Downloaded from www.microbiologyresearch.org by IP: 54.162.190.106 On: Fri, 18 Mar 2016 18:02:19 The Candida albicans pH-regulated KER1 gene encodes a lysine/glutamic-acid-rich plasma-membrane protein that is involved in cell aggregation Amparo Gala ´n, 1 3 Manuel Casanova, 1 3 Amelia Murgui, 2 Donna M. MacCallum, 3 Frank C. Odds, 3 Neil A. R. Gow 3 and Jose ´ P. Martı ´nez 1 Correspondence Jose ´ P. Martı ´nez jose.pedro.martinez@uv.es 1,2 Departamento de Microbiologia y Ecologia 1 and Departamento de Bioquı ´mica y Biologı ´a Molecular 2 , Facultad de Farmacia, Universitat de Vale ` ncia, Spain 3 Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK Received 5 November 2003 Revised 25 May 2004 Accepted 27 May 2004 Immunoscreening of a Candida albicans cDNA library with a polyclonal germ-tube-specific antibody (pAb anti-gt) resulted in the isolation of a gene encoding a lysine/glutamic-acid-rich protein, which was consequently designated KER1. The nucleotide and deduced amino acid sequences of this gene displayed no significant homology with any other known sequence. KER1 encodes a 134 kDa lysine (14?5 %)/glutamic acid (16?7 %) protein (Ker1p) that contains two potential transmembrane segments. KER1 was expressed in a pH-conditional manner, with maximal expression at alkaline pH and lower expression at pH 4?0, and was regulated by RIM101. A Dker1/Dker1 null mutant grew normally but was hyperflocculant under germ-tube-inducing conditions, yet this behaviour was also observed in stationary-phase cells grown under other incubation conditions. Western blotting analysis of different subcellular fractions, using as a probe a monospecific polyclonal antibody raised against a highly antigenic domain of Ker1p (pAb anti-Ker1p), revealed the presence of a 134 kDa band in the purified plasma-membrane fraction from the wild-type strain that was absent in the homologous preparation from Dker1/Dker1 mutant. The pattern of cell-wall protein and mannoprotein species released by digestion with b-glucanases, reactive towards pAbs anti-gt and anti-Ker1p, as well as against concanavalin A, was also different in the Dker1/Dker1 mutant. Mutant strains also displayed an increased cell-surface hydrophobicity and sensitivity to Congo red and Calcofluor white. Overall, these findings indicate that the mutant strain was affected in cell-wall composition and/or structure. The fact that the ker1 mutant had attenuated virulence in systemic mouse infections suggests that this surface protein is also important in host–fungus interactions. INTRODUCTION Many of the genes required for virulence in bacterial pathogens are regulated in response to environmental signals (pH, temperature, osmotic pressure, and iron and calcium ion concentrations) indigenous to the host niche (Brown & Gow, 1999; Mekalanos, 1992). In the dimorphic, opportunistic fungal pathogen Candida albicans, the ability to respond to ambient pH appears to play a critical role in growth and virulence (for a review see Pen ˜alva & Arst, 2002). It is known that external pH and temperature influence, at least in vitro, the yeast-to-mycelium transition of C. albicans, which is one of the virulence traits of this organism (Buffo et al., 1984; Odds, 1988). An acidic pH generally encourages growth in the yeast form, whereas a neutral pH promotes hyphal development. The pH response in C. albicans involves the differential expression of at least three genes, PHR1, PHR2 and PRA1 (Mu ¨hlschlegel & Fonzi, 1997; Saporito-Irwin et al., 1995; Sentandreu et al., 1998). PHR1 is expressed at a pH above 5?5 and is required for normal morphology at these pH levels. The gene product plays a key role in systematic infections. PHR2 is expressed at an acidic pH, is required 3These two authors contributed equally. Abbreviations: CSH, cell-surface hydrophobicity; Con A, concanavalin A. The GenBank accession number for the sequence determined in this work is AF337555. KER1 has the ORF number 6.8869 in the Stanford database and corresponds to IPF 2795 in the Candida database. 0002-6339 G 2004 SGM Printed in Great Britain 2641 Microbiology (2004), 150, 2641–2651 DOI 10.1099/mic.0.26339-0