Candida glabrata displays pseudohyphal growth

The ability to undergo morphological change has been reported as an advantageous trait in fungal pathogenesis. Here we demonstrate that Candida glabrata ATCC2001, like diploid Saccharomyces cerevisiae strains, forms elongated chains of pseudohyphal cells on solid nitrogen starvation media (SLAD). Constrictions were apparent between adjoining cells; no parallel-sided hyphae were seen and pseudohyphae invaded the agar. When SLAD was supplemented with ammonium sulfate both C. glabrata and diploid S. cerevisiae strains lost their ability to undergo pseudohyphal growth. However, on this media C. glabrata yeast cells invaded the agar in a similar fashion to the invasive growth mode exhibited by haploid strains of S. cerevisiae cultured on rich media such as YPD. C. glabrata was not capable of invading YPD demonstrating that the process of filamentation is distinct in these two fungi. To our knowledge this is the first report to demonstrate that C. glabrata can undergo morphological change and grow as an invasive filamentous organism. (cid:223) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.


Introduction
The incidence of nosocomial fungal infection has increased dramatically over the last few years to the extent that 10% of such infections are now caused by fungi and Candida species are now the fourth leading cause of bloodstream infections in the US with an attributable mortality of 38% [1,2]. While Candida albicans remains the most commonly isolated species, Candida glabrata is now encountered regularly and is responsible for up to 1:5 cases of candidosis [3,4].
C. albicans is a diploid organism with no known sexual cycle. Blastoconidia of this organism readily undergo the morphological change from yeast to hyphal or pseudohyphal growth in response to a wide variety of conditions including nitrogen starvation [5]. Diploid Saccharomyces cerevisiae also switch to pseudohyphal growth when starved for nitrogen [6] and in response to a limited range of other stimuli [7]. Haploid S. cerevisiae cells, on the other hand, form short, invasive pseudohyphal like cells on rich media but not on nitrogen starvation media. This is referred to as haploid invasive growth.
The signalling pathway and transcription factor genes that are known to govern pseudohyphal growth in S. cerevisiae are at least partially conserved in C. albicans. Mutants lacking these genes in C. albicans are reduced in their ability to ¢lament in vitro and exhibit attenuated virulence in systemic mouse models of candidosis [5,8^11]. A double STE12(CaCPH1)/PHD1(CaEFG1) mutant is locked in the yeast form and is avirulent [12]. The ability to ¢lament has therefore become accepted as being advantageous for virulence.
Unlike these two organisms, C. glabrata has never been observed in a ¢lamentous form. This inability to adopt a ¢lamentous growth mode has made C. glabrata relatively easy to distinguish from other Candida species observed in medical practice [13,14]. However, isolates of asexual budding yeast with no known ¢lamentous form such as C. glabrata were originally assigned to the genus Torulopsis and the organism was originally designated Torulopsis glabrata. Despite the merger of Torulopsis into the genus Candida this designation has persisted in the medical liter-ature. The merger of the two genera was debated for many years because of the budding only morphology of C. glabrata and the historical de¢nition of the genus Candida as pseudomycelial'. However, it was ¢nally agreed upon, because the genus Candida includes species for which pseudohyphae are absent or rudimentary and many isolates of Torulopsis form pseudohyphae [14]. Recent DNA sequence data have con¢rmed this relationship and resulted in the reassignment of the genus Candida; family Candidaceae (formerly of the form-order Cryptococcales; formclass Deuteromycetes) to the order Saccharomycetales; phylum Ascomycota. This also includes the fully sequenced model organism S. cerevisiae which is very closely related to C. glabrata (http://www3.ncbi.nlm.nih.gov/Taxonomy/ tax.html).
The ability of these organisms to undergo morphological change in response to nitrogen starvation plus the close taxonomic relationship between these organisms, S. cerevisiae and C. glabrata, in particular, prompted us to investigate the response of C. glabrata to growth on solid nitrogen starvation media. Here we report that the C. glabrata does indeed produce invasive pseudohyphae under nitrogen limiting conditions on solid media and in addition forms invasive yeast cells on the same media supplemented with ammonium sulfate. To our knowledge this is the ¢rst report of morphological transition in this increasingly important pathogenic fungus.

