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1.  Altered Dynamics of Candida albicans Phagocytosis by Macrophages and PMNs When Both Phagocyte Subsets Are Present 
mBio  2013;4(6):e00810-13.
ABSTRACT
An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection.
IMPORTANCE
Extensive work investigating fungal cell phagocytosis by macrophages and PMNs of the innate immune system has been carried out. These studies have been informative but have examined this phenomenon only when one phagocyte subset is present. The current study employed live-cell video microscopy to break down C. albicans phagocytosis into its component parts and examine the effect of a single phagocyte subset, versus a mixed phagocyte population, on these individual stages. Through this approach, we identified that the rate of fungal cell engulfment and rate of phagocyte killing altered significantly when both macrophages and PMNs were incubated in coculture with C. albicans compared to the rate of either phagocyte subset incubated alone with the fungus. This research highlights the significance of studying pathogen-host cell interactions with a combination of phagocytes in order to gain a greater understanding of the interactions that occur between cells of the host immune system in response to fungal invasion.
doi:10.1128/mBio.00810-13
PMCID: PMC3809565  PMID: 24169578
2.  A Drug-Sensitive Genetic Network Masks Fungi from the Immune System 
PLoS Pathogens  2006;2(4):e35.
Fungal pathogens can be recognized by the immune system via their β-glucan, a potent proinflammatory molecule that is present at high levels but is predominantly buried beneath a mannoprotein coat and invisible to the host. To investigate the nature and significance of “masking” this molecule, we characterized the mechanism of masking and consequences of unmasking for immune recognition. We found that the underlying β-glucan in the cell wall of Candida albicans is unmasked by subinhibitory doses of the antifungal drug caspofungin, causing the exposed fungi to elicit a stronger immune response. Using a library of bakers' yeast (Saccharomyces cerevisiae) mutants, we uncovered a conserved genetic network that is required for concealing β-glucan from the immune system and limiting the host response. Perturbation of parts of this network in the pathogen C. albicans caused unmasking of its β-glucan, leading to increased β-glucan receptor-dependent elicitation of key proinflammatory cytokines from primary mouse macrophages. By creating an anti-inflammatory barrier to mask β-glucan, opportunistic fungi may promote commensal colonization and have an increased propensity for causing disease. Targeting the widely conserved gene network required for creating and maintaining this barrier may lead to novel broad-spectrum antimycotics.
Synopsis
Opportunistic fungal pathogens such as Candida albicans often cause fatal infections in patients with a compromised immune system. Unfortunately, current drugs often fail to halt fungal disease, are ineffective against drug-resistant strains, and have severe side effects. Despite the clear clinical significance of fungal infections, it is still not understood how fungi are recognized by the immune system. Candida has high levels of the structural molecule β-glucan in its cell wall, but the majority of its β-glucan is masked by a mannoprotein coat and is therefore invisible to the immune system. Masking of β-glucan may be a fungal virulence factor, because exposed β-glucan provokes a proinflammatory response that is important for mounting an effective immune response against the fungus and clearing the infection. By surveying the genome of the model fungus Saccharomyces cerevisiae (bakers' yeast), the authors discovered a genetic network required for masking β-glucan from the immune system. Mutation of genes in this network in C. albicans caused unmasking of β-glucan and an increased immune response to the fungus. The authors also found that sublethal doses of the antifungal drug caspofungin cause unmasking and lead to a greater immune response. Drugs targeting this fungally conserved masking network may provide new tools to fight fungal infections.
doi:10.1371/journal.ppat.0020035
PMCID: PMC1447670  PMID: 16652171
3.  CO2 Acts as a Signalling Molecule in Populations of the Fungal Pathogen Candida albicans 
PLoS Pathogens  2010;6(11):e1001193.
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 CO2 acts as a communication molecule promoting the switch from yeast to filamentous growth essential for C. albicans pathology. We find that CO2-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 CO2/bicarbonate regulation of Cyr1p. Disruption of the CO2/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 CO2 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 CO2-signalling system.
Author Summary
Pathogenic microorganisms can produce a variety of secondary metabolites and signalling molecules which can affect the host, or provide them with a selective advantage against competing commensal organisms. We demonstrate that gaseous, metabolically generated CO2 can serve as a signalling molecule to enhance the organism's virulence during infection establishment by using the fungal pathogen Candida albicans as a model. Furthermore, we identified a CO2 receptor site within the catalytic domain of the soluble adenylyl cyclase, Cyr1p, which is critical for CO2 sensing and hence virulence of the organism. CO2 sensing is conserved in a variety of pathogenic species, and increased levels have been shown to suppress the host's immune system. Thus, CO2 sensing may represent a mechanism to enhance C. albicans virulence when the host's immune system is suppressed.
doi:10.1371/journal.ppat.1001193
PMCID: PMC2987819  PMID: 21124988
4.  A Novel Pseudopodial Component of the Dendritic Cell Anti-Fungal Response: The Fungipod 
PLoS Pathogens  2010;6(2):e1000760.
Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the “fungipod”, formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7±5.6 µm long and 1.8±0.67 µm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225±55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen Candida parapsilosis induces DC fungipod formation strongly, but the response is species specific since the related fungal pathogens Candida tropicalis and Candida albicans induce very few and no fungipods, respectively. Our findings show that fungipods are dynamic actin-driven cellular structures involved in fungal recognition by DC. They may promote yeast particle phagocytosis by DC and are a specific response to large (i.e., 5 µm) particulate ligands. Our work also highlights the importance of this novel protrusive structure to innate immune recognition of medically significant Candida yeasts in a species specific fashion.
Author Summary
Yeasts are normal microbial commensals of humans and a significant source of opportunistic infections, especially in immunocompromised individuals. We report a novel cellular protrusive structure, the fungipod, which participates in the host-microbe interaction between human immature dendritic cells (DC) and yeasts. The fungipod's structure is based on and propelled by a robust process of local actin cytoskeleton growth at the DC-yeast contact site, and this cytoskeletal remodeling results in a durable tubular structure over 10 µm long connecting the dorsal DC membrane and yeast. The fungal cell wall polysaccharides mannan and chitin trigger fungipod formation by stimulating the carbohydrate pattern recognition receptor CD206. Fungipods are part of a specific response to large particulate objects (i.e., yeast), and they may promote the human immature DC's relatively poor phagocytosis of yeast. The human fungal pathogen, Candida parapsilosis, induces a strong fungipod response from DC, and this response is highly species specific since the related pathogens Candida albicans and Candida tropicalis induce fungipods rarely. Our work highlights a novel cell biological element of fungal recognition by the innate immune system.
doi:10.1371/journal.ppat.1000760
PMCID: PMC2820528  PMID: 20169183
5.  Serological Profiling of a Candida albicans Protein Microarray Reveals Permanent Host-Pathogen Interplay and Stage-Specific Responses during Candidemia 
PLoS Pathogens  2010;6(3):e1000827.
