Geldanamycin-Induced Morphological Changes Require Candida albicans Hyphal Growth Regulatory Machinery.

In Candida albicans, geldanamycin treatment inhibits the essential chaperone Hsp90 and induces a change from yeast to filamentous morphology, likely by impeding cell cycle progression and division. However, filaments formed by wild-type cells upon geldanamycin exposure are quite different in appearance from true hyphae. We have observed that effects on morphology caused by geldanamycin treatment appear to vary in strains with defects in different morphological regulators. These results indicate that the filamentous forms induced by inhibiting Hsp90p, while not true hyphae, nonetheless require some components of the hypha induction machinery for their formation. Furthermore, we have found that BRG1, a known regulator of hypha formation, is also required for pseudohypha induction in response to nitrogen starvation and for the formation of elongated filaments upon exposure to geldanamycin.

Global Transcriptomic Analysis of the Candida albicans Response to Treatment with a Novel Inhibitor of Filamentation.

The opportunistic pathogenic fungus Candida albicans can cause devastating infections in immunocompromised patients. Its ability to undergo a morphogenetic transition from yeast to filamentous forms allows it to penetrate tissues and damage tissues, and the expression of genes associated with a number of pathogenetic mechanisms is also coordinately regulated with the yeast-to-hypha conversion. Therefore, it is widely considered that filamentation represents one of the main virulence factors of C. albicans We have previously identified N-[3-(allyloxy)-phenyl]-4-methoxybenzamide (compound 9029936) as the lead compound in a series of small-molecule inhibitors of C. albicans filamentation and characterized its activity both in vitro and in vivo This compound appears to be a promising candidate for the development of alternative antivirulence strategies for the treatment of C. albicans infections. In this study, we performed RNA sequencing analysis of samples obtained from C. albicans cells grown under filament-inducing conditions in the presence or absence of this compound. Overall, treatment with compound 9029936 resulted in 618 upregulated and 702 downregulated genes. Not surprisingly, some of the most downregulated genes included well-characterized genes associated with filamentation and virulence such as SAP5, ECE1 (candidalysin), and ALS3, as well as genes that impact metal chelation and utilization. Gene ontology analysis revealed an overrepresentation of cell adhesion, iron transport, filamentation, biofilm formation, and pathogenesis processes among the genes downregulated during treatment with this leading compound. Interestingly, the top upregulated genes suggested an enhancement of vesicular transport pathways, particularly those involving SNARE interactions.IMPORTANCE These results from whole-genome transcriptional profiling provide further insights into the biological activity and mode of action of a small-molecule inhibitor of C. albicans filamentation. This information will assist in the development of novel antivirulence strategies against C. albicans infections.

Contributions of Candida albicans Dimorphism, Adhesive Interactions, and Extracellular Matrix to the Formation of Dual-Species Biofilms with Streptococcus gordonii.