Strains
C. glabrata ATCC2001 was obtained from the American Type Culture Collection (Rockville, MD, USA). The diploid S. cerevisiae strains used were L5366 ura3^52/ ura3^52 (41278b background) [15] which has been transformed to prototrophy with the vector pRS426 containing the URA3 gene and the prototrophic pseudohyphal strain L6294. The haploid prototrophic strain was 10560-5A. All S. cerevisiae strains were gifts from the laboratory of G.R. Fink (Whitehead Institute, Cambridge, MA, USA). C. albicans SC5314, a wild-type clinical isolate, was used throughout.

Media and culture conditions
Solid synthetic low ammonia dextrose nitrogen starvation medium (SLAD), was prepared as described by Gimeno et al. [6,16]. SLAD contains 2% (w/v) D-glucose (BDH Inc., Toronto, Canada), 1.7 g l 31 yeast nitrogen base without amino acids or ammonium sulfate (Difco Laboratories, Detroit, MI, USA), 10^50 WM ammonium sulfate (BDH Inc., Toronto, Canada) as a sole nitrogen source, and 2% (w/v) washed Bacto-Difco agar (granulated agar from BBL, Becton Dickinson and Co., Cock-eysville, MD, USA was also used). To prepare the medium a 4U stock of the yeast nitrogen base (6.7 g l 31 ), a 10U (0.5 mM) stock of ammonium sulfate, and a 40% (w/v) glucose stock were ¢lter sterilized through a 0.45 Wm Millipore ¢lter, diluted to 2U the appropriate concentrations, warmed to 50³C and added to an equal volume of 50³C 4% (w/v) agar which had been washed four times with distilled deionized water prior to autoclaving. SLAD supplemented with 7.6 mM ammonium sulfate (SHAD) was also prepared.
Lee's solid medium (SM), developed for hyphal growth of C. albicans, was prepared by mixing autoclaved agar (4% w/v) and ¢lter sterilized 2U media described in Lee et al. [17].
To examine haploid invasive growth on rich medium (YPD) [18] cells were streaked onto the agar in quadrants and incubated at 30³C for 3 days followed by 1 day at room temperature prior to washing of agar surfaces. Agar invasion was investigated by gently scraping plates with a plastic inoculating needle and washing thoroughly under a stream of distilled water.
Cells were incubated at either 30³C (S. cerevisiae) or at 30³C and/or 37³C (C. glabrata and C. albicans). Experiments were duplicated in two independent laboratories.

Calco£uor staining
To determine the nature of budding in C. glabrata, cells were cultured overnight at 37³C on solid YPD plates containing 2% (w/v) agar. Cells were removed into 4% (v/v) formaldehyde, spun down immediately, washed 3U in PBS and resuspended in 100 Wl sterile distilled water. Aliquots of 10 Wl were mixed with 10 Wl 1 mg ml 31 Calco£uor (Sigma) and left at room temperature for 5 min. The cells were washed 3U with sterile distilled water and resuspended in 100 Wl volumes of PBS.

Microscopy
Colonies and cells were photographed using either a Nikon TMS inverted microscope and Kodak TMAX ¢lm and scanned into the computer, or a Zeiss Axiophot microscope and captured using a Hamamatsu CCD camera, and Improvision software. Plates for invasive growth on YPD were scanned directly into Adobe Photoshop (Adobe Systems Inc.) and all images were assembled using this program.
Budding patterns were visualized using a Nikon Eclipse E600 microscope with a 100U £uorescence objective and an excitation wavelength of 330^380 nm. Images were captured using a Nikon CoolPix 950 digital camera.

