THE ENZYME COMPLEXES FOR THE DESTRUCTION OF THE CELL WALL OF FILAMENTOUS FUNGI - PRODUCERS OF INDUSTRIAL ENZYMES

Chitin synthesis contributes to cell wall biogenesis and is essential for invasion of solid substrata and pathogenicity of filamentous fungi. In contrast to yeasts, filamentous fungi contain up to 10 chitin synthases (CHS), which might reflect overlapping functions and indicate their complex lifestyle. Previous studies have shown that a class VI CHS of the maize anthracnose fungus Colletotrichum graminicola is essential for cell wall synthesis of conidia and vegetative hyphae. Here, we report on cloning and characterization of three additional CHS genes, CgChsI, CgChsIII, and CgChsV, encoding class I, III, and V CHS, respectively. All CHS genes are expressed during vegetative and pathogenic development. ΔCgChsI and ΔCgChsIII mutants did not differ significantly from the wild-type isolate with respect to hyphal growth and pathogenicity. In contrast, null mutants in the CgChsV gene, which encodes a CHS with an N-terminal myosin-like motor domain, are strongly impaired in vegetative growth and pathogenicity. Even in osmotically stabilized media, vegetative hyphae of ΔCgChsV mutants exhibited large balloon-like swellings, appressorial walls appeared to disintegrate during maturation, and infection cells were nonfunctional. Surprisingly, ΔCgChsV mutants were able to form dome-shaped hyphopodia that exerted force and showed host cell wall penetration rates comparable with the wild type. However, infection hyphae that formed within the plant cells exhibited severe swellings and were not able to proceed with plant colonization efficiently. Consequently, ΔCgChsV mutants did not develop macroscopically visible anthracnose disease symptoms and, thus, were nonpathogenic.

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Cell Wall Biosynthesis in Filamentous Fungi

Stress Biology of Yeasts and Fungi ◽

10.1007/978-4-431-55248-2_10 ◽

2015 ◽

pp. 151-168 ◽

Cited By ~ 1

Author(s):

Takuji Oka ◽

Taiki Futagami ◽

Masatoshi Goto

Keyword(s):

Cell Wall ◽

Filamentous Fungi ◽

Cell Wall Biosynthesis

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Direct and indirect interactions promote complexes of the lipoprotein LbcA, the CtpA protease and its substrates, and other cell wall proteins in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00393-21 ◽

2021 ◽

Author(s):

Dolonchapa Chakraborty ◽

Andrew J. Darwin

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Protein Complexes ◽

Associated Proteins ◽

Carboxyl Terminal ◽

Multiple Species ◽

Processing Protease ◽

Enzyme Complexes

The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function, and for virulence in a mouse model of acute pneumonia. In this study we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA and ctpA null mutations had strikingly similar effects on the proteome, suggesting that assisting CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate, and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these non-substrates did not. Subsequent experiments suggested that the non substrates might co-purify with LbcA by participating in multi-enzyme complexes containing LbcA-binding CtpA substrates. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are widely conserved and associated with the virulence of several bacteria, including CtpA in Pseudomonas aeruginosa . CtpA copurifies with the uncharacterized lipoprotein, LbcA. This study shows that the most impactful role of LbcA might be to promote CtpA-dependent proteolysis, and that it achieves this as a scaffold for CtpA and its substrates. It also reveals that LbcA copurification partners are enriched for cell wall-associated proteins, one of which is a novel CtpA substrate. Some of the LbcA copurification partners are not cleaved by CtpA, but might copurify with LbcA because they participate in multi-enzyme complexes containing CtpA substrates. These findings are important, because CTPs and their associated proteins affect peptidoglycan remodeling and virulence in multiple species.

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Direct and indirect interactions promote complexes of the lipoprotein LbcA, the CtpA protease and its substrates, and other cell wall proteins in Pseudomonas aeruginosa

10.1101/2021.07.29.454410 ◽

2021 ◽

Author(s):

Dolonchapa Chakraborty ◽

Andrew J Darwin

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Type Iii Secretion ◽

Protein Complexes ◽

Peptidoglycan Hydrolases ◽

Carboxyl Terminal ◽

Processing Protease ◽

Enzyme Complexes

The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function, and for virulence in a mouse model of acute pneumonia. In this study we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA and ctpA null mutations had strikingly similar effects on the proteome, suggesting that facilitating CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate, and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these non-substrates did not. Subsequent experiments suggested that the non substrates might co-purify with LbcA by participating in multi-enzyme complexes containing LbcA-binding CtpA substrates.

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