Fungal and bacterial populations coexist in the oral cavity, frequently forming mixed-species biofilms that complicate treatment against polymicrobial infections. However, despite relevance to oral health, the bidirectional interactions between these microbial populations are poorly understood. In this study, we aimed to elucidate the mechanisms underlying the interactions between the fungal species Candida albicans and the bacterial species Streptococcus gordonii as they coexist in mixed-species biofilms. Specifically, the interactions of different C. albicans mutant strains deficient in filamentation (efg1Δ/Δ and brg1Δ/Δ), adhesive interactions (als3Δ/Δ and bcr1Δ/Δ), and production of matrix exopolymeric substances (EPS) (kre5Δ/Δ, mnn9Δ/Δ, rlm1Δ/Δ, and zap1Δ/Δ) were evaluated with S. gordonii under different conditions mimicking the environment in the oral cavity. Interestingly, our results revealed that growth of the biofilm-deficient C. albicansals3Δ/Δ and bcr1Δ/Δ mutant strains in synthetic saliva or with S. gordonii restored their biofilm-forming ability. Moreover, challenging previous observations indicating an important role of morphogenetic conversions in the interactions between these two species, our results indicated a highly synergistic interaction between S. gordonii and the C. albicans filamentation-deficient efg1Δ/Δ and brg1Δ/Δ deletion mutants, which was particularly noticeable when the mixed biofilms were grown in synthetic saliva. Importantly, dual-species biofilms were found to exhibit increase in antimicrobial resistance, indicating that components of the fungal exopolymeric material confer protection to streptococcal cells against antibacterial treatment. Collectively, these findings unravel a high degree of complexity in the interactions between C. albicans and S. gordonii in mixed-species biofilms, which may impact homeostasis in the oral cavity.IMPORTANCE Microbial communities have a great impact in health and disease. C. albicans interacts with multiple microorganisms in the oral cavity, frequently forming polymicrobial biofilms. We report on the synergistic interactions between C. albicans and the Gram-positive bacterium S. gordonii, for which we have examined the different contributions of adhesive interactions, filamentation, and the extracellular matrix to the formation of dual-species biofilms. Our results demonstrate that growth in the presence of the bacterium can restore the biofilm-forming ability of different C. albicans mutant strains with defects in adhesion and filamentation. The mixed-species biofilms also show high levels of resistance to antibacterial and antifungal antibiotics, and our results indicate that the fungal biofilm matrix protects bacterial cells within these mixed-species biofilms. Our observations add to a growing body of evidence indicating a high level of complexity in the reciprocal interactions and consortial behavior of fungal/bacterial biofilms.

Candida albicans Morphology-Dependent Host FGF-2 Response as a Potential Therapeutic Target.

Angiogenesis mediated by proteins such as Fibroblast Growth Factor-2 (FGF-2) is a vital component of normal physiological processes and has also been implicated in contributing to the disease state associated with various microbial infections. Previous studies by our group and others have shown that Candida albicans, a common agent of candidiasis, induces FGF-2 secretion in vitro and angiogenesis in brains and kidneys during systemic infections. However, the underlying mechanism(s) via which the fungus increases FGF-2 production and the role(s) that FGF-2/angiogenesis plays in C. albicans disease remain unknown. Here we show, for the first time, that C. albicans hyphae (and not yeast cells) increase the FGF-2 response in human endothelial cells. Moreover, Candidalysin, a toxin secreted exclusively by C. albicans in the hyphal state, is required to induce this response. Our in vivo studies show that in the systemic C. albicans infection model, mice treated with FGF-2 exhibit significantly higher mortality rates when compared to untreated mice not given the angiogenic growth factor. Even treatment with fluconazole could not fully rescue infected animals that were administered FGF-2. Our data suggest that the increase of FGF-2 production/angiogenesis induced by Candidalysin contributes to the pathogenicity of C. albicans.

Role of the Fractalkine Receptor in CNS Autoimmune Inflammation: New Approach Utilizing a Mouse Model Expressing the Human CX3CR1I249/M280 Variant.

Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ∼20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and influence on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses due to impaired CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation.

In Vitro Characterization of a Biaryl Amide Anti-virulence Compound Targeting Candida albicans Filamentation and Biofilm Formation.