Results and discussion
On YPD and Lee's solid medium, which promotes radial hyphal growth of C. albicans colonies [19,20], C. glabrata formed smooth and circular colonies ( Fig. 1A (a)) composed of yeast form cells. This smooth circular morphology is characteristic of unicellular yeast growing in the budding form [5,6,20,21].
On the other hand, on nitrogen starvation SLAD medium, C. glabrata developed colonies with regions of lateral growth which extended beyond colony borders ( Fig. 1A (b, c)). This distinctive morphology is typical of S. cerevisiae and C. albicans ¢lamenting colonies and is the result of growth proceeding away from the oldest cell combined with the ability of ¢lamentous cells to penetrate agar [5,6,19,20]. Regions of C. glabrata radial growth occurring on SLAD were composed of chains of cells morphologically comparable to that of diploid S. cerevisiae pseudohyphae (Fig. 1B). Both S. cerevisiae and C. glabrata cells had an elongated cell shape and constrictions separated adjoining cells (Fig. 1B), consistent with the distinct pseudohyphal cell type of S. cerevisiae [6].
To quantify our observations we calculated a`morphology index' (2+1.78 log sl/d 2 where s is the diameter of the septal junction, l is the length of the cell, and d is the maximum diameter of the cell) for the three species growing under various conditions. This index was devised as an objective indicator of cell shape in C. albicans [22].  [22]. The morphology indices for C. glabrata and diploid S. cerevisiae grown on SLAD were 2.4 (range 1.9^3.2) and 2.5 (range 1.6^3.4) respectively, and thus the cells were similar in cell shape and had M i greater than that of spherical yeast cells. Thus our ¢ndings support the existence of a ¢lamentous, pseudohyphal growth mode for C. glabrata.
In C. albicans and diploid S. cerevisiae ¢lamentous growth of SLAD is accompanied by invasion of the media [8,25,28]. In addition haploid S. cerevisiae strains invade rich solid media below the colonies as very short chains of cells, distinct from diploid pseudohyphae. Haploid invasive growth occurs on solid rich media, but not in response to nitrogen starvation and is accompanied by a switch from axial to bipolar, but not unipolar, bud site choice [18]. Haploid S. cerevisiae are capable, however, of undergoing pseudohyphal growth in response to butanol [7]. Each of these developmental alterations involves components of the same MAP kinase cascade [7,18].
We investigated the ability of C. glabrata to undergo invasive growth on both rich YPD media and on nitrogen starvation SLAD medium. On SLAD, both C. glabrata and S. cerevisiae diploids formed pseudohyphal ¢laments that invaded the agar (Fig. 2). On YPD invasive growth was observed only for S. cerevisiae haploids, neither S. cerevisiae diploids nor C. glabrata haploids invaded the agar (Fig. 3). However, when SLAD was supplemented with 7.6 mM ammonium sulfate C. glabrata invaded the agar as clumps of yeast cells beneath the colony (Fig. 2) in a similar manner to the haploid invasive growth of S. cerevisiae (Fig. 3). Therefore the morphogenetic and invasion phenotypes of C. glabrata are distinct and separable from both the pseudohyphal and invasive growth phenotypes observed in S. cerevisiae [6,18]. The molecular mechanisms underlying these phenotypes in C. glabrata are unexplored. However, recent work has demonstrated that it may be di¡erent to that seen in S. cerevisiae. The which in S. cerevisiae help to inhibit the transcription factor until the MAP kinase cascade is activated [23,24].
When growing pseudohyphally S. cerevisiae and C. albicans cells do not undergo cytokinesis but form chains of cells separated by constrictions [6,21,22]. Daughter cells in these chains grow away from the oldest cell in the chain because new buds emerge at the pole opposite the previous mother/daughter junction (unipolar) [6]. Unlike S. cerevisiae, C. albicans has a true hyphal form which undergoes apical growth and is composed of elongated cellular compartments that are separated by perpendicular septa [22,25]. In order to undertake pseudohyphal growth cells must be able to bud in a unipolar fashion [6,21,26]. Budding growth of S. cerevisiae, C. albicans, and C. glabrata results in the formation of a small, spherical, outgrowth which eventually breaks away from the mother cell to form a new individual [4,21]. On rich liquid media diploid S. cerevisiae and C. albicans have a polar budding pattern, whereas haploid S. cerevisiae strains exhibit axial budding which results in the formation of tight clusters of cells (Fig. 4A) [18]. C. glabrata also demonstrated a polar budding pattern (Fig. 4A and B) and the pattern of cell growth appeared consistent with asymmetric and unipolar bud site selection. Daughter cells tended to emerge from the pole distal to the site of emergence of the mother cell resulting in the formation of branched chains (Fig. 4A). The degree of this asymmetrical growth pattern was most evident when cells were stained with Calco£uor White to visualize bud scars (Fig. 4B). C. glabrata therefore demonstrates a budding pattern consistent with the ability to undergo pseudohyphal growth.
