Host mechanisms enabling discrimination between the commensal and pathogenic states of opportunistic pathogens are critical in mucosal defense and homeostasis. Here, we demonstrate that oral epithelial cells orchestrate an innate response to the human fungal pathogen Candida albicans via NF-κB and a bi-phasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to the recognition of fungal cell wall structures. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha-formation and fungal burdens, and correlates with proinflammatory responses. This MAPK-based discriminatory pathway may provide a mechanism for epithelial tissues to remain quiescent in the presence of low fungal burdens whilst responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may thus comprise a `danger response' pathway in vivo and may be critical in identifying when this normally commensal fungus has become pathogenic.
We previously reported that a bi-phasic innate immune MAPK response, constituting activation of the mitogen-activated protein kinase (MAPK) phosphatase MKP1 and c-Fos transcription factor, discriminates between the yeast and hyphal forms of Candida albicans in oral epithelial cells (ECs). Since the vast majority of mucosal Candida infections are vaginal, we sought to determine whether a similar bi-phasic MAPK-based immune response was activated by C. albicans in vaginal ECs. Here, we demonstrate that vaginal ECs orchestrate an innate response to C. albicans via NF-κB and MAPK signaling pathways. However, unlike in oral ECs, the first MAPK response, defined by c-Jun transcription factor activation, is delayed until 2 h in vaginal ECs but is still independent of hypha formation. The ‘second’ or ‘late’ MAPK response, constituting MKP1 and c-Fos transcription factor activation, is identical to oral ECs and is dependent upon both hypha formation and fungal burdens. NF-κB activation is immediate but independent of morphology. Furthermore, the proinflammatory response in vaginal ECs is different to oral ECs, with an absence of G-CSF and CCL20 and low level IL-6 production. Therefore, differences exist in how C. albicans activates signaling mechanisms in oral and vaginal ECs; however, the activation of MAPK-based pathways that discriminate between yeast and hyphal forms is retained between these mucosal sites. We conclude that this MAPK-based signaling pathway is a common mechanism enabling different human epithelial tissues to orchestrate innate immune responses specifically against C. albicans hyphae.
Oral epithelial cells detect the human pathogenic fungus Candida albicans via NF-κB and a bi-phasic mitogen-activated protein kinase (MAPK) signaling response. However, discrimination between C. albicans yeast and hyphal forms is mediated only by the MAPK pathway, which constitutes activation of the MAPK phosphatase MKP1 and the c-Fos transcription factor and is targeted against the hyphal form. Given that C. albicans is not the only Candida species capable of filamentation or causing mucosal infections, we sought to determine whether this MAPK/MKP1/c-Fos mediated response mechanism was activated by other pathogenic Candida species, including C. dubliniensis, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. Although all Candida species activated the NF-κB signaling pathway, only C. albicans and C. dubliniensis were capable of inducing MKP1 and c-Fos activation, which directly correlated with hypha formation. However, only C. albicans strongly induced cytokine production (G-CSF, GM-CSF, IL-6 and IL-1α) and cell damage. Candida dubliniensis, C. tropicalis and C. parapsilosis were also capable of inducing IL-1α and this correlated with mild cell damage and was dependent upon fungal burdens. Our data demonstrate that activation of the MAPK/MKP1/c-Fos pathway in oral epithelial cells is specific to C. dubliniensis and C. albicans hyphae.
Candida albicans; Candida dubliniensis; Hypha formation; MAPK; MKP1; c-Fos; NF-κB; Oral epithelium; Innate immunity
Oral epithelial cells discriminate between the yeast and hyphal forms of Candida albicans via the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether β-glucan and N- or O-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of β-glucan and N- and O-mannan mutants, we found that N-mannosylation (via Δoch1 and Δpmr1 mutants) and O-mannosylation (via Δpmt1 and Δmnt1 Δmnt2 mutants), but not phosphomannan (via a Δmnn4 mutant) or β-1,2 mannosylation (via Δbmt1 to Δbmt6 mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N- and O-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the α-1,6-mannose backbone of the N-linked outer chain (corresponding to a Δmnn9 mutant) may be required for epithelial adhesion, while the α-1,2-mannose component of phospholipomannan (corresponding to a Δmit1 mutant) may contribute to epithelial cell damage. β-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N- and O-mannosylation defects affect the ability of C. albicans to induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.
