CO2 enhances the formation, nutrient scavenging and drug resistance properties of C. albicans biofilms.

C. albicans is the predominant human fungal pathogen and frequently colonises medical devices, such as voice prostheses, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is the elevation of CO2 levels, as observed in exhaled breath for example. However, despite the clear medical relevance, the effect of CO2 on C. albicans biofilm growth has not been investigated to date. Here we show that physiologically relevant CO2 elevation enhances each stage of the C. albicans biofilm-forming process: from attachment through maturation to dispersion. The effects of CO2 are mediated via the Ras/cAMP/PKA signalling pathway and the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown under elevated CO2 conditions also exhibit increased azole resistance, increased Sef1-dependent iron scavenging and enhanced glucose uptake to support their rapid growth. These findings suggest that C. albicans has evolved to utilise the CO2 signal to promote biofilm formation within the host. We investigate the possibility of targeting CO2-activated processes and propose 2-deoxyglucose as a drug that may be repurposed to prevent C. albicans biofilm formation on medical airway management implants. We thus characterise the mechanisms by which CO2 promotes C. albicans biofilm formation and suggest new approaches for future preventative strategies.

Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved.

The spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a > tenfold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. ± NO) and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO.

CO2 sensing in fungi: at the heart of metabolic signaling.

Adaptation to the changing environmental CO2 levels is essential for all living cells. In particular, microorganisms colonizing and infecting the human body are exposed to highly variable concentrations, ranging from atmospheric 0.04 to 5% and more in blood and specific host niches. Carbonic anhydrases are highly conserved metalloenzymes that enable fixation of CO2 by its conversion into bicarbonate. This process is not only crucial to ensure the supply of adequate carbon amounts for cellular metabolism, but also contributes to several signaling processes in fungi, including morphology and communication. The fungal specific carbonic anhydrase gene NCE103 is transcribed in response to CO2 availability. As recently shown, this regulation relies on the ATF/CREB transcription factor Cst6 and the AGC family protein kinase Sch9. Here, we review the regulatory mechanisms which control NCE103 expression in the model organism Saccharomyces cerevisiae and the pathogenic yeasts Candida albicans and Candida glabrata and discuss which additional factors might contribute in this novel CO2 sensing cascade.

Lipid Signaling via Pkh1/2 Regulates Fungal CO2 Sensing through the Kinase Sch9.

Adaptation to alternating CO2 concentrations is crucial for all organisms. Carbonic anhydrases-metalloenzymes that have been found in all domains of life-enable fixation of scarce CO2 by accelerating its conversion to bicarbonate and ensure maintenance of cellular metabolism. In fungi and other eukaryotes, the carbonic anhydrase Nce103 has been shown to be essential for growth in air (~0.04% CO2). Expression of NCE103 is regulated in response to CO2 availability. In Saccharomyces cerevisiae, NCE103 is activated by the transcription factor ScCst6, and in Candida albicans and Candida glabrata, it is activated by its homologues CaRca1 and CgRca1, respectively. To identify the kinase controlling Cst6/Rca1, we screened an S. cerevisiae kinase/phosphatase mutant library for the ability to regulate NCE103 in a CO2-dependent manner. We identified ScSch9 as a potential ScCst6-specific kinase, as the sch9Δ mutant strain showed deregulated NCE103 expression on the RNA and protein levels. Immunoprecipitation revealed the binding capabilities of both proteins, and detection of ScCst6 phosphorylation by ScSch9 in vitro confirmed Sch9 as the Cst6 kinase. We could show that CO2-dependent activation of Sch9, which is part of a kinase cascade, is mediated by lipid/Pkh1/2 signaling but not TORC1. Finally, we tested conservation of the identified regulatory cascade in the pathogenic yeast species C. albicans and C. glabrata Deletion of SCH9 homologues of both species impaired CO2-dependent regulation of NCE103 expression, which indicates a conservation of the CO2 adaptation mechanism among yeasts. Thus, Sch9 is a Cst6/Rca1 kinase that links CO2 adaptation to lipid signaling via Pkh1/2 in fungi. IMPORTANCE: All living organisms have to cope with alternating CO2 concentrations as CO2 levels range from very low in the atmosphere (0.04%) to high (5% and more) in other niches, including the human body. In fungi, CO2 is sensed via two pathways. The first regulates virulence in pathogenic yeast by direct activation of adenylyl cyclase. The second pathway, although playing a fundamental role in fungal metabolism, is much less understood. Here the transcription factor Cst6/Rca1 controls carbon homeostasis by regulating carbonic anhydrase expression. Upstream signaling in this pathway remains elusive. We identify Sch9 as the kinase controlling Cst6/Rca1 activity in yeast and demonstrate that this pathway is conserved in pathogenic yeast species, which highlights identified key players as potential pharmacological targets. Furthermore, we provide a direct link between adaptation to changing CO2 conditions and lipid/Pkh1/2 signaling in yeast, thus establishing a new signaling cascade central to metabolic adaptation.

Ras signalling in pathogenic yeasts.

The small GTPase Ras acts as a master regulator of growth, stress response and cell death in eukaryotic cells. The control of Ras activity is fundamental, as highlighted by the oncogenic properties of constitutive forms of Ras proteins. Ras also plays a crucial role in the pathogenicity of fungal pathogens where it has been found to regulate a number of adaptions required for virulence. The importance of Ras in fungal disease raises the possibility that it may provide a useful target for the development of new treatments at a time when resistance to available antifungals is increasing. New findings suggest that important regulatory sequences found within fungal Ras proteins that are not conserved may prove useful in the development of new antifungals. Here we review the roles of Ras protein function and signalling in the major human yeast pathogens Candida albicans and Cryptococcus neoformans and discuss the potential for targeting Ras as a novel approach to anti-fungal therapy.

Invasive rhino-orbito-cerebral mucormycosis in a diabetic patient - the need for prompt treatment.

Mucormycosis is a rare life threatening fungal infection predominately seen in immunocompromised or diabetic patients. The following case is of a known type II diabetic patient who presented with sepsis and sudden unilateral loss of vision secondary to infective rhino-orbito-cerebral mucormycosis. Treatment of the condition required extensive surgical intervention and medical management for a life saving outcome.

Candida biofilm formation on voice prostheses.

Laryngopharyngeal malignancy is treated with radiotherapy and/or surgery. When total laryngectomy is required, major laryngeal functions (phonation, airway control, swallowing and coughing) are affected. The insertion of a silicone rubber voice prosthesis in a surgically created tracheoesophageal puncture is the most effective method for voice rehabilitation. Silicone, as is the case with other synthetic materials such as polymethylmethacrylate, polyurethane, polyvinyl chloride, polypropylene and polystyrene, has the propensity to become rapidly colonized by micro-organisms (mainly Candida albicans) forming a biofilm, which leads to the failure of the devices. Silicone is used within voice prosthetic devices because of its flexible properties, which are essential for valve function. Valve failure, as well as compromising speech, may result in aspiration pneumonia, and repeated valve replacement may lead to either tract stenosis or insufficiency. Prevention and control of biofilm formation are therefore crucial for the lifespan of the prosthesis and promotion of tracheoesophageal tissue and lung health. To date, the mechanisms of biofilm formation on voice prostheses are not fully understood. Further studies are therefore required to identify factors influencing Candida biofilm formation. This review describes the factors known to influence biofilm formation on voice prostheses and current strategies employed to prolong their life by interfering with microbial colonization.

A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity.

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.

Carbonic anhydrase inhibitors: inhibition of the ß-class enzyme from the pathogenic yeast Candida glabrata with sulfonamides, sulfamates and sulfamides.

The fungal pathogen Candida glabrata encodes for a ß-carbonic anhydrase (CA, EC 4.2.1.1), CgNce103, recently discovered. Only anions have been investigated as CgNce103 inhibitors up until now. Here we report the first sulfonamides inhibition study of this enzyme. Simple sulfonamides showed weak or moderate CgNce103 inhibitory properties, whereas acetazolamide, and a series of 4-substituted ureido-benzene-sulfonamides, sulfamates and sulfamides showed effective CgNce103 inhibitory properties, with KIs in the range of 4.1-115 nM, being also ineffective as human CA II inhibitors. As there is significant resistance of C. glabrata clinical isolates to many classical antifungal agents, inhibition of the ß-CA from this organism may allow an interesting means of controlling the pathogen growth, eventually leading to antifungals with a novel mechanism of action.

Carbonic anhydrase regulation and CO(2) sensing in the fungal pathogen Candida glabrata involves a novel Rca1p ortholog.