It has been suggested that ¢lament formation in S. cerevisiae may have been used to seek out new nutrient supplies [6]. Indeed, recent evidence has demonstrated that one of the genes induced by the heteromeric ¢lamentation transcription factor (Tec1p/Ste12p) encodes a secreted endopolygalacturonase (Pgu1p) that is capable of degrading the plant speci¢c polysaccharide pectin [27]. Nitrogen starvation induces a MAPK cascade in S. cerevisiae which results in the`activation' of this heteromeric ¢lamentation transcription factor (Tec1p/Ste12p). In the human pathogen C. albicans, ¢lamentous growth has been linked to virulence [12]. Strains lacking functional copies of CPH1 (the homologue of STE12) and EFG1 (the homologue of PHD1) are unable to form hyphae in vitro and are attenuated in animal models of candidosis. However, Fig. 2. C. glabrata ATCC2001 exhibits invasive growth as pseudohyphae on nitrogen starvation media and as yeast cells on de¢ned media containing 7.6 mM ammonium sulfate. Cells were cultured on low ammonia nitrogen starvation media (SLAD) or SLAD supplemented with 7.6 mM ammonium sulfate (SHAD) for 15 days at 30³C (diploid S. cerevisiae L5366 (transformed with pRS426)) or 37³C (C. glabrata ATCC2001). Colonies growing on agar were photographed with a 10U objective and then plates were washed thoroughly with distilled water and photographed again. As expected the diploid S. cerevisiae strain produced pseudohyphae on SLAD which were capable of invading the agar as they were not removed by washing. In contrast, when cultured on SHAD S. cerevisiae L5366 (transformed with pRS426) grew as non-invasive spherical yeast cells which could be removed from the agar by washing. Similarly C. glabrata ATCC2001 produced invasive pseudohyphae on SLAD. However, when cultured on SHAD, this C. glabrata strain invaded the agar directly below the colony as yeast cells which could not be removed by washing. recent work suggests that e¡ectors involved in processes other than ¢lamentation may play a part in this attenuation as ¢lamentous forms of this mutant were seen in infected gnotobiotic pigs [28]. The role of ¢lamentation in C. glabrata disease remains unexplored, however to our knowledge no description of C. glabrata growing in vivo as anything other than a budding yeast exists.
The morphological similarities between C. glabrata and S. cerevisiae pseudohyphae, and the similar environmental conditions required for inducing the yeast to pseudohyphal switch, suggest that the molecular mechanisms underlying these alterations may be similar. In both S. cerevisiae and C. albicans, at least two independently regulated biochemical cascades induce the yeast to hyphal transition and are required for the virulence of C. albicans [12]. Whether invasion of tissues by pseudohyphae plays a role in the virulence of C. glabrata is not yet known, although only budding growth has been observed in animal tissues [4]. The answer to this and many other interesting aspects of C. glabrata pathobiology will require the cloning and disruption of C. glabrata homologues of S. cerevisiae and C. albicans ¢lamentation genes. In addition the haploid nature of C. glabrata renders certain genetic screens practical [29]. It is anticipated that further analysis will reveal novel genes involved in ¢lamentation and pathogenicity. The demonstration, here, of dimorphism and invasive behaviors for this genetically tractable haploid fungal pathogen, enhances the reputation of C. glabrata as a model organism for the analysis of fungal virulence. Fig. 3. C. glabrata ATCC2001 does not exhibit invasive growth on YPD. Cells were incubated at either 30³C (S. cerevisiae 10560-5A, S. cerevisiae L6294) or 37³C (C. glabrata ATCC2001 and C. albicans SC5314) for 2 days on YPD. Colonies growing on agar were photographed and then plates were washed thoroughly with distilled water and photographed again. As expected haploid S. cerevisiae and C. albicans were able to invade YPD, while diploid S. cerevisiae did not invade the agar. C. glabrata ATCC2001 grew as yeast cells on YPD but did not invade the agar at either 30³C or 37³C. Fig. 4. C. glabrata ATCC2001 exhibits a polar budding pattern. A: Cells were incubated at either 30³C (S. cerevisiae 10560-5A, S. cerevisiae L6294) or 37³C (C. glabrata ATCC2001, C. albicans SC5314) overnight in liquid YPD. They were then inoculated on to solid YPD and incubated for 2 days at 30³C or 37³C, prior to 1 day at room temperature. Growing cells were then viewed using Nomarski optics (63U objective). C. glabrata ATCC2001 had a distinct polar budding pattern (arrow), similar to that seen in C. albicans and diploid S. cerevisiae but distinct from the axial budding pattern seen in haploid S. cerevisiae. B: C. glabrata cells prepared as in A were stained with Calco£uor White and observed with a £uorescent microscope (excitation wavelength 330^380 nm, 100U objective). The presence of bud scars at opposite poles of the yeast cells (arrows) indicates that C. glabrata ATCC2001 undergoes polar budding. ful discussions and support. Very special thanks to Julia Kohler. This is NRCC manuscript number 42945. Work in the K.H. laboratory is supported by the MRC, Action Research and Chronic Granulomatous Disorder Research Trust.