Characterization of Cell Wall Proteins in Saccharomyces cerevisiae Clinical Isolates Elucidates Hsp150p in Virulence

ABSTRACT We have isolated several Saccharomyces cerevisiaemutants resistant to calcofluor that contain mutations in thePBS2 or HOG1 genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway. We report that blockage of either of the two activation branches of the pathway, namely, SHO1 and SLN1, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance. However, chitin biosynthesis is independent of the HOG pathway. Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected. Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway. Characterization of one of the mutants isolated, pbs2-14, revealed that resistance to calcofluor and HOG-dependent osmoadaptation are two different physiological processes. Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance. Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.

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Saccharomyces cerevisiae HOC1, a Suppressor of pkc1, Encodes a Putative Glycosyltransferase

Genetics ◽

10.1093/genetics/145.3.637 ◽

1997 ◽

Vol 145 (3) ◽

pp. 637-645 ◽

Cited By ~ 12

Author(s):

Aaron M Neiman ◽

Vijay Mhaiskar ◽

Vladimir Manus ◽

Francis Galibert ◽

Neta Dean

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Lysis ◽

Integral Membrane Protein ◽

Protein Glycosylation ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Calcofluor White ◽

Kinase C

The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall.

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MP1 Encodes an Abundant and Highly Antigenic Cell Wall Mannoprotein in the Pathogenic Fungus Penicillium marneffei

Infection and Immunity ◽

10.1128/iai.66.3.966-973.1998 ◽

1998 ◽

Vol 66 (3) ◽

pp. 966-973 ◽

Cited By ~ 57

Author(s):

Liang Cao ◽

Che-man Chan ◽

Cindy Lee ◽

Samson Sai-yin Wong ◽

Kwok-yung Yuen

Keyword(s):

Cell Wall ◽

Signal Sequence ◽

Pathogenic Fungus ◽

Penicillium Marneffei ◽

Cell Wall Proteins ◽

Amino Acid Residues ◽

Immunogold Staining ◽

Glycosylation Sites ◽

Molecular Masses

ABSTRACT We cloned the MP1 gene, which encodes an abundant antigenic cell wall mannoprotein from the dimorphic pathogenic fungusPenicillium marneffei. MP1 is a unique gene without homologs in sequence databases. It codes for a protein, Mp1p, of 462 amino acid residues, with a few sequence features that are present in several cell wall proteins of Saccharomyces cerevisiae andCandida albicans. It contains two putative N glycosylation sites, a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Mp1p antibody was generated with recombinant Mp1p protein purified from Escherichia coli to allow further characterization of Mp1p. Western blot analysis with anti-Mp1p antibody revealed that Mp1p has predominant bands with molecular masses of 58 and 90 kDa and that it belongs to a group of cell wall proteins that can be readily removed from yeast cell surfaces by glucanase digestion. In addition, Mp1p is an abundant yeast glycoprotein and has high affinity for concanavalin A, a characteristic indicative of a mannoprotein. Furthermore, ultrastructural analysis with immunogold staining indicated that Mp1p is present in the cell walls of the yeast, hyphae, and conidia of P. marneffei. Finally, it was observed that infected patients develop a specific antibody response against Mp1p, suggesting that this protein represents a good cell surface target for host humoral immunity.

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Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase.

Journal of Bacteriology ◽

10.1128/jb.175.7.2102-2106.1993 ◽

1993 ◽

Vol 175 (7) ◽

pp. 2102-2106 ◽

Cited By ~ 97

Author(s):

V Mrsa ◽

F Klebl ◽

W Tanner

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Gene Product ◽

Purification And Characterization ◽

A Cell

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Functional Characterization of Clinical Isolates of the Opportunistic Fungal Pathogen Aspergillus nidulans

mSphere ◽

10.1128/msphere.00153-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 6

Author(s):

Rafael Wesley Bastos ◽

Clara Valero ◽

Lilian Pereira Silva ◽

Taylor Schoen ◽

Milton Drott ◽

...

Keyword(s):

Cell Wall ◽

Reference Strain ◽

Carbon Sources ◽

Neutrophil Function ◽

Clinical Isolates ◽

Biological Characterization ◽

Content Type ◽

Virulence Traits ◽

Opportunistic Fungal Pathogen

ABSTRACT Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency, and virulence of A. nidulans isolates has mainly been studied in the context of chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, nonclinical FGSC A4 strain. Both CIs presented increased growth in comparison to that of the reference strain in the presence of physiologically relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall-perturbing agents but not to other physiologically relevant stresses. Genome analyses identified several frameshift variants in genes encoding cell wall integrity (CWI) signaling components. Significant differences in CWI signaling were confirmed by Western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function than the other strains. In summary, this study presents detailed biological characterization of two A. nidulans sensu stricto clinical isolates. Just as in Aspergillus fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity. IMPORTANCE Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus. Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans, are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.

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