Alexandra Brand

Ectopic Expression of URA3 Can Influence the Virulence Phenotypes and Proteome of Candida albicans but Can Be Overcome by Targeted Reintegration of URA3 at the RPS10 Locus

ABSTRACT Uridine auxotrophy, based on disruption of both URA3 alleles in diploid Candida albicans strain SC5314, has been widely used to select gene deletion mutants created in this fungus by “Ura-blasting” and PCR-mediated disruption. We compared wild-type URA3 expression with levels in mutant strains where URA3 was positioned either within deleted genes or at the highly expressed RPS10 locus. URA3 expression levels differed significantly and correlated with the specific activity of Ura3p, orotidine 5′-monophosphate decarboxylase. Reduced URA3 expression following integration at the GCN4 locus was associated with an attenuation of virulence. Furthermore, a comparison of the SC5314 (URA3) and CAI-4 (ura3) proteomes revealed that inactivation of URA3 caused significant changes in the levels of 14 other proteins. The protein levels of all except one were partially or fully restored by the reintegration of a single copy of URA3 at the RPS10 locus. Transcript levels of genes expressed ectopically at this locus in reconstituted heterozygous mutants also matched the levels found when the genes were expressed at their native loci. Therefore, phenotypic changes in C. albicans can be associated with the selectable marker rather than the target gene. Reintegration of URA3 at an appropriate expression locus such as RPS10 can offset most problems related to the phenotypic changes associated with gene knockout methodologies.

Mnt1p and Mnt2p of Candida albicans Are Partially Redundant α-1,2-Mannosyltransferases That Participate in O-Linked Mannosylation and Are Required for Adhesion and Virulence

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant α-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Δ and mnt1Δ single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Δmnt2Δ mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections.

Hyphal Orientation of Candida albicans Is Regulated by a Calcium-Dependent Mechanism

Summary Eukaryotic cells from fungal hyphae to neurites that grow by polarized extension must coordinate cell growth and cell orientation to enable them to exhibit growth tropisms and to respond to relevant environmental cues. Such cells generally maintain a tip-high Ca2+ cytoplasmic gradient, which is correlated with their ability to exhibit polarized tip growth and to respond to growth-directing extracellular signals [1–5]. In yeast and other fungi, the polarisome, exocyst, Arp2/3, and Spitzenkörper protein complexes collectively orchestrate tip growth and cell polarity, but it is not clear whether these molecular complexes also regulate cell orientation or whether they are influenced by cytoplasmic Ca2+ gradients. Hyphae of the human pathogenic fungus Candida albicans reorient their growth axis in response to underlying surface topography (thigmotropism) [6] and imposed electric fields (galvanotropism) [7]. The establishment and maintenance of directional growth in relation to these environmental cues was Ca2+ dependent. Tropisms were attenuated in media containing low Ca2+, or calcium-channel blockers, and in mutants where calcium channels or elements of the calcium signaling pathway were deleted. Therefore galvanotropism and thigmotropism may both be mediated by localized Ca2+ influx at sites of polarized growth via Ca2+ channels that are activated by appropriate environmental signals.

Mechanisms of hypha orientation of fungi.

Hypha orientation is an essential aspect of polarised growth and the morphogenesis, spatial ecology and pathogenesis of fungi. The ability to re-orient tip growth in response to environmental cues is critical for colony ramification, the penetration of diverse host tissues and the formation of mating structures. Recent studies have begun to describe the molecular machinery regulating hypha orientation. Calcium signalling, the polarisome Bud1-GTPase module and the Tea cell-end marker proteins of the microtubule cytoskeleton, along with specific kinesins and sterol-rich apical microdomains, are involved in hypha orientation. Mutations that affect these processes generate normal-shaped, growing hyphae that have either abnormal meandering trajectories or attenuated tropic responses. Hyphal tip orientation and tip extension are, therefore, distinct regulatory mechanisms that operate in parallel during filamentous growth, thereby allowing fungi to orchestrate their reproduction in relation to gradients of effectors in their environments.

A multifunctional mannosyltransferase family in Candida albicans determines cell wall mannan structure and host-fungus interactions.

The cell wall proteins of fungi are modified by N- and O-linked mannosylation and phosphomannosylation, resulting in changes to the physical and immunological properties of the cell. Glycosylation of cell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnTΔ null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of β-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.

Calcineurin Controls Drug Tolerance, Hyphal Growth, and Virulence in Candida dubliniensis

ABSTRACT Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.

Cell wall glycans and soluble factors determine the interactions between the hyphae of Candida albicans and Pseudomonas aeruginosa

The fungus, Candida albicans, and the bacterium, Pseudomonas aeruginosa, are opportunistic human pathogens that have been coisolated from diverse body sites. Pseudomonas aeruginosa suppresses C. albicans proliferation in vitro and potentially in vivo but it is the C. albicans hyphae that are killed while yeast cells are not. We show that hyphal killing involves both contact-mediated and soluble factors. Bacterial culture filtrates contained heat-labile soluble factors that killed C. albicans hyphae. In cocultures, localized points of hyphal lysis were observed, suggesting that adhesion and subsequent bacteria-mediated cell wall lysis is involved in the killing of C. albicans hyphae. The glycosylation status of the C. albicans cell wall affected the rate of contact-dependent killing because mutants with severely truncated O-linked, but not N-linked, glycans were hypersensitive to Pseudomonas-mediated killing. Deletion of HWP1, ALS3 or HYR1, which encode major hypha-associated cell wall proteins, had no effect on fungal susceptibility.

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.

Calcium homeostasis is required for contact-dependent helical and sinusoidal tip growth in Candida albicans hyphae

Hyphae of the dimorphic fungus, Candida albicans, exhibit directional tip responses when grown in contact with surfaces. On hard surfaces or in liquid media, the trajectory of hyphal growth is typically linear, with tip re‐orientation events limited to encounters with topographical features (thigmotropism). In contrast, when grown on semisolid surfaces, the tips of C. albicans hyphae grow in an oscillatory manner to form regular two‐dimensional sinusoidal curves and three‐dimensional helices. We show that, like thigmotropism, initiation of directional tip oscillation in C. albicans hyphae is severely attenuated when Ca2+ homeostasis is perturbed. Chelation of extracellular Ca2+ or deletion of the Ca2+ transporters that modulate cytosolic [Ca2+] (Mid1, Cch1 or Pmr1) did not affect hyphal length but curve formation was severely reduced in mid1Δ and cch1Δ and abolished in pmr1Δ. Sinusoidal hypha morphology was altered in the mid1Δ, chs3Δ and heterozygous pmr1Δ/PMR1 strains. Treatments that affect cell wall integrity, changes in surface mannosylation or the provision of additional carbon sources had significant but less pronounced effects on oscillatory growth. The induction of two‐ and three‐dimensional sinusoidal growth in wild‐type C. albicans hyphae is therefore the consequence of mechanisms that involve Ca2+ influx and signalling rather than gross changes in the cell wall architecture.

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.