The opportunistic fungal pathogen Candida albicans is both a benign gut commensal and a frequently fatal systemic pathogen. The interaction of C. albicans with the host's innate immune system is the primary factor in this balance; defects in innate immunity predispose the patient to disseminated candidiasis. Because of the central importance of phagocytic cells in defense against fungal infections, we have investigated the response of C. albicans to phagocytosis by mammalian macrophages using genomic transcript profiling. This analysis reveals a dramatic reprogramming of transcription in C. albicans that occurs in two successive steps. In the early phase cells shift to a starvation mode, including gluconeogenic growth, activation of fatty acid degradation, and downregulation of translation. In a later phase, as hyphal growth enables C. albicans to escape from the macrophage, cells quickly resume glycolytic growth. In addition, there is a substantial nonmetabolic response imbedded in the early phase, including machinery for DNA damage repair, oxidative stress responses, peptide uptake systems, and arginine biosynthesis. Further, a surprising percentage of the genes that respond specifically to macrophage contact have no known homologs, suggesting that the organism has undergone substantial evolutionary adaptations to the commensal or pathogen lifestyle. This transcriptional reprogramming is almost wholly absent in the related, but nonpathogenic, yeast Saccharomyces cerevisiae, suggesting that these large-scale and coordinated changes contribute significantly to the ability of this organism to survive and cause disease in vivo.
The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.
The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin αXβ2 in the pathogenesis of fungal infection was assessed. Both purified αXβ2 and αXβ2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by β-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of αMβ2. Mice deficient in αXβ2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in αXβ2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of αXβ2-deficient mice within 9 d. Organs taken from αXβ2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that αXβ2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use αXβ2 to control fungal invasion.
Candida albicans is a commensal fungus of the normal flora yet causes opportunistic infection following trauma or surgery and during immunosuppression. C. albicans virulence factors include morphogenesis into invasive filaments, adherence to host cells, and secretion of proteases. This study evaluated the role of fungal hyphal extension in experimental C. albicans keratitis using genetically altered yeast strains. Scarified corneas of adult BALB/c mice were topically inoculated with wild-type (SC5314) or 10 transposon-induced mutant strains of C. albicans and monitored for 4 days post inoculation. In vitro growth kinetics and the yeast strains’ ability to bud into pseudohyphae or hyphae were also compared. The wild-type human isolate had a high degree of virulence in the murine cornea, and four fungal strains deficient in genes regulating adherence or encoding membrane proteins did not significantly differ from the parental strain (P > 0.3). Five yeast strains deficient in genes involved in filamentation resulted in fully or partially attenuated keratomycosis (P < 0.0001). The overall growth kinetics of wild-type and mutant strains were similar in rich media (P > 0.9), but mutants with deficient morphogenesis had reduced filamentation in vitro. Phenotypic switching from yeasts to filamentous forms facilitates the establishment and progression of experimental corneal disease by C. albicans.
Candida albicans; keratitis; keratomycosis; microbial pathogenesis; ocular infection
The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin αXβ2 in the pathogenesis of fungal infection was assessed. Both purified αXβ2 and αXβ2-expressing HEK293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by β-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of αMβ2. Mice deficient in αXβ2 were more prone to systemic infection with the LD50 fungal inoculum decreasing three-fold in αXβ2 deficient mice compared to wild-type mice. After challenging intravenously with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 days compared to 100% mortality of αXβ2-deficient mice within 9 days. Organs taken from αXβ2-deficient mice 16 hrs after infection revealed a 10-fold increase in fungal invasion into brain and 2-fold increase into liver. These data indicate that αXβ2 is important for protection against systemic C. albicans infections and macrophage subsets in liver, Kupffer cells, while in brain, microglial cells employ αXβ2 to control fungal invasion.
integrin αXβ2; Pra1; C. albicans; systemic fungal infections; protection; murine model; adhesion molecules; macrophages; microglia; Kupffer cells
► First demonstration of post-mitotic fusion of macrophages infected with Candida albicans. ► Post-mitotic fusion was attributed to C. albicans hyphae but not yeast form cells. ► Post-mitotic fusion may inhibit macrophage proliferation and the formation of new uninfected phagocytes.
The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to inhibit effective destruction by host phagocytes. Using live cell video microscopy, we show here for the first time that C. albicans inhibits cell division in macrophages undergoing mitosis. Inhibition of macrophage cell division is dependent on the ability of C. albicans to form hyphae, as it is rarely observed following phagocytosis of UV-killed or morphogenesis-defective mutant Candida. Interestingly, failed cell division following phagocytosis of hyphal C. albicans is surprisingly common, and leads to the formation of large multinuclear macrophages. This raises question as to whether inhibition of macrophage cell division is another virulence attribute of C. albicans or enables host macrophages to contain the pathogen.
