Propeptide genesis by Kex2-dependent cleavage of yeast wall protein 1 (Ywp1) of Candida albicans

Candida albicans is a prevalent fungal resident and opportunistic pathogen of humans, exhibiting a variety of ovoid and filamentous morphologies. Anchored within the cell wall of the ovoid yeast form of C. albicans is an abundant glycoprotein termed yeast wall protein 1 (Ywp1). Ywp1 has an antiadhesive effect that may facilitate yeast cell dispersal; it also contributes to the masking of the glucan matrix of the yeast cell wall, potentially providing shielding from recognition by the human immune system. Mature Ywp1 consists of an O-glycosylated core of 378 amino acids associated with an N-glycosylated propeptide that originates from an N-terminal segment of Ywp1. A tribasic (-RRR-) sequence in the immature Ywp1 polypeptide is separated by 8 amino acids from a dibasic (-KR-) sequence that is a canonical site for cleavage by the intracellular endopeptidase Kex2, and cleavage occurs at both of these sites to generate an 11 kilodalton (kDa) propeptide that remains strongly associated with the mature core of Ywp1. Previous studies demonstrated an absence of the 11 kDa propeptide in strains lacking Kex2, but the presence of lesser amounts of a 12 kDa propeptide ostensibly (and paradoxically) arising from cleavage at the dibasic site. Subsequent studies of wild type strains, however, suggested that post-secretion cleavages were carried out in vitro by acid proteases in unbuffered cultures to generate the 12 kDa propeptide. Here, intact and Gfp-tagged Ywp1 are utilized to show that neither of the two multibasic sites is normally cleaved in the absence of Kex2, but that uncleaved Ywp1 is still N-glycosylated and subsequently anchored to the cell wall. This furthers our understanding of the multistep cleavage of this highly conserved sequence, as well as the possible contributions of the cleaved propeptide to the maturation and functioning of Ywp1.

Microbe cultures are often routinely grown in batches, in aerated, orbitally-shaking flasks. Initially, oxygen may be the limiting nutrient, as better aeration often leads to faster growth. When allowed to proceed to stationary phase, growth conceivably ceases because of a buildup of toxic metabolites or because an essential nutrient is used up and no longer available for continued growth. In Candida albicans, YWP1 expression in such batch cultures is induced when phosphate becomes the limiting nutrient; the starting concentration of phosphate can thus determine the final cell mass and the time at which phosphate limitation is sensed and responded to. Yeast cultures are sometimes grown or maintained in a rich, partly-defined medium such as YPD (1% yeast extract, 2% peptone, 2% dextrose) or GYEP (0.3% yeast extract, 1% peptone, 2% dextrose). Early investigations [1] indicated that phosphate might become limiting during growth of C. albicans in GYEP batch cultures, but not YPD batch cultures, as GFP reporting the expression of YWP1 or PHO100 (an inducible acid phosphatase) was sometimes found to have been induced when stationary phase cells were examined.
Difco reports the phosphate composition of Bacto yeast extract and peptone as 3.270% and 0.445%, respectively, resulting in a final concentration of 1.50 mM in GYEP and 4.38 mM in YPD (3.44 mM from yeast extract and 0.94 mM from peptone). Since at least 2 mM phosphate is needed in Medium 13 to avoid phosphate limitation, GYEP may indeed be phosphate limited.
To determine what might be ultimately limiting the growth of C. albicans in YPD cultures, components of Medium 13 [1] were individually added. Only magnesium ion noticeably increased the final cell mass. Supplementing the starting culture with 1 mM magnesium ion was found to increase the final optical density by 20-40%. Adding magnesium to an unsupplemented YPD culture at stationary phase allowed additional growth to occur, suggesting that growth had not ceased because of a buildup of any growth inhibitor.
Similar results had previously been found for Saccharomyces cerevisiae: When grown in YEPD (0.5% yeast extract, 1% peptone, 20% dextrose) batch cultures, magnesium ion was found to be the limiting nutrient [2]. Difco reports the Mg composition of Bacto yeast extract and peptone as 0.075% and 0.007%, respectively, resulting in a final concentration of 0.18 mM in YEPD and 0.37 mM in YPD (0.31 mM from yeast extract and 0.06 mM from peptone). A direct analysis of YPD has indicated a magnesium concentration of 0.49 mM [3]. Thus, relative to the other components of the medium, this quantity of magnesium is evidently insufficient for maximal growth.
Upon supplementation of YPD with magnesium, a different essential nutrient may become limiting in batch cultures. In line with the GYEP observations mentioned above, this nutrient appears to be phosphate, as YWP1 expression appears to be induced under these conditions. Thus, when studying cellular responses in batch culture, one should be aware of confounding starvation responses that may arise as a function of the initial composition of the medium, regardless of how rich it is.

Additional note of interest
Candida albicans can utilize ethanol as a carbon source, even as its sole carbon source. A preliminary experiment has indicated that ethanol can even be assimilated from the vapor phase by broth cultures. Specifically, cells were inoculated into a Petri plate containing carbon-free medium (Medium 13 without glucose) and placed in a chamber surrounded by a reservoir of water containing 1% ethanol (172 mM); the cells grew slowly, but ultimately attained an optical density comparable to that of complete Medium 13. The ethanol presumably steadily partitioned from the surrounding solution into the cell culture medium to support cell growth. There was little growth of control cultures surrounded by water without the ethanol. A separate set of experiments suggested significant mortality if the surrounding water contained 10% ethanol. Exemplifying what is already well known, volatile nutrients and catabolites can be difficult to account for, but can have significant effects on cells.