Candida albicans in the immunocompetent host is a benign member of the human microbiota. Though, when host physiology is disrupted, this commensal-host interaction can degenerate and lead to an opportunistic infection. Relatively little is known regarding the dynamics of C. albicans colonization and pathogenesis. We developed a C. albicans cell surface protein microarray to profile the immunoglobulin G response during commensal colonization and candidemia. The antibody response from the sera of patients with candidemia and our negative control groups indicate that the immunocompetent host exists in permanent host-pathogen interplay with commensal C. albicans. This report also identifies cell surface antigens that are specific to different phases (i.e. acute, early and mid convalescence) of candidemia. We identified a set of thirteen cell surface antigens capable of distinguishing acute candidemia from healthy individuals and uninfected hospital patients with commensal colonization. Interestingly, a large proportion of these cell surface antigens are involved in either oxidative stress or drug resistance. In addition, we identified 33 antigenic proteins that are enriched in convalescent sera of the candidemia patients. Intriguingly, we found within this subset an increase in antigens associated with heme-associated iron acquisition. These findings have important implications for the mechanisms of C. albicans colonization as well as the development of systemic infection.
Author Summary
Candida albicans has both a benign and pathogenic association with the human host. Previous to this study, little was known in regard to how the host humoral system responds to the commensal colonization of C. albicans, as well as the development of hematogenously disseminated candidiasis. We show using a C. albicans cell surface protein microarray that the immunocompetent host exists in permanent host-pathogen interplay with commensal C. albicans, and undergoes stage-specific antibody responses as the yeast transitions from a benign microbe to an opportunistic fungal pathogen. Also identified were serological signatures specific for acute and convalescent stages of candidemia. Our findings provide new insight in the characterization of potential serodiagnostic antigens and vaccine candidates to the opportunistic pathogen C. albicans.
doi:10.1371/journal.ppat.1000827
PMCID: PMC2845659  PMID: 20361054
6.  A Virtual Infection Model Quantifies Innate Effector Mechanisms and Candida albicans Immune Escape in Human Blood 
PLoS Computational Biology  2014;10(2):e1003479.
Candida albicans bloodstream infection is increasingly frequent and can result in disseminated candidiasis associated with high mortality rates. To analyze the innate immune response against C. albicans, fungal cells were added to human whole-blood samples. After inoculation, C. albicans started to filament and predominantly associate with neutrophils, whereas only a minority of fungal cells became attached to monocytes. While many parameters of host-pathogen interaction were accessible to direct experimental quantification in the whole-blood infection assay, others were not. To overcome these limitations, we generated a virtual infection model that allowed detailed and quantitative predictions on the dynamics of host-pathogen interaction. Experimental time-resolved data were simulated using a state-based modeling approach combined with the Monte Carlo method of simulated annealing to obtain quantitative predictions on a priori unknown transition rates and to identify the main axis of antifungal immunity. Results clearly demonstrated a predominant role of neutrophils, mediated by phagocytosis and intracellular killing as well as the release of antifungal effector molecules upon activation, resulting in extracellular fungicidal activity. Both mechanisms together account for almost of C. albicans killing, clearly proving that beside being present in larger numbers than other leukocytes, neutrophils functionally dominate the immune response against C. albicans in human blood. A fraction of C. albicans cells escaped phagocytosis and remained extracellular and viable for up to four hours. This immune escape was independent of filamentation and fungal activity and not linked to exhaustion or inactivation of innate immune cells. The occurrence of C. albicans cells being resistant against phagocytosis may account for the high proportion of dissemination in C. albicans bloodstream infection. Taken together, iterative experiment–model–experiment cycles allowed quantitative analyses of the interplay between host and pathogen in a complex environment like human blood.
Author Summary
Candida albicans is the most important fungal pathogen in nosocomial bloodstream infections. So far little is known about the interplay of different cellular and non-cellular immune mechanisms mediating the protective response against C. albicans in blood. The in vivo scenario of C. albicans infection can be mimicked by human whole-blood infection assays to analyze the innate immune response against this pathogen. These experiments reveal the time-evolution of certain mechanisms while leaving the values of other quantities in the dark. To shed light on quantities that are not experimentally accessible, we exploited the descriptive and predictive power of mathematical models to estimate these parameters. The combination of experiment and theory enabled us to identify and quantify the main course of the immune response against C. albicans in human blood. We quantified the central role of neutrophils in the defence against this fungal pathogen, both directly by phagocytosis and indirectly by secreting antimicrobial factors inducing extracellular killing. Other findings include the distribution of C. albicans cells in neutrophils and monocytes as well as the immune escape of C. albicans cells in the course of infection.
doi:10.1371/journal.pcbi.1003479
PMCID: PMC3930496  PMID: 24586131
7.  Cell Cycle-Independent Phospho-Regulation of Fkh2 during Hyphal Growth Regulates Candida albicans Pathogenesis 
PLoS Pathogens  2015;11(1):e1004630.
The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Although previous gene-knockout studies have identified many factors involved in this transformation, it remains unclear how these factors are regulated to coordinate the switch. Investigating morphogenetic control by post-translational phosphorylation has generated important regulatory insights into this process, especially focusing on coordinated control by the cyclin-dependent kinase Cdc28. Here we have identified the Fkh2 transcription factor as a regulatory target of both Cdc28 and the cell wall biosynthesis kinase Cbk1, in a role distinct from its conserved function in cell cycle progression. In stationary phase yeast cells 2D gel electrophoresis shows that there is a diverse pool of Fkh2 phospho-isoforms. For a short window on hyphal induction, far before START in the cell cycle, the phosphorylation profile is transformed before reverting to the yeast profile. This transformation does not occur when stationary phase cells are reinoculated into fresh medium supporting yeast growth. Mass spectrometry and mutational analyses identified residues phosphorylated by Cdc28 and Cbk1. Substitution of these residues with non-phosphorylatable alanine altered the yeast phosphorylation profile and abrogated the characteristic transformation to the hyphal profile. Transcript profiling of the phosphorylation site mutant revealed that the hyphal phosphorylation profile is required for the expression of genes involved in pathogenesis, host interaction and biofilm formation. We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes. Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity. Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity.
