Mass spectrometric quantification of the adaptations in the wall proteome of Candida albicans in response to ambient pH

Abstract

The mucosal layers colonized by the pathogenic fungus Candida albicans differ widely in ambient pH. Because the properties and functions of wall proteins are probably pH dependent, we hypothesized that C. albicans adapts its wall proteome to the external pH. We developed an in vitro system that mimics colonization of mucosal surfaces by growing biomats at pH 7 and 4 on semi-solid agarose containing mucin as the sole nitrogen source. The biomats expanded radially for at least 8 days at a rate of~30 mm h "1. At pH 7, hyphal growth predominated and growth was invasive, whereas at pH 4 only yeast and pseudohyphal cells were present and growth was noninvasive. Both qualitative mass spectrometric analysis of the wall proteome by tandem mass spectrometry and relative quantification of individual wall proteins (pH 7/pH 4), using Fourier transform mass spectrometry (FT-MS) and a reference mixture of 15 N-labelled yeast and hyphal walls, identified similar sets of .20 covalently linked wall proteins. The adhesion proteins Als1 and Als3, Hyr1, the transglucosidase Phr1, the detoxification enzyme Sod5 and the mammalian transglutaminase substrate Hwp1 (immunological detection) were only present at pH 7, whereas at pH 4 the level of the transglucosidase Phr2 was .35-fold higher than at pH 7. Sixteen out of the 22 proteins identified by FT-MS showed a greater than twofold change. These results demonstrate that ambient pH strongly affects the wall proteome of C. albicans, show that our quantitative approach can give detailed insights into the dynamics of the wall proteome, and point to potential vaccine targets.

Hyphal induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein profile

“…These can originate from highly different growth conditions and sources such as planktonic cultures, biofilms, and infected tissues and organs, irrespective of nitrogen sources. To obtain a wide representation of wall proteins, and since the culturing pH is known to have a strong effect on the mode of growth and the composition of the wall proteome (Sosinska et al, 2011), we used an acidic pH (pH 4) and a pH close to neutral (pH 7.4) for metabolic 15 N-labelling of C. albicans. This allows for systematically collecting query : reference ratios of individual wall proteins belonging to wall proteomes obtained in this and later studies and for a future meta-analysis of the data.…”

“…Ion abundances in the exported array of monoisotopic mass lists were the spectral intensities of the most abundant isotope summed over all charge states for each peptide. The exported .mgf file was imported into the in-house-developed CoolToolBox software program (de Koning et al, 2006;Heijnis et al, 2011;Kasper et al, 2007;Müller et al, 2009;Popolo et al, 2008;Sosinska et al, 2011). From the imported array of up to 1800 monoisotopic mass spectra the CoolToolBox program constructed up to 1000 peptide ion chromatograms.…”

“…Wall proteins show a variety of functions ranging from adhesion (Hoyer et al, 1998;Staab et al, 1999) and iron acquisition (Almeida et al, 2008) to tissue invasion and defence against the immune response (Frohner et al, 2009). Recently, the response of the wall proteome to changes in ambient pH has been described (Sosinska et al, 2011). The adhesins Als1 and Als3 as well as Hwp1 and Hyr1 have been used to vaccinate mice against C. albicans infections, resulting in reduced fungal burden and strongly improved survival (Luo et al, 2010;Spellberg et al, 2006).…”

Iron restriction-induced adaptations in the wall proteome of Candida albicans

“…Following injection, a linear gradient (from 0.1 % formic acid/100 % H 2 O to 0.1 % formic acid/40 % CH 3 CN/60 % H 2 O) was applied over a period of 120 min at a flow rate of 2 ml min 21 . During elution a chromatogram of up to 1850 high-resolution ESI-FT-MS spectra was recorded using an MS duty cycle of about 3 s. Processing the data and identification and quantification of the proteins has been described previously (Sosinska et al, 2011) and is based on accurate tryptic peptide mass tags and LC retention tags. The resulting protein 14 N/ 15 N isotopic ratios are listed in Table S1 (available in Microbiology Online) under YPS R1 and R2, BPS-16 R1 and R2 and BPS-26 R1.…”

“…Weissman & Kornitzer (2004) identified Rbt5, Csa1, and Pga10 as haem/haemoglobin receptors in C. albicans. These proteins are GPI-proteins and have been previously shown to be membrane-and cell-wall-localized (Heilmann et al, 2011(Heilmann et al, , 2013Sosinska et al, 2008Sosinska et al, , 2011Weissman & Kornitzer, 2004;Weissman et al, 2008). Together with the GPI-modified wall protein Pga7 and the secreted protein Csa2 they belong to the CFEM family (common in fungal extracellular membranes), which is very commonly found among fungi and is characterized by one or more 8-cysteine-containing CFEM domains (Kulkarni et al, 2003).…”