*For correspondence. E-mail: yyang@mail.nctu.edu.tw; Tel.: +886-3-571- 2121; Fax: +886-3-572-9288 Hui-Ching Ko 1 , Ting-Yin Hsiao 2 , Chiung-Tong Chen 3 , and Yun-Liang Yang 1,2 * 1 Department of Biological Science and Technology, 2 Institute for Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, 300, Taiwan 3 Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Miaoli, 350, Taiwan (Received October 22, 2012 / Accepted February 16, 2013) Journal of Microbiology (2013) Vol. 51, No. 3, pp. 345–351 Copyright 2013, The Microbiological Society of Korea DOI 10.1007/s12275-013-2577-z Candida albicans ENO1 Null Mutants Exhibit Altered Drug Susceptibility, Hyphal Formation, and Virulence We previously showed that the expression of ENO1 (enolase) in the fungal pathogen Candida albicans is critical for cell growth. In this study, we investigate the contribution of the ENO1 gene to virulence. We conducted our functional study of ENO1 in C. albicans by constructing an eno1/eno1 null mutant strain in which both ENO1 alleles in the genome were knockouted with the SAT1 flipper cassette that con- tains the nourseothricin-resistance marker. Although the null mutant failed to grow on synthetic media containing glucose, it was capable of growth on media containing yeast extract, peptone, and non-fermentable carbon sources. The null mutant was more susceptible to certain antifungal drugs. It also exhibited defective hyphal formation, and was avirulent in BALB/c mice. Keywords: enolase, opportunistic human pathogen, drug susceptibility, hyphal formation, virulence Introduction Enolase catalyzes the conversion of 2-phospho-D-glycerate to phosphoenolpyruvate, and is critical for both glycolysis and gluconeogenesis. The enzymatic functions of enolase are highly conserved across virtually all taxa, including bac- teria, plants, animals, and humans (Van der Straeten et al., 1991). Enolase has alternative cellular functions. Human enolase acts as a plasminogen receptor in inflammation (Plow et al., 1986; Plow and Das, 2009), and is closely asso- ciated with several disease processes, including various can- cers (Nesland et al., 1988; Sung and Cho, 2008). It is also a component of the avian eye lens (Rudner et al., 1990; Kim and Wistow, 1993), and is a transcription repressor of the proto-oncogene c-myc (Feo et al., 2000). Although several ENO1 homologues have been identified in humans, only the non-tandem enolase structural genes ENO1 and ENO2 are contained in the Saccharomyces cer- evisiae genome. Mutations in one, but not both, of the S. cerevisiae enolase genes are viable in the presence of glucose (McAlister and Holland, 1982; Niedenthal et al., 1999), in- dicating redundancy in enolase enzymatic activity. One ex- planation for the observation that mutations in both ENO1 and ENO2 inhibit S. cerevisiae from growing in the presence of glucose is glucose repression (Trumbly, 1992). DNA se- quencing and southern blotting analyses in C. albicans indi- cated that only ENO1 encodes the enolase activity (Sundstrom and Aliaga, 1992), and RNA knock-down of ENO1 expre- ssion reduced the growth rate of C. albicans (De Backer et al., 2001). Enolase comprises approximately 0.7% of the total protein in yeast-form cells and 2% of that in the hyphae (Sundstrom and Aliaga, 1994). Enolase is the major antigen in candidiasis patients (Klingspor et al., 1997), and it binds plasmin to in- duce fibrinolysis. The plasmin-bound fungal cells demons- trate increased cellular invasion (Jong et al., 2003). Immuni- zation with enolase and interleukin-12 (Montagnoli et al., 2004) or β-mannan trisaccharide and enolase (Xin et al., 2008) significantly reduced the kidney fungal burden in a murine model, but increased survival only moderately. In the presence of fluconazole, the expression of enolase in flu- conazole-susceptible strains of C. albicans is substantially reduced (Angiolella et al., 2002), but the amount of enolase secreted into the medium is increased (Angiolella et al., 2002) compared with that in the absence of fluconazole, suggesting that enolase may be involved in mechanisms of drug susce- ptibility. The mechanisms of drug susceptibility may also affect hy- phal formation and virulence (Lo et al., 2005). Therefore, we introduced mutations in ENO1 in C. albicans to investigate the role of enolase in virulence-related processes. We used a tetracycline-regulated (TR) expression system (Baron et al., 1997, 1999; Tremblay et al., 1998) to investigate the function of eno1 mutants in the C. albicans BWP17 strain. We con- structed the TR-ENO1/eno1 strain, in which one copy of the ENO1 gene is replaced by the ARG4 selective nutrition marker and the other is under the control of a TR promoter (Nakayama et al., 2000). We showed that the expression of ENO1 in C. albicans is critical to cell growth in glucose-con- taining media (Yang et al., 2006a). Our TR expression system is, however, suboptimal for the evaluation of the null phenotype because mutant pheno- types may be affected by the use of nutrient markers in gene- replacement experiments (Chibana et al., 2005; Sharkey et al., 2005). And, in the presence of doxycycline, the meta- bolic activity of the C. albicans biofilm was reduced by as much as 85%, and the combined high levels of doxycycline and fluconazole demonstrated synergistic antifungal activity