Cheryl A. Gale

Candida albicans hyphae have a Spitzenkörper that is distinct from the polarisome found in yeast and pseudohyphae.

Fungi grow with a variety of morphologies: oval yeast cells, chains of elongated cells called pseudohyphae and long, narrow, tube-like filaments called hyphae. In filamentous fungi, hyphal growth is strongly polarised to the tip and is mediated by the Spitzenkörper, which acts as a supply centre to concentrate the delivery of secretory vesicles to the tip. In the budding yeast Saccharomyces cerevisiae, polarised growth is mediated by the polarisome, a surface cap of proteins that nucleates the formation of actin cables delivering secretory vesicles to the growing tip. The human fungal pathogen, Candida albicans, can grow in all three morphological forms. Here we show the presence of a Spitzenkörper at the tip of C. albicans hyphae as a ball-like localisation of secretory vesicles, together with the formin Bni1 and Mlc1, an ortholog of an S. cerevisiae myosin regulatory light chain. In contrast, in C. albicans yeast cells, pseudohyphae and hyphae Spa2 and Bud6, orthologs of S. cerevisiae polarisome components, as well as the master morphology regulator Cdc42, localise predominantly, but not exclusively, to a surface cap resembling the polarisome of S. cerevisiae yeast cells. A small amount of Cdc42 also localises to the Spitzenkörper. Furthermore, we show differences in the genetic and cytoskeletal requirements, and cell cycle dynamics of polarity determinants in yeast, pseudohyphae and hyphae. These results, together with the cytological differences between the cell types, suggest that the Spitzenkörper and polarisome are distinct structures, that the polarisome and Spitzenkörper coexist in hyphae, and that polarised growth in hyphae is driven by a fundamentally different mechanism to that in yeast and pseudohyphae.

PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance

Protein O‐mannosyltransferases (Pmt proteins) initiate O‐mannosylation of secretory proteins. The PMT gene family of the human fungal pathogen Candida albicans consists of PMT1 and PMT6, as well as three additional PMT genes encoding Pmt2, Pmt4 and Pmt5 isoforms described here. Both PMT2 alleles could not be deleted and growth of conditional strains, containing PMT2 controlled by the MET3‐ or tetOScHOP1‐promoters, was blocked in non‐permissive conditions, indicating that PMT2 is essential for growth. A homozygous pmt4 mutant was viable, but synthetic lethality of pmt4 was observed in combination with pmt1 mutations. Hyphal morphogenesis of a pmt4 mutant was defective under aerobic induction conditions, yet increased in embedded or hypoxic conditions, suggesting a role of Pmt4p‐mediated O‐glycosylation for environment‐specific morphogenetic signalling. Although a PMT5 transcript was detected, a homozygous pmt5 mutant was phenotypically silent. All other pmt mutants showed variable degrees of supersensitivity to antifungals and to cell wall‐destabilizing agents. Cell wall composition was markedly affected in pmt1 and pmt4 mutants, showing a significant decrease in wall mannoproteins. In a mouse model of haematogenously disseminated infection, PMT4 was required for full virulence of C. albicans. Functional analysis of the first complete PMT gene family in a fungal pathogen indicates that Pmt isoforms have variable and specific roles for in vitro and in vivo growth, morphogenesis and antifungal resistance.

Hyphal Guidance and Invasive Growth in Candida albicans Require the Ras-Like GTPase Rsr1p and Its GTPase-Activating Protein Bud2p

ABSTRACT Candida albicans, the most prevalent fungal pathogen of humans, causes superficial mycoses, invasive mucosal infections, and disseminated systemic disease. Many studies have shown an intriguing association between C. albicans morphogenesis and the pathogenesis process. For example, hyphal cells have been observed to penetrate host epithelial cells at sites of wounds and between cell junctions. Ras- and Rho-type GTPases regulate many morphogenetic processes in eukaryotes, including polarity establishment, cell proliferation, and directed growth in response to extracellular stimuli. We found that the C. albicans Ras-like GTPase Rsr1p and its predicted GTPase-activating protein Bud2p localized to the cell cortex, at sites of incipient daughter cell growth, and provided landmarks for the positioning of daughter yeast cells and hyphal cell branches, similar to the paradigm in the model yeast Saccharomyces cerevisiae. However, in contrast to S. cerevisiae, CaRsr1p and CaBud2p were important for morphogenesis: C. albicans strains lacking Rsr1p or Bud2p had abnormal yeast and hyphal cell shapes and frequent bends and promiscuous branching along the hypha and were unable to invade agar. These defects were associated with abnormal actin patch polarization, unstable polarisome localization at hyphal tips, and mislocalized septin rings, consistent with the idea that GTP cycling of Rsr1p stabilizes the axis of polarity primarily to a single focus, thus ensuring normal cell shape and a focused direction of polarized growth. We conclude that the Rsr1p GTPase functions as a polarity landmark for hyphal guidance and may be an important mediator of extracellular signals during processes such as host invasion.