We have previously identified a small molecule compound, N-[3-(allyloxy)-phenyl]-4-methoxybenzamide (9029936), that exerts potent inhibitory activity against filamentation and biofilm formation by the Candida albicans SC5314 strain and represents a lead candidate for the development of anti-virulence approaches against C. albicans infections. Here we present data from a series of experiments to further characterize its in vitro activity and drug-like characteristics. We demonstrate the activity of this compound against a panel of C. albicans clinical isolates, including several displaying resistance to current antifungals; as well as against a set of C. albicans gain of function strains in key transcriptional regulators of antifungal drug resistance. The compound also inhibits filamentation and biofilm formation in the closely related species C. dubliniensis, but not C. glabrata or C. tropicalis. Combinatorial studies reveal the potential of compound 9029936 to be used together with currently available conventional antifungals. Results of serial passage experiments indicate that repeated exposure to this compound does not elicit resistance. Viability staining of C. albicans in the presence of high concentrations of compound 9029936 confirms that the compound is not toxic to fungal cells, and cytological staining using image flow cytometry analysis reveals that treatment with the lead compound affects hyphal length, with additional effects on cell wall and integrity of the membrane system. In vitro pharmacological profiling provides further evidence that the lead compound displays a safe profile, underscoring its excellent "drug-like" characteristics. Altogether these results confirm the potential of this compound to be further developed as a true anti-virulence agent for the treatment of C. albicans infections, including those refractory to treatment with conventional antifungal agents.

Development of Anti-Virulence Approaches for Candidiasis via a Novel Series of Small-Molecule Inhibitors of Candida albicans Filamentation.

Candida albicans remains the main etiologic agent of candidiasis, the most common fungal infection and now the third most frequent infection in U.S. hospitals. The scarcity of antifungal agents and their limited efficacy contribute to the unacceptably high morbidity and mortality rates associated with these infections. The yeast-to-hypha transition represents the main virulence factor associated with the pathogenesis of C. albicans infections. In addition, filamentation is pivotal for robust biofilm development, which represents another major virulence factor for candidiasis and further complicates treatment. Targeting pathogenic mechanisms rather than growth represents an attractive yet clinically unexploited approach in the development of novel antifungal agents. Here, we performed large-scale phenotypic screening assays with 30,000 drug-like small-molecule compounds within ChemBridge's DIVERSet chemical library in order to identify small-molecule inhibitors of C. albicans filamentation, and our efforts led to the identification of a novel series of bioactive compounds with a common biaryl amide core structure. The leading compound of this series, N-[3-(allyloxy)-phenyl]-4-methoxybenzamide, was able to prevent filamentation under all liquid and solid medium conditions tested, suggesting that it impacts a common core component of the cellular machinery that mediates hypha formation under different environmental conditions. In addition to filamentation, this compound also inhibited C. albicans biofilm formation. This leading compound also demonstrated in vivo activity in clinically relevant murine models of invasive and oral candidiasis. Overall, our results indicate that compounds within this series represent promising candidates for the development of novel anti-virulence approaches to combat C. albicans infections.IMPORTANCE Since fungi are eukaryotes, there is a limited number of fungus-specific targets and, as a result, the antifungal arsenal is exceedingly small. Furthermore, the efficacy of antifungal treatment is compromised by toxicity and development of resistance. As a consequence, fungal infections carry high morbidity and mortality rates, and there is an urgent but unmet need for novel antifungal agents. One appealing strategy for antifungal drug development is to target pathogenetic mechanisms associated with infection. In Candida albicans, one of the most common pathogenic fungi, morphogenetic transitions between yeast cells and filamentous hyphae represent a key virulence factor associated with the ability of fungal cells to invade tissues, cause damage, and form biofilms. Here, we describe and characterize a novel small-molecule compound capable of inhibiting C. albicans filamentation both in vitro and in vivo; as such, this compound represents a leading candidate for the development of anti-virulence therapies against candidiasis.

Targeting Candida albicans filamentation for antifungal drug development.

Candida albicans remains the main etiological agent of candidiasis, as this otherwise normal commensal of humans is capable of causing active infection in immune- and medically-compromised patients. The high morbidity and mortality rates associated with candidiasis, coupled with the emergence of drug resistance demand the development of novel therapeutic strategies. However, there is a paucity of selective targets that can be exploited in the development of new antifungals. Contrary to conventional antibiotics that kill or curtail growth, specifically targeting virulence mechanisms represents an attractive option for antifungal drug development. In C. albicans, a growing body of research over the last few decades has provided important insights into its virulence factors and their contribution to the pathogenesis of candidiasis. Of these, filamentation is the one that has received the most attention and perhaps shows the most promise as a target for new anti-virulence strategies to combat C. albicans infections.