Candida albicans; Macrophage; Phagocytosis; Mitosis; Innate immunology
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.
The common fungal pathogen Candida albicans has the ability to grow as a yeast or as a hypha and can alternate between these morphotypes. The overall biomass of both morphotypes increases with growth. However, only yeasts, but not hyphae, exist as discrete cellular entities. Multiplicity of infection (MOI) is a useful parameter to determine the initial inoculum of yeasts for in vitro infection assays. Since the amount of hyphae is difficult to quantify, comparable starting conditions in such assays cannot be determined accurately for yeasts and hyphae using MOI. To circumvent this problem, we have established a set of correlation coefficients to convert fungal metabolic activity and optical density to dry mass. Using these correlations, we were able to accurately compare ROS production and IL-8 release by polymorphonuclear neutrophils upon infection with equal dry mass amounts of yeast and hyphal morphotypes. Neutrophil responses depended on the initial form of infection, irrespective of C. albicans wild-type yeasts transforming to hyphal growth during the assay. Infection with a high mass of live C. albicans yeasts resulted in lower neutrophil ROS and this decrease stems from efficient ROS detoxification by C. albicans without directly affecting the phagocyte ROS machinery. Moreover, we show that dead C. albicans induces significantly less ROS and IL-8 release than live fungi, but thimerosal-killed C. albicans were still able to detoxify neutrophil ROS. Thus, the dry mass approach presented in this study reveals neutrophil responses to different amounts and morphotypes of C. albicans and serves as a template for studies that aim to identify morphotype-specific responses in a variety of immune cells.
Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification.
Many organisms are able to change their phenotype in response to changes in the environment, a phenomenon referred to as plasticity. Candida albicans, a major opportunistic fungal pathogen of humans, can undergo reversible morphological changes between yeast (spherical) and hyphal (filamentous) forms of growth in response to environmental cues. This morphological plasticity is essential for its pathogenesis and survival in its hosts. In this study, we show that hyphal development is initiated and maintained by two major nutrient-sensing cellular growth pathways that act by removing the inhibition provided by the transcriptional repressor Nrg1. While initiation requires a rapid but temporary disappearance of Nrg1 via activation of the cAMP-dependent protein kinase A pathway, maintenance requires the recruitment to promoters of the Hda1 histone deacetylase under conditions of reduced signaling by the target of rapamycin (TOR) kinase, leading to chromatin remodeling that blocks Nrg1 access to the promoters of hypha-specific genes. We observed that recruitment of Hda1 to promoters happens only during the time window when Nrg1 is absent. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals in the regulation of gene expression and phenotypic plasticity during development and cell fate specification.
Candida albicans is an opportunistic and polymorphic fungal pathogen that causes mucosal, disseminated and invasive infections in humans. Transition from the yeast form to the hyphal form is one of the key virulence factors in C. albicans contributing to macrophage evasion, tissue invasion and biofilm formation. Nontoxic small molecules that inhibit C. albicans yeast-to-hypha conversion and hyphal growth could represent a valuable source for understanding pathogenic fungal morphogenesis, identifying drug targets and serving as templates for the development of novel antifungal agents. Here, we have identified the triterpenoid saponin family of gymnemic acids (GAs) as inhibitor of C. albicans morphogenesis. GAs were isolated and purified from Gymnema sylvestre leaves, the Ayurvedic traditional medicinal plant used to treat diabetes. Purified GAs had no effect on the growth and viability of C. albicans yeast cells but inhibited its yeast-to-hypha conversion under several hypha-inducing conditions, including the presence of serum. Moreover, GAs promoted the conversion of C. albicans hyphae into yeast cells under hypha inducing conditions. They also inhibited conidial germination and hyphal growth of Aspergillus sp. Finally, GAs inhibited the formation of invasive hyphae from C. albicans-infected Caenorhabditis elegans worms and rescued them from killing by C. albicans. Hence, GAs could be useful for various antifungal applications due to their traditional use in herbal medicine.
The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects of C. albicans innate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion of C. albicans by macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4Δ, pmr1Δ, and mnt3 mnt5Δ), whereas O- and N-linked mannan defects (mnt1Δ mnt2Δ and mns1Δ) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1Δ, hwp1Δ, and als3Δ) and yeast-locked mutants (clb2Δ, hgc1Δ, cph1Δ, efg1Δ, and efg1Δ cph1Δ), was similar to that observed for wild-type C. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and process C. albicans.
Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.
Host defence against systemic candidiasis relies mainly on the ingestion and elimination of fungal cells by cells of the innate immune system, especially neutrophils and macrophages. Here we have used live cell video microscopy coupled with sophisticated image analysis to generate a temporal and spatial analysis in unprecedented detail of the specific effects of C. albicans viability, cell wall composition, morphogenesis and spatial orientation on two distinct stages (macrophage migration and engulfment of bound C. albicans) of the phagocytosis process. The novel methods employed here to study phagocytosis of C. albicans could be applied to study other pathogens and uptake of dying host cells. Thus, our studies have direct implications for a much broader community and provide a blueprint for future studies with other phagocytes/microorganisms that would significantly enhance our understanding of the mechanisms that govern effective phagocytosis and ultimately the innate immune response to infection.
Candida albicans is an opportunistic human fungal pathogen that causes systemic candidiasis as well as superficial mucosal candidiasis. In response to the host environment, C. albicans transitions between yeast and hyphal forms. In particular, hyphal growth is important in facilitating adhesion and invasion of host tissues, concomitant with the expression of various hypha-specific virulence factors. In previous work, we showed that the cyclic AMP (cAMP) signaling pathway plays a crucial role in morphogenic transitions and virulence of C. albicans by studying genes encoding adenylate cyclase-associated protein (CAP1) and high-affinity phosphodiesterase (PDE2) (Y. S. Bahn, J. Staab, and P. Sundstrom, Mol. Microbiol. 50:391-409, 2003; and Y. S. Bahn and P. Sundstrom, J. Bacteriol. 183:3211-3223, 2001). However, little is known about the downstream targets of the cAMP signaling pathway that are responsible for morphological transitions and the expression of virulence factors. Here, microarrays were probed with RNA from strains with hypoactive (cap1/cap1 null mutant), hyperactive (pde2/pde2 null mutant), and wild-type cAMP signaling pathways to provide insight into the molecular mechanisms of virulence that are regulated by cAMP and that are related to the morphogenesis of C. albicans. Genes controlling metabolic specialization, cell wall structure, ergosterol/lipid biosynthesis, and stress responses were modulated by cAMP during hypha formation. Phenotypic traits predicted to be regulated by cAMP from the profiling results correlated with the relative strengths of the mutants when tested for resistance to azoles and subjected to heat shock stress and oxidative/nitrosative stress. The results from this study provide important insights into the role of the cAMP signaling pathway not only in morphogenic transitions of C. albicans but also for adaptation to stress and for survival during host infections.
The ability of Candida albicans, the most common human fungal pathogen, to transition from yeast to hyphae is essential for pathogenicity. While a variety of transcription factors important for filamentation have been identified and characterized, links between transcriptional regulators of C. albicans morphogenesis and molecular mechanisms that drive hyphal growth are not well defined. We have previously observed that constitutive expression of UME6, which encodes a filament-specific transcriptional regulator, is sufficient to direct hyphal growth in the absence of filament-inducing conditions. Here we show that HGC1, encoding a cyclin-related protein necessary for hyphal growth under filament-inducing conditions, is specifically important for agar invasion, hyphal extension, and formation of true septa in response to constitutive UME6 expression under non-filament-inducing conditions. HGC1-dependent inactivation of Rga2, a Cdc42 GTPase activating protein (GAP), also appears to be important for these processes. In response to filament-inducing conditions, HGC1 is induced prior to UME6 although UME6 controls the level and duration of HGC1 expression, which are likely to be important for hyphal extension. Interestingly, an epistasis analysis suggests that UME6 and HGC1 play distinct roles during early filament formation. These findings establish a link between a key regulator of filamentation and a downstream mechanism important for hyphal formation. In addition, this study demonstrates that a strain expressing constitutive high levels of UME6 provides a powerful strategy to specifically dissect downstream mechanisms important for hyphal development in the absence of complex filament-inducing conditions.