An Internal Polarity Landmark Is Important for Externally Induced Hyphal Behaviors in Candida albicans

ABSTRACT Directional growth is a function of polarized cells such as neurites, pollen tubes, and fungal hyphae. Correct orientation of the extending cell tip depends on signaling pathways and effectors that mediate asymmetric responses to specific environmental cues. In the hyphal form of the eukaryotic fungal pathogen Candida albicans, these responses include thigmotropism and galvanotropism (hyphal turning in response to changes in substrate topography and imposed electrical fields, respectively) and penetration into semisolid substrates. During vegetative growth in C. albicans, as in the model yeast Saccharomyces cerevisiae, the Ras-like GTPase Rsr1 mediates internal cellular cues to position new buds in a prespecified pattern on the mother cell cortex. Here, we demonstrate that Rsr1 is also important for hyphal tip orientation in response to the external environmental cues that induce thigmotropic and galvanotropic growth. In addition, Rsr1 is involved in hyphal interactions with epithelial cells in vitro and its deletion diminishes the hyphal invasion of kidney tissue during systemic infection. Thus, Rsr1, an internal polarity landmark in yeast, is also involved in polarized growth responses to asymmetric environmental signals, a paradigm that is different from that described for the homologous protein in S. cerevisiae. Rsr1 may thereby contribute to the pathogenesis of C. albicans infections by influencing hyphal tip responses triggered by interaction with host tissues.

Cassettes for the PCR-mediated construction of regulatable alleles in Candida albicans

The recent availability of genome sequence information for the opportunistic pathogen Candida albicans has greatly facilitated the ability to perform genetic manipulations in this organism. Two important molecular tools for studying gene function are regulatable promoters for generating conditional mutants and fluorescent proteins for determining the subcellular localization of fusion gene products. We describe a set of plasmids containing promoter–GFP cassettes (PMET3–GFP, PGAL1–GFP, and PPCK1–GFP), linked to a selectable nutritional marker gene (URA3). PCR‐mediated gene modification generates gene‐specific promoter, or gene‐specific promoter–GFP, fusions at the 5′‐end of the gene of interest. One set of primers can be used to generate three strains expressing a native protein of interest, or an amino‐terminal GFP‐tagged version, from three different regulatable promoters. Thus, these promoter cassette plasmids facilitate construction of conditional mutant strains, overexpression alleles and/or inducible amino‐terminal GFP fusion proteins. Copyright

Candida albicans INT1-Induced Filamentation in Saccharomyces cerevisiae Depends on Sla2p

ABSTRACT The Candida albicans INT1 gene is important for hyphal morphogenesis, adherence, and virulence (C. Gale, C. Bendel, M. McClellan, M. Hauser, J. M. Becker, J. Berman, and M. Hostetter, Science 279:1355–1358, 1998). The ability to switch between yeast and hyphal morphologies is an important virulence factor in this fungal pathogen. When INT1 is expressed in Saccharomyces cerevisiae, cells grow with a filamentous morphology that we exploited to gain insights into how C. albicans regulates hyphal growth. In S. cerevisiae, INT1-induced filamentous growth was affected by a small subset of actin mutations and a limited set of actin-interacting proteins including Sla2p, anS. cerevisiae protein with similarity in its C terminus to mouse talin. Interestingly, while SLA2 was required forINT1-induced filamentous growth, it was not required for polarized growth in response to several other conditions, suggesting that Sla2p is not required for polarized growth per se. The morphogenesis checkpoint, mediated by Swe1p, contributes toINT1-induced filamentous growth; however, epistasis analysis suggests that Sla2p and Swe1p contribute toINT1-induced filamentous growth through independent pathways. The C. albicans SLA2 homolog (CaSLA2) complements S. cerevisiae sla2Δ mutants for growth at 37°C and INT1-induced filamentous growth. Furthermore, in a C. albicans Casla2/Casla2 strain, hyphal growth did not occur in response to either nutrient deprivation or to potent stimuli, such as mammalian serum. Thus, through analysis ofINT1-induced filamentous growth in S. cerevisiae, we have identified a C. albicans gene,SLA2, that is required for hyphal growth in C. albicans.