Examination of the pathogenic potential of Candida albicans filamentous cells in an animal model of haematogenously disseminated candidiasis.

The opportunistic fungal pathogen Candida albicans is an increasingly common threat to human health. Candida albicans grows in several morphologies and mutant strains locked in yeast or filamentous forms have attenuated virulence in the murine model of disseminated candidiasis. Thus, the ability to change shape is important for virulence. The transcriptional repressors Nrg1p and Tup1p are required for normal regulation of C. albicans morphology. Strains lacking either NRG1 or TUP1 are constitutively pseudohyphal under yeast growth conditions, and display attenuated virulence in the disseminated model. To dissect the relative importance of hyphae and pseudohyphae during an infection, we used strains in which the morphological transition could be externally manipulated through controlled expression of NRG1 or TUP1. Remarkably, hyphal form inocula retain the capacity to cause disease. Whilst induction of a pseudohyphal morphology through depletion of TUP1 did result in attenuated virulence, this was not due to a defect in the ability to escape the bloodstream. Instead, we observed that pseudohyphal cells are cleared from tissues much more efficiently than either hyphal (virulent) or yeast form (avirulent) cells, indicating that different C. albicans morphologies have distinct interactions with host cells during an infection.

A Novel Small Molecule Inhibitor of Candida albicans Biofilm Formation, Filamentation and Virulence with Low Potential for the Development of Resistance.

BACKGROUND/OBJECTIVES: Candida albicans is the principal causative agent of candidiasis, the most common fungal infection in humans. Candidiasis represents the third-to-fourth most frequent nosocomial infection worldwide, as this normal commensal of humans causes opportunistic infections in an expanding population of immune- and medically-compromised patients. These infections are frequently associated with biofilm formation, which complicates treatment and contributes to unacceptably high mortality rates. METHODS: To address the pressing need for new antifungals we have performed a high content screen of 20,000 small molecules in a chemical library (NOVACore™) to identify compounds that inhibit C. albicans biofilm formation, and conducted a series of follow-up studies to examine the in vitro and in vivo activity of the identified compounds. RESULTS: The screen identified a novel series of diazaspiro-decane structural analogs which were largely represented among the bioactive compounds. Characterization of the leading compound from this series indicated that it inhibits processes associated with C. albicans virulence, most notably biofilm formation and filamentation, without having an effect on overall growth or eliciting resistance. This compound demonstrated in vivo activity in clinically-relevant murine models of both invasive and oral candidiasis and as such represents a promising lead for antifungal drug development. Furthermore, these results provide proof of concept for the implementation of anti-virulence approaches against C. albicans and other fungal infections that would be less likely to foster the emergence of resistance.

High-content phenotypic screenings to identify inhibitors of Candida albicans biofilm formation and filamentation.

Candida species represent the main cause of opportunistic fungal infections worldwide, and Candida albicans remains the most common etiological agent of candidiasis, now the third to fourth most common nosocomial infection. These infections are typically associated with high morbidity and mortality, mainly due to the limited efficacy of current antifungal drugs. In C. albicans, morphogenetic conversions between yeast and filamentous forms and biofilm formation represent two important biological processes that are intimately associated with the biology of this fungus and also play important roles during the pathogenesis of candidiasis. We have performed cell-based phenotypic screens using three different chemical libraries from the National Cancer Institute's Open Chemical Repository collection and identified several compounds with inhibitory activity against C. albicans biofilm formation and/or filamentation. These phenotype-based approaches represent a prosperous alternative to conventional genetics and genomics techniques to address experimentally challenging and complex biological phenomena, such as biofilm formation and filamentation, while at the same time opening new possibilities for the development of new antifungal agents.