Carbon dioxide (CO2) is a ubiquitous gas present at 0.0391% in atmospheric air and 5.5% in human blood. It forms part of numerous carboxylation and decarboxylation reactions carried out in every cell. Carbonic anhydrases (CA) enhance the hydration of CO2 to generate bicarbonate, which is subsequently used in cellular metabolism. In microorganisms, including the yeasts Candida albicans and Saccharomyces cerevisiae, inactivation of CA leads to a growth defect in air, which is complemented in an atmosphere enriched with CO2. In this study we characterize the CA from the fungal pathogen of humans Candida glabrata, CgNce103p, and report a comparable phenotype following its inactivation. Furthermore, we show that expression of the C. glabrata CA is strongly regulated by environmental CO2 at both the protein and transcript level. Similar to what we have previously reported for C. albicans and S. cerevisiae, C. glabrata CA regulation by CO2 is independent from the cAMP-PKA pathway and requires the novel bZIP transcription factor CgRca1p. We show that CgRca1p is an ortholog of the transcription factors Rca1p from C. albicans and Cst6p from S. cerevisiae and prove that CA induction in low CO2 involves the conserved DNA-binding motif TGACGTCA located on this C. glabrata promoter. However, in contrast to what is found in C. albicans CgRca1p expression itself is not affected by CO2. Although our results suggest a high level of similarity between the CO2 sensing pathways from C. glabrata, S. cerevisiae and C. albicans, they also point out significant intrinsic differences.

Functional characterization of the small heat shock protein Hsp12p from Candida albicans.

Hsp12p is considered to be a small heat shock protein and conserved among fungal species. To investigate the expression of this heat shock protein in the fungal pathogen Candida albicans we developed an anti-CaHsp12p antibody. We show that this protein is induced during stationary phase growth and under stress conditions including heat shock, osmotic, oxidative and heavy metal stress. Furthermore, we find that CaHsp12p expression is influenced by the quorum sensing molecule farnesol, the change of CO(2) concentration and pH. Notably we show that the key transcription factor Efg1p acts as a positive regulator of CaHsp12p in response to heat shock and oxidative stress and demonstrate that CaHsp12p expression is additionally modulated by Hog1p and the cAMP-PKA signaling pathway. To study the function of Hsp12p in C. albicans we generated a null mutant, in which all four CaHSP12 genes have been deleted. Phenotypic analysis of the strain shows that CaHSP12 is not essential for stress resistance, morphogenesis or virulence when tested in a Drosophila model of infection. However, when overexpressed, CaHSP12 significantly enhanced cell-cell adhesion, germ tube formation and susceptibility to azole antifungal agents whilst desensitizing C. albicans to the quorum sensing molecule farnesol.

The transcription factor Flo8 mediates CO2 sensing in the human fungal pathogen Candida albicans.

Physiological levels of CO(2) have a profound impact on prominent biological attributes of the major fungal pathogen of humans, Candida albicans. Elevated CO(2) induces filamentous growth and promotes white-to-opaque switching. However, the underlying molecular mechanisms of CO(2) sensing in C. albicans are insufficiently understood. Here we identify the transcription factor Flo8 as a key regulator of CO(2)-induced morphogenesis in C. albicans by screening a gene null mutant library. We show that Flo8 is required for CO(2)-induced white-to-opaque switching, as well as for filamentous growth. Ectopic expression of FLO8 hypersensitizes C. albicans cells to the elevated CO(2) levels. Furthermore, we demonstrate that CO(2) signaling in C. albicans involves two pathways: the already reported cAMP/protein kinase A and another major one that is unidentified. The two pathways converge on the transcription factor Flo8, which is the master regulator of CO(2) sensing in C. albicans and plays a critical role in regulation of white-to-opaque switching and filamentous growth. Our findings provide new insights into the understanding of CO(2) sensing in pathogenic fungi that have important implications for higher organisms.

Dithiocarbamates are strong inhibitors of the beta-class fungal carbonic anhydrases from Cryptococcus neoformans, Candida albicans and Candida glabrata.

A series of N-mono- and N,N-disubstituted dithiocarbamates have been investigated as inhibitors of three ß-carbonic anhydrases (CAs, EC 4.2.1.1) from the fungal pathogens Cryptococcus neoformans, Candida albicans and Candida glabrata, that is, Can2, CaNce103 and CgNce103, respectively. These enzymes were inhibited with efficacies between the subnanomolar to the micromolar range, depending on the substitution pattern at the nitrogen atom from the dithiocarbamate zinc-binding group. This new class of ß-CA inhibitors may have the potential for developing antifungal agents with a diverse mechanism of action compared to the clinically used drugs for which drug resistance was reported, and may also explain the efficacy of dithiocarbamates as agricultural antifungal agents.

The bZIP transcription factor Rca1p is a central regulator of a novel CO2 sensing pathway in yeast.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO(2) concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO(2) and identify the bZIP transcription factor Rca1p as the first CO(2) regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO(2) build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO(2) sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO(2) availability in environments as diverse as the phagosome, yeast communities or liquid culture.

Communication in fungi.