Molecular Genetic and Genomic Approaches to the Study of Medically Important Fungi

Since the early work of Beadle and Tatum ([6][1]), fungi have been important model organisms for solving genetic problems, largely because of their ease of culture, their defined life cycles, and their short generation times. However, with the development of aggressive and successful medical

Human Milk Oligosaccharides Inhibit Candida albicans Invasion of Human Premature Intestinal Epithelial Cells

BACKGROUND Human milk oligosaccharides (HMOs) are a highly abundant, diverse group of unique glycans that are postulated to promote the development of a protective bacterial microbiota in the intestine and prevent adhesive and invasive interactions of pathogenic bacteria with mucosal epithelia. Candida albicans, a prevalent fungal colonizer of the neonatal gut, causes the majority of fungal disease in premature infants and is highly associated with life-threatening intestinal disorders. OBJECTIVE The objective of the current study was to test the hypothesis that HMOs protect human premature intestinal epithelial cells (pIECs) from invasion by C. albicans. METHODS To study fungal invasion, a quantitative immunocytochemical assay was used to distinguish invading from noninvading C. albicans cells in the presence and absence of HMOs. To understand how HMOs affect C. albicans invasion of pIECs, the expression of C. albicans virulence traits that are important for invasiveness (hyphal morphogenesis and ability to associate with host cells) were quantified. RESULTS Treatment with HMOs reduced invasion of pIECs by C. albicans in a dose-dependent manner by 14-67%, with a physiologic concentration (15mg/mL) of HMOs causing a 52% reduction in invasion (P < 0.05). The decreased invasive ability of C. albicans was associated with hyphal lengths that were ∼30% shorter (P < 0.05), likely because of a delay in the induction of hyphal morphogenesis after inoculation of yeast onto pIECs, which correlated with a 23% reduction in the combined expression level of hyphal-specific genes (P < 0.05). In addition, HMOs caused a 40% decrease in the number of C. albicans cells able to associate with pIECs at the time of hyphal induction (P < 0.05). CONCLUSIONS These results, obtained with the use of a primary pIEC model, indicate that HMOs reduce virulence characteristics of C. albicans and suggest a role for HMOs in protecting the premature infant intestine from invasion and damage by C. albicans hyphae.

Cdc42 GTPase dynamics control directional growth responses

Significance The growth of many cell types combines polarized elongation with directional responses to external cues. We have previously linked Ca2+ influx with directional growth in fungi and show here that Ca2+ influx can rescue phenotypes caused by genetic disruption of two Cdc42 GTPase plasma-membrane trafficking pathways that are required for polarity establishment, hence restoring directional polarization. Constitutive activation of Cdc42 reversed the direction of polarization, which was also enhanced by the provision of exogenous Ca2+. Our model proposes that Ca2+ transport amplifies weak directional growth signals specified by activated Cdc42 by promoting Cdc42 trafficking to the plasma membrane, thereby enhancing its directional regulation of polarized growth. Polarized cells reorient their direction of growth in response to environmental cues. In the fungus Candida albicans, the Rho-family small GTPase, Cdc42, is essential for polarized hyphal growth and Ca2+ influx is required for the tropic responses of hyphae to environmental cues, but the regulatory link between these systems is unclear. In this study, the interaction between Ca2+ influx and Cdc42 polarity-complex dynamics was investigated using hyphal galvanotropic and thigmotropic responses as reporter systems. During polarity establishment in an applied electric field, cathodal emergence of hyphae was lost when either of the two Cdc42 apical recycling pathways was disrupted by deletion of Rdi1, a guanine nucleotide dissociation inhibitor, or Bnr1, a formin, but was completely restored by extracellular Ca2+. Loss of the Cdc42 GTPase activating proteins, Rga2 and Bem3, also abolished cathodal polarization, but this was not rescued by Ca2+. Expression of GTP-locked Cdc42 reversed the polarity of hypha emergence from cathodal to anodal, an effect augmented by Ca2+. The cathodal directional cue therefore requires Cdc42 GTP hydrolysis. Ca2+ influx amplifies Cdc42-mediated directional growth signals, in part by augmenting Cdc42 apical trafficking. The Ca2+-binding EF-hand motif in Cdc24, the Cdc42 activator, was essential for growth in yeast cells but not in established hyphae. The Cdc24 EF-hand motif is therefore essential for polarity establishment but not for polarity maintenance.

Rsr1 Focuses Cdc42 Activity at Hyphal Tips and Promotes Maintenance of Hyphal Development in Candida albicans

ABSTRACT The extremely elongated morphology of fungal hyphae is dependent on the cells ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cells ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 “fine-tunes” the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis.