Comprehensive Analysis of Proteins Secreted by Trichophyton rubrum and Trichophyton violaceum under in Vitro Conditions

Giddey, Karin; Monod, Michel; Barblan, Jachen; Potts, Alexandra; Waridel, Patrice; Zaugg, Christophe; Quadroni, Manfredo

doi:10.1021/pr070153m

Cited by 52 publications

(40 citation statements)

References 43 publications

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“…For instance, in other fungi, such as Aspergillus spp. and dermatophytes, multiple proteases are concomitantly secreted in culture supernatant and are not degraded (13,33). In addition, the acquisition of the correct three-dimensional structure may protect secreted proteins from proteolysis.…”

Section: Discussionmentioning

confidence: 99%

Secretion of an Endogenous Subtilisin by Pichia pastoris Strains GS115 and KM71

Salamin

Sriranganadane

Léchenne

et al. 2010

Appl Environ Microbiol

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The methylotrophic yeast Pichia pastoris is widely used for the expression of heterologous enzymes. While the purity of the desired expression product is of major importance for many applications, we found that recombinant enzymes produced in methanol medium were contaminated by a 37-kDa endogenous yeast protease. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) but not by 1,10-phenanthroline, EDTA, and pepstatin A, suggesting the nature of a serine protease. Its secretion was abolished in P. pastoris strains GS115 and KM71 by specific mutagenesis of a subtilisin gene (SUB2) but not by inactivation of the gene encoding vacuolar proteinase B (PRB). Bioinformatic comparisons of Sub2 protein with subtilisins from other fungal genomes and phylogenetic analyses indicated that this enzyme is not an orthologue of the vacuolar protease cerevisin generally present in yeasts but is more closely related to another putative subtilisin found in a small number of yeast genomes. During growth of P. pastoris, Sub2 was produced as a secreted enzyme at a concentration of 10 g/ml of culture supernatant after overexpression of the full-length SUB2 gene. During fermentative production of recombinant enzymes in methanol medium, 1 ml of P. pastoris culture supernatant was found to contain approximately 3 ng of Sub2, while the enzyme was not detected during growth in a medium containing glycerol as a carbon source. The mutant strain GS115-sub2 was subsequently used as a host for the production of recombinant proteases without endogenous subtilisin contamination.The methylotrophic yeast Pichia pastoris has been used successfully to express a wide range of heterologous proteins. Pichia pastoris has two genes that encode alcohol oxidase, AOX1 and AOX2. The transcription of the former gene is tightly regulated by methanol, while the latter is expressed in small quantities (7, 11). Alcohol oxidase is not found in the cell when P. pastoris grows in the presence of glycerol, glucose, or ethanol, but in the presence of methanol, alcohol oxidase enzyme 1 (Aox1) amounts to 5% of total cellular proteins in shake-flask cultures and over 30% of total cellular proteins in fermenter cultures (6). The procedure for producing a secreted recombinant protein by using P. pastoris consists of cloning the cDNA encoding the protein of interest downstream of a signal sequence under the control of the AOX1 promoter in a P. pastoris expression vector. In general, the P. pastoris acid phosphatase gene (PHO1) signal sequence or the ␣-factor signal peptide sequence is used for entry of the secretory pathway of the yeast (19). The construct, which carries in addition to the cloned coding sequence of interest a gene for selection after transformation of P. pastoris, is inserted into the P. pastoris genome at the AOX1 locus via homologous recombination. Selected transformants are screened for recombinant protein production after induction in a medium containing methanol.A fundamental objective of our research on fungi (Aspergillus spp. an...

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Section: Discussionmentioning

confidence: 99%

Secretion of an Endogenous Subtilisin by Pichia pastoris Strains GS115 and KM71

Salamin

Sriranganadane

Léchenne

et al. 2010

Appl Environ Microbiol

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“…By two-dimensional polyacrylamide gel electrophoresis and a shotgun mass spectrometry approach, secreted proteins from T. rubrum and T. violaceum were identified in soy culture supernatants, including endo-and exoproteases and other hydrolases (8). Such analyses of proteolytic dermatophyte supernatants, however, are limited to the identification of secreted proteins.…”