We will discuss fungal communication in the context of fundamental biological functions including mating, growth, morphogenesis, and the regulation of fungal virulence determinants. We will address intraspecies but also interkingdom signaling by systematically discussing the sender of the message, the molecular message, and receiver. Analyzing communication shows the close coevolution of fungi with organisms present in their environment giving insights into multispecies communication. A better understanding of the molecular mechanisms underlying microbial communication will promote our understanding of the "fungal communicome."

Candida albicans developmental regulation: adenylyl cyclase as a coincidence detector of parallel signals.

In the healthy individual, Candida albicans is frequently found as a harmless commensal residing in the gastrointestinal tract. However, in the compromised patient, C. albicans may invade the body and cause disease that is associated with poor prognosis and high mortality. The C. albicans adenylyl cyclase, Cyr1, which is required for virulence in animal models, regulates three developmental programs, including invasive filamentous growth, phenotypic switching to a mating-competent cell type, and biofilm formation. Evidence suggests that Cyr1 controls these phenotypes in response to various environmental cues that are present within microbial populations. Additionally, C. albicans secretes an autoregulatory molecule, farnesol, which was recently shown to directly inhibit Cyr1 activity. Below, we summarize recent advances in our understanding of Cyr1-regulated development and discuss the multiple inputs known to positively and negatively regulate cAMP synthesis. We discuss the possibility that Cyr1 acts as a coincidence detector that tightly regulates fungal development in response to parallel environmental stimuli, and highlight ways in which this might occur.

The quorum-sensing molecules farnesol/homoserine lactone and dodecanol operate via distinct modes of action in Candida albicans.

Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C(12)-homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C(12)-homoserine lactone, may be used by other quorum-sensing molecules.

Carbonic anhydrase inhibitors. Inhibition of the ß-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with branched aliphatic/aromatic carboxylates and their derivatives.

The inhibition of the ß-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with a series of 25 branched aliphatic and aromatic carboxylates has been investigated. Human isoforms hCA I and II were also included in the study for comparison. Aliphatic carboxylates were generally millimolar hCA I and II inhibitors and low micromolar/submicromolar ß-CA inhibitors. Aromatic carboxylates were micromolar inhibitors of the four enzymes but some of them showed low nanomolar activity against the fungal pathogenic enzymes. 4-Hydroxy- and 4-methoxy-benzoate inhibited Can2 with K(I)s of 9.5-9.9 nM. The methyl esters, hydroxamates, hydrazides and carboxamides of some of these derivatives were also effective inhibitors of the α- and ß-CAs investigated here.

Natural product-based phenols as novel probes for mycobacterial and fungal carbonic anhydrases.

In order to discover novel probes that may help in the investigation and control of infectious diseases through a new mechanism of action, we have evaluated a library of phenol-based natural products (NPs) for enzyme inhibition against four recently characterized pathogen ß-family carbonic anhydrases (CAs). These include CAs from Mycobacterium tuberculosis, Candida albicans, and Cryptococcus neoformans as well as α-family human CA I and CA II for comparison. Many of the NPs selectively inhibited the mycobacterial and fungal ß-CAs, with the two best performing compounds displaying submicromolar inhibition with a preference for fungal over human CA inhibition of more than 2 orders of magnitude. These compounds provide the first example of non-sulfonamide inhibitors that display ß over α CA enzyme selectivity. Structural characterization of the library compounds in complex with human CA II revealed a novel binding mode whereby a methyl ester interacts via a water molecule with the active site zinc.

CO(2) acts as a signalling molecule in populations of the fungal pathogen Candida albicans.

When colonising host-niches or non-animated medical devices, individual cells of the fungal pathogen Candida albicans expand into significant biomasses. Here we show that within such biomasses, fungal metabolically generated CO(2) acts as a communication molecule promoting the switch from yeast to filamentous growth essential for C. albicans pathology. We find that CO(2)-mediated intra-colony signalling involves the adenylyl cyclase protein (Cyr1p), a multi-sensor recently found to coordinate fungal responses to serum and bacterial peptidoglycan. We further identify Lys 1373 as essential for CO(2)/bicarbonate regulation of Cyr1p. Disruption of the CO(2)/bicarbonate receptor-site interferes selectively with C. albicans filamentation within fungal biomasses. Comparisons between the Drosophila melanogaster infection model and the mouse model of disseminated candidiasis, suggest that metabolic CO(2) sensing may be important for initial colonisation and epithelial invasion. Our results reveal the existence of a gaseous Candida signalling pathway and its molecular mechanism and provide insights into an evolutionary conserved CO(2)-signalling system.