Author Summary
The fungus Candida albicans is a commensal in the human microbiota, responsible for superficial infections such as oral and vaginal thrush. However, it can become highly virulent, causing life-threatening systemic candidemia in severely immunocompromised patients, including those taking immunosuppressive drugs for transplantation, sufferers of AIDS and neutropenia, and individuals undergoing chemotherapy or at extremes of age. With a rapidly increasing ageing population worldwide, C. albicans and other fungal pathogens will become more prevalent, demanding a greater understanding of their pathogenesis for the development of effective therapeutics. Fungal pathogenicity requires a coordinated change in the pattern of gene expression orchestrated by a set of transcription factors. Here we have discovered that a transcription factor, Fkh2, is modified by phosphorylation under the control of the kinases Cdc28 and Cbk1 in response to conditions that activate virulence factor expression. Fkh2 is involved in a wide variety of cellular processes including cell proliferation, but this phosphorylation endows it with a specialized function in promoting the expression of genes required for tissue invasion, biofilm formation, and pathogenesis in the host. This study highlights the role of protein phosphorylation in regulating pathogenesis and furthers our understanding of the pathogenic switch in this important opportunistic fungal pathogen.
doi:10.1371/journal.ppat.1004630
PMCID: PMC4305328  PMID: 25617770
8.  Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells 
PLoS Biology  2007;5(3):e64.
Candida albicans is the most common cause of hematogenously disseminated and oropharyngeal candidiasis. Both of these diseases are characterized by fungal invasion of host cells. Previously, we have found that C. albicans hyphae invade endothelial cells and oral epithelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the fungal surface protein and host cell receptors that mediate this process. We found that the C. albicans Als3 is required for the organism to be endocytosed by human umbilical vein endothelial cells and two different human oral epithelial lines. Affinity purification experiments with wild-type and an als3Δ/als3Δ mutant strain of C. albicans demonstrated that Als3 was required for C. albicans to bind to multiple host cell surface proteins, including N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. Furthermore, latex beads coated with the recombinant N-terminal portion of Als3 were endocytosed by Chinese hamster ovary cells expressing human N-cadherin or E-cadherin, whereas control beads coated with bovine serum albumin were not. Molecular modeling of the interactions of the N-terminal region of Als3 with the ectodomains of N-cadherin and E-cadherin indicated that the binding parameters of Als3 to either cadherin are similar to those of cadherin–cadherin binding. Therefore, Als3 is a fungal invasin that mimics host cell cadherins and induces endocytosis by binding to N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. These results uncover the first known fungal invasin and provide evidence that C. albicans Als3 is a molecular mimic of human cadherins.
Author Summary
The fungus Candida albicans is usually a harmless colonizer of human mucosal surfaces. In the mouth, it can cause oropharyngeal candidiasis, also called thrush. In hospitalized and immunocompromised patients, C. albicans can enter the blood stream and be carried throughout the body to cause a disseminated infection, which is associated with a mortality rate of up to 40%. The organism invades the epithelial cell lining of the mouth during oropharyngeal candidiasis and invades the endothelial cell lining of the blood vessels during disseminated candidiasis. We discovered that Als3, a protein expressed on the surface of C. albicans, is required for this invasion process. Cadherins on the surface of human cells normally bind other cadherins for adhesion and signaling; however, we found that Als3 also binds to cadherins on endothelial cells and oral epithelial cells, and this binding induces these host cells to take up the fungus. The structure of Als3 is predicted to be quite similar to that of the two cadherins studied, and the parameters of the binding of Als3 to either cadherin are similar to those of cadherin–cadherin binding. These results suggest that Als3 is a functional and structural mimic of human cadherins, and provide new insights into how C. albicans invades host cells.
Als3 aids the invasion of the fungal pathogenCandida albicans into human host cells by mimicking human cadherins to induce endocytosis.
doi:10.1371/journal.pbio.0050064
PMCID: PMC1802757  PMID: 17311474
9.  Dynamic, Morphotype-Specific Candida albicans β-Glucan Exposure during Infection and Drug Treatment 
PLoS Pathogens  2008;4(12):e1000227.
Candida albicans, a clinically important dimorphic fungal pathogen that can evade immune attack by masking its cell wall β-glucan from immune recognition, mutes protective host responses mediated by the Dectin-1 β-glucan receptor on innate immune cells. Although the ability of C. albicans to switch between a yeast- or hyphal-form is a key virulence determinant, the role of each morphotype in β-glucan masking during infection and treatment has not been addressed. Here, we show that during infection of mice, the C. albicans β-glucan is masked initially but becomes exposed later in several organs. At all measured stages of infection, there is no difference in β-glucan exposure between yeast-form and hyphal cells. We have previously shown that sub-inhibitory doses of the anti-fungal drug caspofungin can expose β-glucan in vitro, suggesting that the drug may enhance immune activity during therapy. This report shows that caspofungin also mediates β-glucan unmasking in vivo. Surprisingly, caspofungin preferentially unmasks filamentous cells, as opposed to yeast form cells, both in vivo and in vitro. The fungicidal activity of caspofungin in vitro is also filament-biased, as corroborated using yeast-locked and hyphal-locked mutants. The uncloaking of filaments is not a general effect of anti-fungal drugs, as another anti-fungal agent does not have this effect. These results highlight the advantage of studying host–pathogen interaction in vivo and suggest new avenues for drug development.
Author Summary
Candida is a common human commensal but disseminated candidiasis is a serious clinical problem, especially among immunocompromised patients. The innate immune system controls Candida infection, in part through the germline-encoded β-glucan receptor Dectin-1. However, during in vitro growth, Candida albicans mutes Dectin-1 recognition by cloaking its β-glucan underneath a layer of mannan. Bridging these two seemingly contradictory observations, we demonstrate that C. albicans masks β-glucan early during infection, but it becomes exposed later, allowing Dectin-1 to recognize the fungi and mediate immunity. Remarkably, treatment of mice with sub-therapeutic doses of the antifungal drug caspofungin causes exposure of β-glucan on C. albicans even when it would not be exposed naturally. We introduce a new technique for monitoring of epitope exposure during infection, which can be used to monitor the availability of any epitope for immune recognition. This technique allowed us to show that natural unmasking of β-glucan is not morphotype-specific, but drug-mediated unmasking is biased towards the invasive filamentous form of C. albicans. These results highlight the unexplored area of dynamic epitope exposure during infection and therapy, which might be targetable to enhance immune recognition and fungal clearance.
doi:10.1371/journal.ppat.1000227
PMCID: PMC2587227  PMID: 19057660
10.  Candida albicans Scavenges Host Zinc via Pra1 during Endothelial Invasion 
PLoS Pathogens  2012;8(6):e1002777.