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confidence: 99%

Gene Expression Profiling in the Human Pathogenic Dermatophyte Trichophyton rubrum during Growth on Proteins

et al. 2009

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Dermatophytes are highly specialized filamentous fungi which cause the majority of superficial mycoses in humans and animals. The high secreted proteolytic activity of these microorganisms during growth on proteins is assumed to be linked to their particular ability to exclusively infect keratinized host structures such as the skin stratum corneum, hair, and nails. Individual secreted dermatophyte proteases were recently described and linked with the in vitro digestion of keratin. However, the overall adaptation and transcriptional response of dermatophytes during protein degradation are largely unknown. To address this question, we constructed a cDNA microarray for the human pathogenic dermatophyte Trichophyton rubrum that was based on transcripts of the fungus grown on proteins. Profiles of gene expression during the growth of T. rubrum on soy and keratin protein displayed the activation of a large set of genes that encode secreted endo-and exoproteases. In addition, other specifically induced factors potentially implicated in protein utilization were identified, including heat shock proteins, transporters, metabolic enzymes, transcription factors, and hypothetical proteins with unknown functions. Of particular interest is the strong upregulation of key enzymes of the glyoxylate cycle in T. rubrum during growth on soy and keratin, namely, isocitrate lyase and malate synthase. This broad-scale transcriptional analysis of dermatophytes during growth on proteins reveals new putative pathogenicityrelated host adaptation mechanisms of these human pathogenic fungi.Dermatophytes are highly specialized fungi which are the most common agents of superficial mycoses in humans and animals (13, 34). Among the human pathogenic dermatophytes, the anthropophilic species Trichophyton rubrum is clinically the most commonly observed. In contrast to most other medically important fungi, dermatophytes are not opportunists but are obligate pathogens, infecting exclusively the skin stratum corneum, nails, or hair. The ability of dermatophytes to degrade and utilize compact hard keratin within such host structures is presumably related to their common secreted keratinolytic activity, which is therefore a major putative virulence attribute of these fungi. Individual endo-and exoproteases secreted by dermatophytes are similar to those of species of the genus Aspergillus. However, in contrast to those of Aspergillus spp., dermatophyte-secreted endoproteases are multiple and members of two large protein families, the subtilisins (Subs) S8 (serine proteases) and the fungalysins M36 (metalloproteases) (see the MEROPS peptidase database, http://merops.sanger.ac.uk) (10, 11). More than 20 genes that encode secreted proteases have been identified in dermatophytes, some of which have been characterized in detail on the molecular level (reviewed in reference 23).In order to better understand the basic mechanisms of protein degradation by dermatophytes, the secretome of different dermatophyte species was previously analyzed during in vitro growt...

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“…Although we cannot overlook the facts that alkaline proteins are more difficult to process than acidic ones and that small proteins are easily lost, we can still hypothesize that secreted proteins are preferentially acidic and of high MW. Indeed, published secretomic results on various fungal species have confirmed that most protein spots resolve within these acidic ranges [15-19, 21, 22], with few exceptions reporting secreted proteins that were well resolved across the entire 3-10 pH range [14,23]. Instead of reducing the volume of biological material as previously achieved though UF, we increased the volume capacity of the centrifuge tubes.…”

Section: Optimizing the Extraction Of Highly Diluted Secreted Proteinmentioning

confidence: 85%

“…Yet, several studies on fungal secretomics used TCA/acetone method to precipitate proteins from liquid media [14,18,19,23], with one study reporting the use of TCA in water followed by multiple ethanol washes [15]. Here, protein content could not be assayed; however, 1-and 2-D profiles were improved and showed intense bands corresponding to 266 spots (Fig.…”

Section: Optimizing the Extraction Of Highly Diluted Secreted Proteinmentioning

confidence: 99%

Hunting down fungal secretomes using liquid‐phase IEF prior to high resolution 2‐DE

et al. 2009

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The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, beta-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.

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Comprehensive Analysis of Proteins Secreted by Trichophyton rubrum and Trichophyton violaceum under in Vitro Conditions

Cited by 52 publications

References 43 publications

Secretion of an Endogenous Subtilisin by Pichia pastoris Strains GS115 and KM71

Secretion of an Endogenous Subtilisin by Pichia pastoris Strains GS115 and KM71

Gene Expression Profiling in the Human Pathogenic Dermatophyte Trichophyton rubrum during Growth on Proteins

Hunting down fungal secretomes using liquid‐phase IEF prior to high resolution 2‐DE

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