic system of Aspergillus fumigatus

Cited by 42 publications

References 29 publications

Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin.

Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin.

PcchiB1, encoding a class V chitinase, is affected by PcVelA and PcLaeA, and is responsible for cell wall integrity in Penicillium chrysogenum

Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product

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