The ability of pathogenic microorganisms to assimilate essential nutrients from their hosts is critical for pathogenesis. Here we report endothelial zinc sequestration by the major human fungal pathogen, Candida albicans. We hypothesised that, analogous to siderophore-mediated iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this essential metal. We postulated that such a “zincophore” system would consist of a secreted factor with zinc-binding properties, which can specifically reassociate with the fungal cell surface. In silico analysis of the C. albicans secretome for proteins with zinc binding motifs identified the pH-regulated antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited zinc binding properties in vitro. Deletion of PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and specifically blocked host cell damage in the absence of exogenous zinc. Phylogenetic analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated physical interaction between Pra1 and Zrt1 and we confirmed this experimentally by demonstrating that Zrt1 was essential for binding of soluble Pra1 to the cell surface of C. albicans. Therefore, we have identified a novel metal acquisition system consisting of a secreted zinc scavenger (“zincophore”), which reassociates with the fungal cell. Furthermore, functional similarities with phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition loci have been independently selected during evolution.
Author Summary
The capacity of disease-causing microbes to acquire nutrients from their host is one of the most fundamental aspects of infection. Host organisms therefore restrict microbial access to certain key nutrients in a process known as nutritional immunity. Recently, it was found that infected vertebrates sequester zinc from invading microorganisms to control infection. Therefore, the mechanisms of microbial zinc acquisition represent potential virulence attributes. Here we report the molecular mechanism of host-derived zinc acquisition by the major human fungal pathogen, Candida albicans. We show that C. albicans utilises a secreted protein, the pH-regulated antigen 1 (Pra1), to bind zinc from its environment. Pra1 then reassociates with the fungal cell via a syntenically encoded (genetically-linked) membrane transporter (Zrt1) to acquire this essential metal. Deletion of PRA1 prevented utilisation of host zinc and damage of host cells in the absence of exogenous zinc. Finally, we demonstrate that this zinc-scavenging locus arose in an ancient fungal lineage and remains conserved in many contemporary species. Syntenically arranged zinc acquisition systems have evolved independently in the fungal and bacterial kingdoms, suggesting that such an arrangement is evolutionary beneficial for microorganisms.
doi:10.1371/journal.ppat.1002777
PMCID: PMC3386192  PMID: 22761575
11.  Interactions of Candida albicans with host epithelial surfaces 
Journal of Oral Microbiology  2013;5:10.3402/jom.v5i0.22434.
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.
doi:10.3402/jom.v5i0.22434
PMCID: PMC3805843  PMID: 24155995
oral microbiology; biofilm; virulence factors; pathogenesis
12.  Bruton's Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent Phagocytosis of Candida albicans in Macrophages 
PLoS Pathogens  2013;9(6):e1003446.
Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.
Author Summary
The opportunistic yeast Candida albicans is a commensal organism of the human digestive tract, but also the most common cause of human fungal infections. Phagocytosis, the process by which innate immune cells engulf pathogens, is vital to prevent C. albicans infections. The major phagocytic receptor involved in anti-fungal immunity is Dectin-1. We identify two new interacting proteins of Dectin-1 in macrophages: Bruton's Tyrosine Kinase (BTK) and Vav1. In the course of phagocytosis, different phosphoinositides (PIs) are formed in the phagosomal membrane to allow the recruitment of proteins equipped with specialized lipid-interaction domains. We show that BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 at the phagocytic cup, but not with diacylglycerol (DAG), which marks more mature phagosomal membranes. Inhibition of BTK affects the production of DAG and the recruitment of DAG-interacting proteins. BTK and Vav1 are essential for C. albicans immune responses, as BTK- or Vav1-deficient macrophages show reduced uptake of C. albicans and BTK- or Vav1-deficient deficient mice are more susceptible to systemic C. albicans infection. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.
doi:10.1371/journal.ppat.1003446
PMCID: PMC3694848  PMID: 23825946
13.  Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans 
mBio  2013;4(6):e00542-13.
ABSTRACT
Candida albicans invades endothelial cells by binding to N-cadherin and other cell surface receptors. This binding induces rearrangement of endothelial cell actin microfilaments, which results in the formation of pseudopods that surround the organism and pull it into the endothelial cell. Here, we investigated the role of endothelial cell septin 7 (SEPT7) in the endocytosis of C. albicans hyphae. Using confocal microscopy, we determined that SEPT7 accumulated with N-cadherin and actin microfilaments around C. albicans as it was endocytosed by endothelial cells. Affinity purification studies indicated that a complex containing N-cadherin and SEPT7 was recruited by C. albicans and that formation of this complex around C. albicans was mediated by the fungal Als3 and Ssa1 invasins. Knockdown of N-cadherin by small interfering RNA (siRNA) reduced recruitment of SEPT7 to C. albicans, suggesting that N-cadherin functions as a link between SEPT7 and the fungus. Also, depolymerization of actin microfilaments with cytochalasin D decreased the association between SEPT7 and N-cadherin and inhibited recruitment of both SEPT7 and N-cadherin to C. albicans, indicating the necessity of an intact cytoskeleton in the functional interaction between SEPT7 and N-cadherin. Importantly, knockdown of SEPT7 decreased accumulation of N-cadherin around C. albicans in intact endothelial cells and reduced binding of N-cadherin to this organism, as revealed by the affinity purification assay. Furthermore, SEPT7 knockdown significantly inhibited the endocytosis of C. albicans. Therefore, in response to C. albicans infection, SEPT7 forms a complex with endothelial cell N-cadherin, is required for normal accumulation of N-cadherin around C. albicans hyphae, and is necessary for maximal endocytosis of the organism.