BRG1 and NRG1 form a novel feedback circuit regulating Candida albicans hypha formation and virulence.

In the opportunistic fungal pathogen Candida albicans both cellular morphology and the capacity to cause disease are regulated by the transcriptional repressor Nrg1p. One of the genes repressed by Nrg1p is BRG1, which encodes a putative GATA family transcription factor. Deletion of both copies of this gene prevents hypha formation. We discovered that BRG1 overexpression is sufficient to overcome Nrg1p-mediated repression and drive the morphogenetic shift from yeast to hyphae even in the absence of environmental stimuli. We further observed that expression of BRG1 influences the stability of the NRG1 transcript, thus controlling filamentation through a feedback loop. Analysis of this phenomenon revealed that BRG1 expression is required for the induction of an antisense NRG1 transcript. This is the first demonstration of a role for mRNA stability in regulating the key C. albicans virulence trait: the ability to form hyphae.

Validation of the tetracycline regulatable gene expression system for the study of the pathogenesis of infectious disease.

Understanding the pathogenesis of infectious disease requires the examination and successful integration of parameters related to both microbial virulence and host responses. As a practical and powerful method to control microbial gene expression, including in vivo, the tetracycline-regulatable system has recently gained the favor of many investigative groups. However, some immunomodulatory effects of the tetracyclines, including doxycycline, could potentially limit its use to evaluate host responses during infection. Here we have used a well-established murine model of disseminated candidiasis, which is highly dependent on both the virulence displayed by the fungal cells and on the host immune status, to validate the use of this system. We demonstrate that the pathogenesis of the wild type C. albicans CAF2-1 strain, which does not contain any tet-regulatable element, is not affected by the presence of doxycycline. Moreover levels of key cytokines, chemokines and many other biomarkers, as determined by multi-analyte profiling, remain essentially unaltered by the presence of the antibiotic during infection. Our results indicate that the levels of doxycycline needed to control the tetracycline regulatable promoter gene expression system have no detectable effect on global host responses during candidiasis. Because tet-regulatable systems are now being increasingly used in a variety of pathogenic microorganisms, these observations have wide implications in the field of infectious diseases.

Candida albicans adhesin Als3p is dispensable for virulence in the mouse model of disseminated candidiasis.

The presence of specific proteins, including Ece1p, Hwp1p and Als3p, distinguishes the Candida albicans hyphal cell wall from that of yeast-form cells. These proteins are thought to be important for the ability of C. albicans cells to adhere to living and non-living surfaces and for the cell-to-cell adhesion necessary for biofilm formation, and also to be pivotal in mediating C. albicans interactions with endothelial cells. Using an in vitro flow adhesion assay, we previously observed that yeast cells bind in greater numbers to human microvascular endothelial cells than do hyphal or pseudohyphal cells. This is consistent with previous observations that, in a murine model of disseminated candidiasis, cells locked in the yeast form can efficiently escape the bloodstream and invade host tissues. To more precisely explore the role of Als3p in adhesion and virulence, we deleted both copies of ALS3 in a wild-type C. albicans strain. In agreement with previous studies, our als3Δ null strain formed hyphae normally but was defective in biofilm formation. Whilst ALS3 was not expressed in our null strain, hypha-specific genes such as ECE1 and HWP1 were still induced appropriately. Both the yeast form and the hyphal form of the als3Δ strain adhered to microvascular endothelial cells to the same extent as a wild-type strain under conditions of flow, indicating that Als3p is not a significant mediator of the initial interaction between fungal cells and the endothelium. Finally, in a murine model of haematogenously disseminated candidiasis the mutant als3Δ remained as virulent as the wild-type parent strain.

Evaluation of tissue engineered models of the oral mucosa to investigate oral candidiasis.

