exceeded 30. However, Northern analysis showed that CaADH2 mRNA was present in the stationary phase cells grown in YPD. This may reflect the greater sensitivity of Northern analysis. Having established that CaADH1 and CaADH2 mRNAs are expressed in C. albicans cells grown under a variety of conditions, both genes were cloned and expressed in S. cerevisiae (Fig. 2). Assays of enzyme activity in affinity-purified extracts from Adh1p- or Adh2p-expressing cells demonstrated only the CaAdh1p-expressing strain exhibited greater activity than the empty cassette vector pABC3-his control (Fig. 3). Although the presence of CaAdh2p in the purified cell extract from the AD/CaADH2-his strain was confirmed by Western blot using an anti-His antibody (Fig. 2), extracts from this strain showed enzyme activities similar to those in extracts from the control empty vector strain (Fig. 3). This would suggest that CaAdh2p is inactive under the conditions tested, and that the activity observed for the AD/CaADH2-his strain represents the background Adh activities of the host AD strain. In order to confirm that the ethanol-utilising activity in extracts from the AD/CaADH1-his recombinant strain was mediated by the cloned CaAdh1p rather than contaminating ScAdh2p, CaAdh1p was expressed in a strain in which ScADH2 was disrupted. ScADH2 was successfully disrupted in the parental AD strain. In this recombinant strain (AD/ScADH2) no significant NAD reduction was detected in extracts when ethanol was given as the substrate (Fig. 4) confirming that ScAdh2p catalyses the ethanol utilising activity in S. cerevisiae (Ciriacy 1975a). When ScADH2 was disrupted in a recombinant strain expressing CaAdh1p, ethanol utilising activity was present (Fig. 4) confirming that CaAdh1p catalyses the utilisation of ethanol which, under the in vitro conditions tested, would result in production of acetaldehyde.
We have previously shown that acetaldehyde is produced in vitro by C. albicans (Collings et al., 1991) and more recent studies have suggested that oral colonisation with C. albicans and other yeast can contribute to the presence of acetaldehyde in saliva (Nieminen et al., 2009, et al., 2013, Marttila et al., 2013a, Marttila et al., 2013b). Intriguingly, Marttila et al (2013a) reported that under low oxygen tension in vitro there was no correlation between acealdehyde concentration and ADH1 RNA expression, but rather with expression of ADH6 RNA (encoding an acetaldehyde utilising enzyme). However, as noted by these authors, Adh1p levels do not correlate closely with ADH1 mRNA levels (Bertram et al., 1996), possibly due to or post-transcriptional control. Indeed, Adh1p is upregulated in biofilm formation (Martinez-Gomariz et al., 2009) and oral biofilms can contain C. albicans (Zijnge et al., 2010). We conclude that of two members of the ADH gene family (ADH1 and ADH2) were expressed under growth conditions replicating both replete and minimal nutritional conditions. Furthermore expression of C. albicans Adh1p, but not Adh2p, conferred ethanol utilisation activity when heterologously expressed in S. cerevisiae. Although regulation of other enzymes involved in acetaldehyde metabolism, such as Adh6p, may be involved in the accumulation of acetaldehyde in cultures or in vivo, we suggest that the high expression of Adh1p by C. albicans, and its ethanol utilising activity, can contribute to the generation of the carcinogen acetaldehyde in the saliva of colonised individuals. ACKNOWLEDGEMENTS MM Bakri was supported by a postgraduate scholarship from the University of Malaya and this work was also supported by the New Zealand Dental Association Research Foundation. We are grateful for the free availability of the Candida and Saccharomyces genome databases (CGD; http://www.candidagenome.org/ and SGD; http://www.yeastgenome.org/).
Figure 3. Adh enzyme activities of Ni-NTA-purified cell-free extracts of recombinant S. cerevisiae strains AD/CaADH1-his, AD/CaADH2-his and the control strain AD/pABC3-his. Enzyme activities were determined with ethanol as a substrate and the assays were carried out at pH 8.5 with 2 mM NAD and 0.3 mM ethanol. All assays were carried out in triplicate. *P < 0.01.
Figure 4. Adh enzyme activities of cell-free extracts of S. cerevisiae strains AD∆/CaADH1(pABC5)/∆ScADH2 and the controls AD∆ and AD∆/∆ScADH2. Enzyme activities were determined with ethanol as a substrate and the assays were carried out at pH 8.5 with 2 mM NAD and 0.3 mM ethanol. All assays were carried out in triplicate. *P < 0.001.