IMPORTANCE
During hematogenously disseminated infection, Candida albicans invades the endothelial cell lining of the blood vessels to invade the deep tissues. C. albicans can invade endothelial cells by inducing its own endocytosis, which is triggered when the C. albicans Als3 and Ssa1 invasins bind to N-cadherin on the endothelial cell surface. How this binding induces endocytosis is incompletely understood. Septins are intracellular GTP-binding proteins that influence the function and localization of cell surface proteins. We found that C. albicans Als3 and Ssa1 bind to a complex containing N-cadherin and septin 7, which in turn interacts with endothelial cell microfilaments, thereby inducing endocytosis of the organism. The key role of septin 7 in governing receptor-mediated endocytosis is likely relevant to host cell invasion by other microbial pathogens, in addition to C. albicans.
doi:10.1128/mBio.00542-13
PMCID: PMC3870263  PMID: 24345743
14.  Hsp90 Governs Echinocandin Resistance in the Pathogenic Yeast Candida albicans via Calcineurin 
PLoS Pathogens  2009;5(7):e1000532.
Candida albicans is the leading fungal pathogen of humans, causing life-threatening disease in immunocompromised individuals. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. We previously established that the molecular chaperone Hsp90, which regulates the form and function of diverse client proteins, potentiates resistance to the azoles in C. albicans and in the model yeast Saccharomyces cerevisiae. Genetic studies in S. cerevisiae revealed that Hsp90's role in azole resistance is to enable crucial cellular responses to the membrane stress exerted by azoles via the client protein calcineurin. Here, we demonstrate that Hsp90 governs cellular circuitry required for resistance to the only new class of antifungals to reach the clinic in decades, the echinocandins, which inhibit biosynthesis of a critical component of the fungal cell wall. Pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to the echinocandins and created a fungicidal combination. Compromising calcineurin function phenocopied compromising Hsp90 function. We established that calcineurin is an Hsp90 client protein in C. albicans: reciprocal co-immunoprecipitation validated physical interaction; Hsp90 inhibition blocked calcineurin activation; and calcineurin levels were depleted upon genetic reduction of Hsp90. The downstream effector of calcineurin, Crz1, played a partial role in mediating calcineurin-dependent stress responses activated by echinocandins. Hsp90's role in echinocandin resistance has therapeutic potential given that genetic compromise of C. albicans HSP90 expression enhanced the efficacy of an echinocandin in a murine model of disseminated candidiasis. Our results identify the first Hsp90 client protein in C. albicans, establish an entirely new role for Hsp90 in mediating resistance to echinocandins, and demonstrate that targeting Hsp90 provides a promising therapeutic strategy for the treatment of life-threatening fungal disease.
Author Summary
Fungal pathogens pose a serious threat to people with compromised immune systems. Chief among the opportunistic fungal pathogens is Candida albicans. Treatment of C. albicans infections remains challenging because there are very few effective drugs and the pathogen has evolved many strategies to survive drug exposure. The echinocandins are the only new class of antifungal drug to reach the clinic in decades and they block biosynthesis of an essential component of the fungal cell wall. We discovered that the molecular chaperone Hsp90, which is required for its client proteins in the cell to fold and function, governs the ability of C. albicans to survive exposure to echinocandins. Compromising Hsp90 function renders the echinocandins more effective at killing C. albicans laboratory strains and clinical isolates. Hsp90 orchestrates the crucial responses to cell wall stress exerted by the echinocandins by enabling the function of its client protein calcineurin, which allows the fungus to survive otherwise lethal conditions. Our results suggest that compromising Hsp90 function provides a powerful and much-needed strategy to render existing antifungal drugs more effective in the treatment of life-threatening fungal infections.
doi:10.1371/journal.ppat.1000532
PMCID: PMC2712069  PMID: 19649312
15.  The Pathogen Candida albicans Hijacks Pyroptosis for Escape from Macrophages 
mBio  2014;5(2):e00003-14.
ABSTRACT
The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection.
IMPORTANCE
Upon phagocytosis by macrophages, Candida albicans can transition to the hyphal form, which causes macrophage death and enables fungal escape. The current model is that the highly polarized growth of hyphae results in macrophage piercing. This model is challenged by recent reports of C. albicans mutants that form hyphae of wild-type morphology but are defective in killing macrophages. We show that C. albicans causes macrophage cell death by at least two mechanisms. Phase 1 killing (first 6 to 8 h) depends on the activation of the pyroptotic programmed host cell death by fungal hyphae. Phase 2 (up to 24 h) is rapid and depends on robust hyphal formation but is independent of pyroptosis. Our data provide a new model for how the interplay between fungal morphogenesis and activation of a host cell death pathway mediates macrophage killing by C. albicans hyphae.
doi:10.1128/mBio.00003-14
PMCID: PMC3977349  PMID: 24667705
16.  Variation in Candida albicans EFG1 Expression Enables Host-Dependent Changes in Colonizing Fungal Populations 
mBio  2012;3(4):e00117-12.
ABSTRACT
To understand differences in host-Candida albicans interactions that occur during colonization of healthy or compromised hosts, production of phenotypic variants and colonization of healthy or immunodeficient mice by C. albicans were studied. We showed that activity of the transcription factor Efg1p exhibited cell-to-cell variability and identified Efg1p as a major regulator of colonization. In C. albicans populations colonizing the murine gastrointestinal tract, average expression of EFG1 differed depending on the immune status of the host. We propose that cellular heterogeneity in Efg1p activity allows the C. albicans colonizing population to differ depending on the immune status of the host, because selective pressure from a healthy host alters the composition of the population. These data are the first demonstration that differences in host immune status are associated with differences in gene expression in colonizing C. albicans cells. Altered gene expression in organisms colonizing immunocompromised hosts may begin the transition of C. albicans from a commensal to a pathogen.
IMPORTANCE
In healthy people, the fungus Candida albicans colonizes the gastrointestinal tract and other sites without producing obvious pathology. In an immunocompromised patient, the organism can cause serious disease. The demonstration that the expression and activity of the C. albicans transcription factor Efg1p differs during colonization of healthy or immunocompromised mice shows that the organism adjusts its physiology when colonizing different hosts. Further, the effects of a healthy host on a heterogeneous C. albicans population containing cells with different levels of Efg1p activity show that selective pressure in the host can change the makeup of the population, allowing the population to respond to host immune status. The ability to sense host status may be key to the ability of C. albicans to colonize as a harmless commensal in some hosts but become a deadly pathogen in others.
doi:10.1128/mBio.00117-12
PMCID: PMC3413400  PMID: 22829676
17.  Candida albicans Infection of Caenorhabditis elegans Induces Antifungal Immune Defenses 
PLoS Pathogens  2011;7(6):e1002074.
