Candida albicans Switches Mates

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Candida albicans Switches Mates The “asexual” human fungal pathogen Candida albicans has recently been engineered to be able to mate. A paper in the August 9, 2002 issue of Cell (Miller and Johnson, 2002) shows that mating competence is increased dramatically when mating partners are in a rare switch variant cell type that does not normally occur at body temperature. Candida albicans is the most important and most common agent of serious fungal infection in humans. This fungus is classified among the large group of so-called imperfect fungi, which have no recognized sexual cycle. To lose the ability to mate and undergo meiosis would seem to be a potentially cataclysmic event for any fungal species striving to maintain the genetic diversity required to cope with environmental pressures such as the vigilant surveillance of the human immune system. Apparently, a second unrelated phenomenon in C. albicans, termed phenotypic switching (Slutsky et al., 1987), evolved to generate high-frequency switch variants that provided a surrogate driver of phenotypic variability that enabled this fungus to compete in the absence of sex. However, not only can C. albicans mate, but mating competence and phenotypic switching are both regulated by a functional mating-type locus (MTL). The recent paper by Miller and Johnson in Cell (2002) demonstrates the surprising dual-regulation of both mating and switching by the C. albicans MTL loci and shows that mating efficiency increases dramatically if strains of opposite mating type are also in a mating-competent switch form. All natural strains of C. albicans are apparently constitutively diploid and therefore recalcitrant to sexual genetics. This suggests that the fungus should be characterized by a clonal population structure, which is for the most part confirmed by recent studies in population genetics (Gra¨ser et al., 1996; Anderson et al., 2001). However, these studies also hint at a background of low frequency recombination. This leaves the door open to the possibility that C. albicans undergoes infrequent meioses in nature or that strains have sexually recombined in recent evolutionary history. Moreover, the C. albicans genome contains homologs of virtually all of the meiosis genes found in the sexually active yeast Saccharomyces cerevisiae (Tzung et al., 2001). The absence of sex imposes severe practical constraints on possible experimental approaches to investigate important aspects of this organism’s unique biology and ability to cause human disease. Identification or reconstitution of sexual recombination in this organism would herald a new era in medical mycology and would influence profoundly our global view of the evolution and ecology of other imperfect fungi. First steps in this direction came from the recognition of a mating-type locus (MTL) in the genome of C. albicans that was structurally similar to that in S. cerevisiae (Hull and Johnson, 1999). This led to the engineering of laboratory strains of the complementary “MTLa” and “MTL␣” mating types (Hull et al., 2000; Magee and

Magee, 2000). These matable strains were created either by targeted deletion of one or the other MTL alleles (Hull et al., 2000) or by inducing loss of one or the other chromatids of the MTL-carrying chromosome 5 (Magee and Magee, 2000). Couplings between all such matable strains occur only at extremely low efficiency and resulted in stable tetraploid progeny. So far, such tetraploids have not been found to complete the sexual cycle and restore the diploid state via meiosis. These studies raised questions about why mating was so inefficient in C. albicans and what else might be regulated by the MTL loci. The apparent absence of meiosis in C. albicans does not mean the fungus is straightjacketed phenotypically. Soll and colleagues had shown that this pathogen has several switching systems that generate at high frequency a variety of heritable colony morphotypes with associated transcriptional signatures. For example, in the well-characterized white/opaque switching system of strain WO-1, white colonies composed of normal ovoid budding yeasts interconverts with a duller opaque colonial form comprised of budding bean-shaped cells (Slutsky et al., 1987). Miller and Johnson (2002) found that the “MTLa” and “MTL␣” strains that had been created by deleting individual MTL alleles in a different strain were able to form opaque sectors in colonies that contained bean-shaped cells with a typical opaque gene expression profile. Notably, the MTLa and MTL␣ strains mated with more than 106 times the efficiency if both partners were in the opaque form rather than the white form. They show that phenotypic switching and mating are both regulated by the Mtla and Mtl␣2 homeodomain proteins that together repress both the switch from the white form to the mating-competent opaque form and the mating reaction. Increasing the efficiency of mating frequency allowed elongated MTLa cells with mating projections to be observed for the first time in the microscope. Importantly, in both the wild-type WO-1 strain, which was shown to be a natural “MTL␣” strain, and in the engineered MTLa and MTL␣ strains, opaque cells are only stable at temperatures well below 37⬚C and they “mass convert” to white cells at body temperature. This implies that mating in natural isolates of C. albicans may only occur efficiently outside the human body—an observation that presents obvious new opportunities in efforts to discover or engineer a sexual cycle in Candida. In nature, the advantages of outbreeding and sexual recombination drive evolution. Self-fertilization (homothallism) in fungi is thought to be a rarer specialization of species in which gene shuffling would either be negatively selected because of the organism’s specialized ecology, for example within a symbiotic partnership (Murtagh et al., 2000), or because potential partners for mating are only encountered rarely. Mixed infection of patients harboring more than one C. albicans strain is well documented (Soll, 2000). Hence, the natural mating potential of the fungus is finite but limited by the requirement for mating competence in the opaque form. It remains to be discovered whether naturally mated progeny recombine sexually. While in the past this seemed a remote possibility, there now seem to be few insurmountable barriers to establish a sexual cycle in this asexual fungus.

Molecular Cell 218

Neil A. R. Gow Department of Molecular and Cell Biology Institute of Medical Science University of Aberdeen Aberdeen AB25 2ZD United Kingdom Selected Reading Anderson, J.B., Wickens, C., Khan, M., Cowen, L.E., Federspiel, N., Jones, T., and Kohn, L.M. (2001). J. Bacteriol. 183, 865–872.