Candida albicans is an opportunistic fungal pathogen that exists as normal flora in healthy human bodies but causes life-threatening infections in immunocompromised patients. In addition to innate and adaptive immunities, hosts also resist microbial infections by developing a mechanism of “natural resistance” that maintains a low level of free iron to restrict the growth of invading pathogens. C. albicans must overcome this iron-deprived environment to cause infections. There are three types of iron-responsive transcriptional regulators in fungi; Aft1/Aft2 activators in yeast, GATA-type repressors in many fungi, and HapX/Php4 in Schizosaccharomyces pombe and Aspergillus species. In this study, we characterized the iron-responsive regulator Hap43, which is the C. albicans homolog of HapX/Php4 and is repressed by the GATA-type repressor Sfu1 under iron-sufficient conditions. We provide evidence that Hap43 is essential for the growth of C. albicans under low-iron conditions and for C. albicans virulence in a mouse model of infection. Hap43 was not required for iron acquisition under low-iron conditions. Instead, it was responsible for repression of genes that encode iron-dependent proteins involved in mitochondrial respiration and iron-sulfur cluster assembly. We also demonstrated that Hap43 executes its function by becoming a transcriptional repressor and accumulating in the nucleus in response to iron deprivation. Finally, we found a connection between Hap43 and the global corepressor Tup1 in low-iron-induced flavinogenesis. Taken together, our data suggest a complex interplay among Hap43, Sfu1, and Tup1 to coordinately regulate iron acquisition, iron utilization, and other iron-responsive metabolic activities.
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.
The opportunistic fungal pathogen Candida albicans is a part of the normal flora but it also causes systemic candidiasis if it reaches the bloodstream. Upon being phagocytized by macrophages, an important component of innate immunity, C. albicans rapidly upregulates a set of arginine biosynthetic genes. Arginine, urea, and CO2 induced hyphae in a density-dependent manner in wild-type, cph1/cph1, and rim101/rim101 strains but not in efg1/efg1 or cph1/cph1 efg1/efg1 strains. Arginase (Car1p) converts arginine to urea, which in turn is degraded by urea amidolyase (Dur1,2p) to produce CO2, a signal for hyphal switching. We used a dur1,2/dur1,2 mutant (KWN6) and the complemented strain, KWN8 (dur1,2/dur1,2::DUR1,2/DUR1,2) to study germ tube formation. KWN6 could not make germ tubes in the presence of arginine or urea but did in the presence of 5% CO2, which bypasses Dur1,2p. We also tested the effect of arginine on the interaction between the macrophage line RAW 264.7 and several strains of C. albicans. Arginine activated an Efg1p-dependent yeast-to-hypha switch, enabling wild-type C. albicans and KWN8 to escape from macrophages within 6 h, whereas KWN6 was defective in this regard. Additionally, two mutants that cannot synthesize arginine, BWP17 and SN152, were defective in making hyphae inside the macrophages, whereas the corresponding arginine prototrophs, DAY286 and SN87, formed germ tubes and escaped from macrophages. Therefore, metabolism of arginine by C. albicans controls hyphal switching and provides an important mechanism for escaping host defense.
Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps), involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap) family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the first defined mechanism behind the enhanced susceptibility of HIV+ individuals to oral candidiasis since the emergence of HIV.
In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.
Candida albicans is an opportunistic fungal pathogen that is found in the normal gastrointestinal flora of most healthy humans. However, under certain environmental conditions, it can become a life-threatening pathogen. The shift from commensal organism to pathogen is often correlated with the capacity to undergo morphogenesis. Indeed, under certain conditions, including growth at ambient temperature, the presence of serum or N-acetylglucosamine, neutral pH, and nutrient starvation, C. albicans can undergo reversible transitions from the yeast form to the mycelial form. This morphological plasticity reflects the interplay of various signal transduction pathways, either stimulating or repressing hyphal formation. In this review, we provide an overview of the different sensing and signaling pathways involved in the morphogenesis and pathogenesis of C. albicans. Where appropriate, we compare the analogous pathways/genes in Saccharomyces cerevisiae in an attempt to highlight the evolution of the different components of the two organisms. The downstream components of these pathways, some of which may be interesting antifungal targets, are also discussed.
Candida albicans is an opportunistic, fungal pathogen of humans that frequently causes superficial infections of oral and vaginal mucosal surfaces of debilitated and susceptible individuals. The organism is however, commonly encountered as a commensal in healthy individuals where it is a component of the normal microflora. The key determinant in the type of relationship that Candida has with its host is how it interacts with the epithelial surface it colonises. A delicate balance clearly exists between the potentially damaging effects of Candida virulence factors and the nature of the immune response elicited by the host. Frequently, it is changes in host factors that lead to Candida seemingly changing from a commensal to pathogenic existence. However, given the often reported heterogeneity in morphological and biochemical factors that exist between Candida species and indeed strains of C. albicans, it may also be the fact that colonising strains differ in the way they exploit resources to allow persistence at mucosal surfaces and as a consequence this too may affect the way Candida interacts with epithelial cells. The aim of this review is to provide an overview of some of the possible interactions that may occur between C. albicans and host epithelial surfaces that may in turn dictate whether Candida removal, its commensal persistence or infection follows.
oral microbiology; biofilm; virulence factors; pathogenesis