Proteomic analysis of Candida albicans yeast and hyphal cell wall and associated proteins

Abstract

Candida albicans is an important human pathogen that causes systemic infections, predominantly among populations with weakened immune systems. The morphological transition from the yeast to the hyphal state is one of the key factors in C. albicans pathogenesis. Owing to their location at the host-pathogen interface, the cell wall and associated proteins are of interest, especially with respect to the yeast to hyphal transition. This study entailed the proteomic analysis of differentially regulated proteins involved in this transition. The protein profiles of C. albicans DTT/SDS-extractible proteins and the cyanogen bromide (CNBr)/trypsin-extractable proteins of a cell wall-enriched fraction from yeast and hyphae were compared. In total, 107 spots were identified from the DTT/SDS-extractible cell wall-enriched fraction, corresponding to 82 unique proteins. Of these DTT/SDS-extractible proteins, 14 proteins were upregulated and 10 were downregulated in response to hyphal induction. Approximately 6-9% of total cell wall-protein-enriched fraction was found to be resistant to DTT/SDS extraction. Analysis of the DTT/SDS-resistant fraction using a CNBr/trypsin extraction resulted in the identification of 29 proteins. Of these, 17 were identified only in the hyphae, four were identified only in the yeast, and eight were identified in both the yeast and hyphae.

Candida albicansCell Wall Proteins

“…In biofilms, ALS1 was also a major differentially expressed gene compared to what was seen for planktonic gene expression by microarray analysis (119) and real-time RT-PCR (280). If protein abundance parallels gene expression, then some of the Als proteins may be present at very low levels and perhaps not detected, as in the studies by de Groot et al (73), who detected only Als1p and Als4p, and Ebanks et al (87), who detected either Als3p or Als6p (Table 4). The ALS genes as well as EAP1 are genes of the broader class of flocculation genes found in yeasts (see the review in reference 395).…”

“…The differences in procedures for and analysis of the proteins of the fractions and multiple protein species (discussed below) and the same protein in more than one fraction make it difficult to assess the number of unique proteins from the cell wall (Table 4). The most striking difference from Table 3 is in the fraction that is released by glucanase or mild alkali from cell walls previously washed with NaCl and boiled in buffer containing SDS and reducing agent (73,87,303). Generally one protocol employs washes of isolated cell walls with 1 M NaCl; boiling in 2% SDS, 40 mm ␤ME (␤-mercaptoethanol), 10 mM EDTA, Tris (pH 7.8) twice for 10 min; and then washing in water before enzyme treatment (73).…”

“…Several studies have investigated the subproteome of the cell surface (73,87,301,303,380,391; M. Martínez-Gomariz, P. Perumal, S. Mekala, C. Nombela, C. Gil, and W. L. Chaffin, unpublished data). Fractionation of this subproteome employed various extraction procedures from either intact organisms or isolated cell walls.…”

“…Generally one protocol employs washes of isolated cell walls with 1 M NaCl; boiling in 2% SDS, 40 mm ␤ME (␤-mercaptoethanol), 10 mM EDTA, Tris (pH 7.8) twice for 10 min; and then washing in water before enzyme treatment (73). The other procedure uses washing with a step series (0.86 M, 0.34 M, and 0.17 M) of NaCl, boiling using 10 mM DTT and Tris (pH 8) along with the SDS and EDTA, and then washing with sodium acetate (87,303). It seems that either the difference in washes or in the application of the isolation procedure in different laboratories has a profound difference on the proteins found in this fraction.…”

Dihydrolipoamide Dehydrogenase of Pseudomonas aeruginosa Is a Surface-Exposed Immune Evasion Protein That Binds Three Members of the Factor H Family and Plasminogen

“…Lpd was also identified at the surface of the 5 analyzed laboratory strains and on 80% of the clinical P. aeruginosa isolates when analyzed by wholecell ELISA. Similar to P. aeruginosa Lpd, Lpd derived from the pathogenic microbes Trypanosoma brucei and C. albicans is also surface localized (40,41). Lpd is a moonlighting protein that lacks a typical secretion signal (42).…”