Candida albicans yeast cells are found in the intestine of most humans, yet this opportunist can invade host tissues and cause life-threatening infections in susceptible individuals. To better understand the host factors that underlie susceptibility to candidiasis, we developed a new model to study antifungal innate immunity. We demonstrate that the yeast form of C. albicans establishes an intestinal infection in Caenorhabditis elegans, whereas heat-killed yeast are avirulent. Genome-wide, transcription-profiling analysis of C. elegans infected with C. albicans yeast showed that exposure to C. albicans stimulated a rapid host response involving 313 genes (124 upregulated and 189 downregulated, ∼1.6% of the genome) many of which encode antimicrobial, secreted or detoxification proteins. Interestingly, the host genes affected by C. albicans exposure overlapped only to a small extent with the distinct transcriptional responses to the pathogenic bacteria Pseudomonas aeruginosa or Staphylococcus aureus, indicating that there is a high degree of immune specificity toward different bacterial species and C. albicans. Furthermore, genes induced by P. aeruginosa and S. aureus were strongly over-represented among the genes downregulated during C. albicans infection, suggesting that in response to fungal pathogens, nematodes selectively repress the transcription of antibacterial immune effectors. A similar phenomenon is well known in the plant immune response, but has not been described previously in metazoans. Finally, 56% of the genes induced by live C. albicans were also upregulated by heat-killed yeast. These data suggest that a large part of the transcriptional response to C. albicans is mediated through “pattern recognition,” an ancient immune surveillance mechanism able to detect conserved microbial molecules (so-called pathogen-associated molecular patterns or PAMPs). This study provides new information on the evolution and regulation of the innate immune response to divergent pathogens and demonstrates that nematodes selectively mount specific antifungal defenses at the expense of antibacterial responses.
Author Summary
Despite being a part of the normal flora of healthy individuals, Candida albicans is the most common fungal pathogen of humans and can cause infections that are associated with staggeringly high mortality rates. Here we devise a model for the study of the host immune response to C. albicans infection using the nematode C. elegans. We found that infection with the yeast form of C. albicans induces rapid and robust transcriptional changes in C. elegans. Analyses of these differentially regulated genes indicate that the nematode mounts antifungal defenses that are remarkably distinct from the host responses to pathogenic bacteria and that the nematode recognizes components possessed by heat-killed C. albicans to initiate this response. Interestingly, during infection with a pathogenic fungus, the nematode downregulates antibacterial immune response genes, which may reflect an evolutionary tradeoff between bacterial and fungal defense.
doi:10.1371/journal.ppat.1002074
PMCID: PMC3121877  PMID: 21731485
18.  Self-Regulation of Candida albicans Population Size during GI Colonization 
PLoS Pathogens  2007;3(12):e184.
Interactions between colonizing commensal microorganisms and their hosts play important roles in health and disease. The opportunistic fungal pathogen Candida albicans is a common component of human intestinal flora. To gain insight into C. albicans colonization, genes expressed by fungi grown within a host were studied. The EFH1 gene, encoding a putative transcription factor, was highly expressed during growth of C. albicans in the intestinal tract. Counterintuitively, an efh1 null mutant exhibited increased colonization of the murine intestinal tract, a model of commensal colonization, whereas an EFH1 overexpressing strain exhibited reduced colonization of the intestinal tract and of the oral cavity of athymic mice, the latter situation modeling human mucosal candidiasis. When inoculated into the bloodstream of mice, both efh1 null and EFH1 overexpressing strains caused lethal infections. In contrast, other mutants are attenuated in virulence following intravenous inoculation but exhibited normal levels of intestinal colonization. Finally, although expression of several genes is dependent on transcription factor Efg1p during laboratory growth, Efg1p-independent expression of these genes was observed during growth within the murine intestinal tract. These results show that expression of EFH1 regulated the level of colonizing fungi, favoring commensalism as opposed to candidiasis. Also, different genes are required in different host niches and the pathway(s) that regulates gene expression during host colonization can differ from well-characterized pathways used during laboratory growth.
Author Summary
Although the fungus Candida albicans commonly colonizes the human gastrointestinal tract as a commensal, the organism is also an opportunistic pathogen, responsible for a wide range of infections in immunocompromised persons. While numerous studies of infection have been conducted, few studies have analyzed the commensal state. The studies described here analyze C. albicans cells colonizing the intestinal tract of immunocompetent mice in the absence of disease, a model for commensalism. Results showed that expression of the putative transcription factor Efh1p by cells colonizing the intestinal tract was relatively high, but paradoxically, expression of Efh1p was associated with lower colonization. Efh1p had no detectable effect on the ability of C. albicans to cause lethal disseminated infection in mice. In contrast, Rbt1p and Rbt4p, two proteins of poorly defined function required for normal disseminated infection, were not required for intestinal colonization. These results argue that the commensal state is distinct from the pathogenic state and that different factors are important in different states. Also, the regulation of expression of genes RBT1, RBT4, and ECE1 during intestinal colonization differed from their well-characterized regulation during laboratory growth. Further studies of commensal colonization are needed to understand this important stage of C. albicans biology.
doi:10.1371/journal.ppat.0030184
PMCID: PMC2134954  PMID: 18069889
19.  Mechanisms Underlying the Exquisite Sensitivity of Candida albicans to Combinatorial Cationic and Oxidative Stress That Enhances the Potent Fungicidal Activity of Phagocytes 
mBio  2014;5(4):e01334-14.
ABSTRACT
Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro. We also show that the high fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as their pharmacological attenuation with apocynin or glibenclamide reduced phagocytic potency to a similar extent. The mechanistic basis for the extreme potency of combinatorial cationic plus oxidative stress—a phenomenon we term stress pathway interference—lies with the inhibition of hydrogen peroxide detoxification by the cations. In C. albicans this causes the intracellular accumulation of ROS, the inhibition of Cap1 (a transcriptional activator that normally drives the transcriptional response to oxidative stress), and altered readouts of the stress-activated protein kinase Hog1. This leads to a loss of oxidative and cationic stress transcriptional outputs, a precipitous collapse in stress adaptation, and cell death. This stress pathway interference can be suppressed by ectopic catalase (Cat1) expression, which inhibits the intracellular accumulation of ROS and the synergistic killing of C. albicans cells by combinatorial cationic plus oxidative stress. Stress pathway interference represents a powerful fungicidal mechanism employed by the host that suggests novel approaches to potentiate antifungal therapy.