Hull, C.M., and Johnson, A.D. (1999). Science 289, 1271–1275. Hull, C.M., Raisner, R.M., and Johnson, A.D. (2000). Science 289, 301–310. Magee, B.B., and Magee, P.T. (2000). Science 289, 310–313. Miller, M.G., and Johnson, A.D. (2002). Cell 110, 293–302. Murtagh, G.J., Dyer, P.S., and Crittenden, P.D. (2000). Nature 404, 564. Slutsky, B., Staebell, M., Anderson, J., Risen, M., Pfaller, M., and Soll, D.R. (1987). J. Bacteriol. 169, 189–197. Soll, D.R. (2000). Clin. Microbiol. Rev. 13, 332–370.

Gra¨ser, Y., Volovsek, M., Arrington, J., Scho¨nian, G., Presber, W., Mitchell, T.G., and Vilgalys, R. (1996). Proc. Natl. Acad. Sci. USA 93, 12473–12477.

Tzung, K.-W., Williams, R.M., Scherer, S., Federspiel, N., Jones, T., Hansen, N., Bivolarevic, V., Huizar, L., Komp, C., Surzycki, R., et al. (2001). Proc. Natl. Acad. Sci. USA 98, 3249–3253.

All Signaling Is Local?

nar flows containing fluorescently conjugated EGF (see Figure, panel A). This method should reproduce more accurately most physiological situations where cells in tissues are stimulated locally. EGF-induced intracellular signaling is visualized by using a fluorescent indicator that monitors tyrosine phosphorylation and a fluorescent indicator that monitors Ras activation. These experiments show that, at normal levels of EGFR expression, both EGF-induced tyrosine phosphorylation and Ras activation are confined to the region of the cell that is exposed to EGF. However, global propagation of tyrosine phosphorylation and Ras activation does occur, but only in cells overexpressing EGF receptors or when endocytosis of EGF receptor is inhibited (see Figure, panel B). This result contradicts the earlier report from Verveer et al. (2000) in which it was demonstrated that application of EGF to cells results in lateral propogation of EGFR activation in the plasma membrane of the entire cell. In this experiment, local stimulation of EGFR activation is achieved by attaching to the cell membrane beads conjugated with EGF. Activation of EGFR was monitored by imaging the fluorescence energy transfer between GFP (green fluorescent proteins)-tagged EGFR molecules. It is important to note that EGFR was overexpressed in this study. It was concluded that local activation of EGFR leads to signal propagation, resulting in the activation of all EGF receptors in the cell membrane. One possible explanation for the global EGFR activation is that unoccupied EGF receptors are preferential substrates of the ligand-activated receptors, initiating a chain reaction that will eventually result in activation of all EGFR on the cell membrane. An alternative mechanism for lateral propagation of EGFR activation is via EGF-induced production of hydrogen peroxide (Lee et al., 1998), which transiently inhibits the activation of an inhibitory protein tyrosine phosphatase (PTPase) that dephosphorylates the phosphosylated residues in the activation loop of EGFR that are responsible for maintaining the PTK domain in an active state (Schlessinger, 2000). It has been shown that PTPases can be inhibited by oxidation of a key cysteine residue in active site that is essential for PTPase activity (Lee et al., 1998). The transient inhibition of an inhibitory protein tyrosine phosphatase may result in a chain reaction that will culminate in the activation of all EGFRs on the cell membrane. This model does not address the question of why EGFinduced global activation of EGFR does not induce the activation of other PTKs, or, in other words, why EGF-

The mechanism of signal transmission following ligand stimulation of receptor tyrosine kinases in living cells is poorly understood. Recent studies have visualized the spatio-temporal pattern of EGF signaling, indicating that receptor density is an important factor in the mechanism of lateral propagation of local EGF signaling. Transmembrane signaling by growth factors, cytokines, and even some peptide hormones is mediated by ligandinduced receptor oligomerization (Lemmon and Schlessinger 1994; Heldin, 1995). The lateral interactions that take place during growth factor-induced activation of receptor tyrosine kinases (RTK) may lead in principle to lateral propagation of receptor activation over a large distance even when growth factor stimulation is confined to a restricted area on the cell membrane. Such a mechanism is potentially possible for RTKs since this family of receptors is activated by intermolecular (trans) autophosphorylation on key tyrosine residues in the activation loop of the protein tyrosine kinase (PTK) domain. Biochemical and structural studies demonstrated that the PTK domain is maintained in an active state as long as the tyrosine residues in the activation loop of the catalytic core are phosphorylated (Schlessinger, 2000). However, the intracellular mechanisms that control the trans-autophosphorylation of RTKs are not known. Furthermore, it is not clear whether only the ligand-occupied RTKs are autophosphorylated and thus activated or whether a ligand-occupied RTK is capable of phosphorylating and activating unoccupied receptors, resulting in lateral propagation of receptor activation. Trans-autophosphorylation may also provide a mechanism for heteromolecular activation of several members of the same family of RTK by ligand-induced stimulation of one member of the family, such as activation of erbB2, erbB3, or erbB4 in response to EGF receptor (EGFR) stimulation. A recent study suggested that lateral propagation of RTK signal may occur under certain conditions (Verveer et al., 2000). In the August, 2002 issue of Developmental Cell, Sawano et al. explore the possibility of whether local stimulation with EGF to a limited area on the cell surface leads to local or global activation of EGFR signaling. Local stimulation in single live cells is achieved by using lami-