IMPORTANCE
The immune system combats infection via phagocytic cells that recognize and kill pathogenic microbes. Human neutrophils combat Candida infections by killing this fungus with a potent mix of chemicals that includes reactive oxygen species (ROS) and cations. Yet, Candida albicans is relatively resistant to these stresses in vitro. We show that it is the combination of oxidative plus cationic stresses that kills yeasts so effectively, and we define the molecular mechanisms that underlie this potency. Cations inhibit catalase. This leads to the accumulation of intracellular ROS and inhibits the transcription factor Cap1, which is critical for the oxidative stress response in C. albicans. This triggers a dramatic collapse in fungal stress adaptation and cell death. Blocking either the oxidative burst or cationic fluxes in human neutrophils significantly reduces their ability to kill this fungal pathogen, indicating that combinatorial stress is pivotal to immune surveillance.
doi:10.1128/mBio.01334-14
PMCID: PMC4161263  PMID: 25028425
20.  Differential Adaptation of Candida albicans In Vivo Modulates Immune Recognition by Dectin-1 
PLoS Pathogens  2013;9(4):e1003315.
The β-glucan receptor Dectin-1 is a member of the C-type lectin family and functions as an innate pattern recognition receptor in antifungal immunity. In both mouse and man, Dectin-1 has been found to play an essential role in controlling infections with Candida albicans, a normally commensal fungus in man which can cause superficial mucocutaneous infections as well as life-threatening invasive diseases. Here, using in vivo models of infection, we show that the requirement for Dectin-1 in the control of systemic Candida albicans infections is fungal strain-specific; a phenotype that only becomes apparent during infection and cannot be recapitulated in vitro. Transcript analysis revealed that this differential requirement for Dectin-1 is due to variable adaptation of C. albicans strains in vivo, and that this results in substantial differences in the composition and nature of their cell walls. In particular, we established that differences in the levels of cell-wall chitin influence the role of Dectin-1, and that these effects can be modulated by antifungal drug treatment. Our results therefore provide substantial new insights into the interaction between C. albicans and the immune system and have significant implications for our understanding of susceptibility and treatment of human infections with this pathogen.
Author Summary
Dectin-1 is a pattern recognition receptor recognising the fungal cell-wall component, β-glucan, and plays an essential role in controlling C. albicans infections in both mouse and man. Candida albicans is part of the normal human microflora, yet is capable of causing superficial mucosal infections as well as life-threatening invasive diseases, particularly in patients whose immune function is compromised. Here we found that the contribution of Dectin-1 is limited to specific strains of C. albicans; effects which are due to the differential adaptation of these pathogens during infection. Importantly, C. albicans strains showed variations in both the composition and nature of their cell walls, and it was these differences which influenced the role of Dectin-1. Crucially, we found that we could alter the fungal cell wall, and subsequent interactions with the host, using antifungal drugs. These findings have substantial implications for our understanding of the factors contributing to human susceptibility to infections with C. albicans, but also treatment strategies.
doi:10.1371/journal.ppat.1003315
PMCID: PMC3630191  PMID: 23637604
21.  The Hyphal-Associated Adhesin and Invasin Als3 of Candida albicans Mediates Iron Acquisition from Host Ferritin 
PLoS Pathogens  2008;4(11):e1000217.
Iron sequestration by host iron-binding proteins is an important mechanism of resistance to microbial infections. Inside oral epithelial cells, iron is stored within ferritin, and is therefore not usually accessible to pathogenic microbes. We observed that the ferritin concentration within oral epithelial cells was directly related to their susceptibility to damage by the human pathogenic fungus, Candida albicans. Thus, we hypothesized that host ferritin is used as an iron source by this organism. We found that C. albicans was able to grow on agar at physiological pH with ferritin as the sole source of iron, while the baker's yeast Saccharomyces cerevisiae could not. A screen of C. albicans mutants lacking components of each of the three known iron acquisition systems revealed that only the reductive pathway is involved in iron utilization from ferritin by this fungus. Additionally, C. albicans hyphae, but not yeast cells, bound ferritin, and this binding was crucial for iron acquisition from ferritin. Transcriptional profiling of wild-type and hyphal-defective C. albicans strains suggested that the C. albicans invasin-like protein Als3 is required for ferritin binding. Hyphae of an Δals3 null mutant had a strongly reduced ability to bind ferritin and these mutant cells grew poorly on agar plates with ferritin as the sole source of iron. Heterologous expression of Als3, but not Als1 or Als5, two closely related members of the Als protein family, allowed S. cerevisiae to bind ferritin. Immunocytochemical localization of ferritin in epithelial cells infected with C. albicans showed ferritin surrounding invading hyphae of the wild-type, but not the Δals3 mutant strain. This mutant was also unable to damage epithelial cells in vitro. Therefore, C. albicans can exploit iron from ferritin via morphology dependent binding through Als3, suggesting that this single protein has multiple virulence attributes.
Author Summary
Iron is an essential nutrient for all microbes. Many human pathogenic microbes have developed sophisticated strategies to acquire iron from the host as most compartments in the body contain little free iron. For example, in oral epithelial cells intracellular iron is bound to ferritin, a protein that is highly resistant to microbial attack. In fact, no microorganism has so far been shown to directly exploit ferritin as an iron source during interaction with host cells. This study demonstrates that the pathogenic fungus Candida albicans can use ferritin as the sole source of iron. Most intriguingly, C. albicans binds ferritin via a receptor that is only exposed on invasive hyphae. This receptor is Als3, which is a member of the Als-protein family. Als3 was previously demonstrated to be an adhesin with invasin-like properties. Mutants lacking Als3 failed to bind ferritin, grew poorly with ferritin as an iron source and were unable to damage epithelial cells. Strains of the baker's yeast expressing C. albicans Als3, but not two closely related proteins, Als1 or Als5, were able to bind ferritin. Therefore, C. albicans uses an additional morphology specific and unique iron uptake strategy based on ferritin while invading into host cells where ferritin is located.
doi:10.1371/journal.ppat.1000217
PMCID: PMC2581891  PMID: 19023418
22.  Mucosal candidiasis elicits NF-κB activation, proinflammatory gene expression and localized neutrophilia in zebrafish 
Disease Models & Mechanisms  2013;6(5):1260-1270.
SUMMARY
The epithelium performs a balancing act at the interface between an animal and its environment to enable both pathogen killing and tolerance of commensal microorganisms. Candida albicans is a clinically important human commensal that colonizes all human mucosal surfaces, yet is largely prevented from causing mucosal infections in immunocompetent individuals. Despite the importance of understanding host-pathogen interactions at the epithelium, no immunocompetent vertebrate model has been used to visualize these dynamics non-invasively. Here we demonstrate important similarities between swimbladder candidiasis in the transparent zebrafish and mucosal infection at the mammalian epithelium. Specifically, in the zebrafish swimmbladder infection model, we show dimorphic fungal growth, both localized and tissue-wide epithelial NF-κB activation, induction of NF-κB -dependent proinflammatory genes, and strong neutrophilia. Consistent with density-dependence models of host response based primarily on tissue culture experiments, we show that only high-level infection provokes widespread activation of NF-κB in epithelial cells and induction of proinflammatory genes. Similar to what has been found using in vitro mammalian models, we find that epithelial NF-κB activation can occur at a distance from the immediate site of contact with epithelial cells. Taking advantage of the ability to non-invasively image infection and host signaling at high resolution, we also report that epithelial NF-κB activation is diminished when phagocytes control the infection. This is the first system to model host response to mucosal infection in the juvenile zebrafish, and offers unique opportunities to investigate the tripartite interactions of C. albicans, epithelium and immune cells in an intact host.
doi:10.1242/dmm.012039
PMCID: PMC3759345  PMID: 23720235
23.  A New Tool to Quantify Receptor Recruitment to Cell Contact Sites during Host-Pathogen Interaction 
PLoS Computational Biology  2014;10(5):e1003639.
To understand the process of innate immune fungal recognition, we developed computational tools for the rigorous quantification and comparison of receptor recruitment and distribution at cell-cell contact sites. We used these tools to quantify pattern recognition receptor spatiotemporal distributions in contacts between primary human dendritic cells and the fungal pathogens C. albicans, C. parapsilosis and the environmental yeast S. cerevisiae, imaged using 3D multichannel laser scanning confocal microscopy. The detailed quantitative analysis of contact sites shows that, despite considerable biochemical similarity in the composition and structure of these species' cell walls, the receptor spatiotemporal distribution in host-microbe contact sites varies significantly between these yeasts. Our findings suggest a model where innate immune cells discriminate fungal microorganisms based on differential mobilization and coordination of receptor networks. Our analysis methods are also broadly applicable to a range of cell-cell interactions central to many biological problems.
Author Summary
Specialized cell-cell contacts are a common theme in cell biology. These structures increase sensitivity and specificity of cellular activation and information flow in contexts ranging from activation of immune responses to transmission of nerve action potentials. Candida species fungal pathogens are responsible for significant morbidity associated with mucocutaneous infections as well as mortality () caused by bloodstream infections. The initial contact between innate immune cells and Candida results in a cell-cell contact between host and microbe. Leukocytes mobilize a network of receptors to these contact sites, and these receptors collaborate to recognize molecular patterns characteristic of microbial surfaces. Receptor recruitment, activation, and cross-talk are critical determinants of the evolution of signaling that directs the activation of downstream immune responses. However, host-pathogen contacts with fungi are complex and variable, and accurate quantification of receptor distribution in space and time is difficult with existing image analysis tools. Therefore, we have developed computational algorithms and a user interface that allows the scientist to both visualize and quantify receptor distribution in and recruitment to cell-cell contacts. We have used this software to show significant differences in contact site receptor accumulation and organization for three different host-fungal contact sites with environmental and pathogenic fungi. We also explored the correlation of contact site characteristics with the important functional outcome of phagocytosis.
doi:10.1371/journal.pcbi.1003639
PMCID: PMC4038466  PMID: 24874253
24.  UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans 
PLoS Pathogens  2011;7(11):e1002384.
Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFNγ and TNFα levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.
Author Summary
Candida albicans is an opportunistic fungal pathogen which infects individuals with debilitated immune system either due to old age, diseases such as AIDS or immune suppressive treatments. The cell wall of C. albicans, like most pathogens, mediates interaction of the pathogen with the host and determines the outcome of the host-pathogen interaction. We discovered that inactivation of GAL102 encoded UDP-glucose 4,6-dehydratase activity in C. albicans causes altered mannosylation of cell wall proteins and loss of cell wall integrity. The mutant cells thus show increased sensitivity to antifungal drugs that target cell wall. Importantly, these mutant cells show significantly lower virulence and reduced ability to elicit inflammatory cytokine responses from the host. Hence, inactivating the enzyme could significantly aid in controlling the infections by C. albicans. Since, the gene encoding the UDP glucose 4, 6-dehydratase is also present in many other fungal genomes, inhibitors of this activity could be useful in effective treatment of candidiasis and other fungal infections.
doi:10.1371/journal.ppat.1002384
PMCID: PMC3219719  PMID: 22114559
25.  Candida albicans Ethanol Stimulates Pseudomonas aeruginosa WspR-Controlled Biofilm Formation as Part of a Cyclic Relationship Involving Phenazines 
PLoS Pathogens  2014;10(10):e1004480.
In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C. albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis.
Author Summary
In many human infections, several species of microbes are often present. This is typically the case with the disease cystic fibrosis, characterized by thick mucus in the lungs that is colonized by bacteria and fungi. Here, we show evidence that interactions between the bacterium Pseudomonas aeruginosa and the fungus Candida albicans result in attributes of infection that are worse for the human host. We found that ethanol, such as that produced by C. albicans, causes increased levels of a signaling molecule in P. aeruginosa that promotes biofilm formation. Biofilm formation by P. aeruginosa is associated with infections that are more difficult to treat. Ethanol stimulated P. aeruginosa colonization of plastic surfaces and airway cells, and we identified components of this mechanism. Fungally-produced ethanol also changes the spectrum of phenazine toxins produced by P. aeruginosa, and phenazines are associated with worse lung function in people with cystic fibrosis. In light of the fact that phenazines interact with C. albicans to promote ethanol production, we propose a positive feedback loop between C. albicans and P. aeruginosa that contributes to worse disease. Our findings could have implications for the study and treatment of multi-species infections.
doi:10.1371/journal.ppat.1004480
PMCID: PMC4207824  PMID: 25340349

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