Candida albicans is a commensal organism that can be isolated from the majority of healthy individuals. However, in certain susceptible individuals C. albicans can become pathogenic leading to the mucocutaneous infection; oral candidiasis. Murine models and in vitro monolayer cultures have generated some data on the likely virulence and host factors that contribute to oral candidiasis but these models have limitations. Recently, tissue engineered oral mucosal models have been developed to mimic the normal oral mucosa but little information is available on their true representation. In this study, we assessed the histological features of three different tissue engineered oral mucosal models compared to the normal oral mucosa and analysed both cell damage and cytokine release following infection with C. albicans. Models comprised of normal oral keratinocytes and a fibroblast-containing matrix displayed more similar immunohistological and proliferation characteristics to normal mucosa, compared to models composed of an oral carcinoma cell line. Although all models were invaded and damaged by C. albicans in a similar manner, the cytokine response was much more pronounced in models containing normal keratinocytes. These data suggest that models based on normal keratinocytes atop a fibroblast-containing connective tissue will significantly aid in dissecting the molecular pathogenesis of oral candidiasis.

The transcriptional regulator Nrg1p controls Candida albicans biofilm formation and dispersion.

The ability of Candida albicans to reversibly switch morphologies is important for biofilm formation and dispersion. In this pathogen, Nrg1p functions as a key negative regulator of the yeast-to-hypha morphogenetic transition. We have previously described a genetically engineered C. albicans tet-NRG1 strain in which NRG1 expression levels can be manipulated by the presence or absence of doxycycline (DOX). Here, we have used this strain to ascertain the role of Nrg1p in regulating the different stages of the C. albicans biofilm developmental cycle. In an in vitro model of biofilm formation, the C. albicans tet-NRG1 strain was able to form mature biofilms only when DOX was present in the medium, but not in the absence of DOX, when high levels of NRG1 expression blocked the yeast-to-hypha transition. However, in a biofilm cell retention assay in which biofilms were developed with mixtures of C. albicans tet-NRG1 and SC5314 strains, tet-NRG1 yeast cells were still incorporated into the mixed biofilms, in which an intricate network of hyphae of the wild-type strain provided for biofilm structural integrity and adhesive interactions. Also, utilizing an in vitro biofilm model under conditions of flow, we demonstrated that C. albicans Nrg1p exerts an exquisite control of the dispersal process, as overexpression of NRG1 leads to increases in dispersion of yeast cells from the biofilms. Our results demonstrate that manipulation of NRG1 gene expression has a profound influence on biofilm formation and biofilm dispersal, thus identifying Nrg1p as a key regulator of the C. albicans biofilm life cycle.

Pseudohyphal regulation by the transcription factor Rfg1p in Candida albicans.

The opportunistic human fungal pathogen Candida albicans is a major cause of nosocomial infections. One of the fundamental features of C. albicans pathogenesis is the yeast-to-hypha transition. Hypha formation is controlled positively by transcription factors such as Efg1p and Cph1p, which are required for hyphal growth, and negatively by Tup1p, Rfg1p, and Nrg1p. Previous work by our group has shown that modulating NRG1 gene expression, hence altering morphology, is intimately linked to the capacity of C. albicans to cause disease. To further dissect these virulence mechanisms, we employed the same strategy to analyze the role of Rfg1p in filamentation and virulence. Studies using a tet-RFG1 strain revealed that RFG1 overexpression does not inhibit hypha formation in vitro or in the mouse model of hematogenously disseminated candidiasis. Interestingly, RFG1 overexpression drives formation of pseudohyphae under yeast growth conditions-a phenotype similar to that of C. albicans strains with mutations in one of several mitotic regulatory genes. Complementation assays and real-time PCR analysis indicate that, although the morphology of the tet-RFG1 strain resembles that of the mitotic regulator mutants, Rfg1p overexpression does not impact expression of these genes.

An analysis of the impact of NRG1 overexpression on the Candida albicans response to specific environmental stimuli.

The ability of the opportunistic fungal pathogen Candida albicans to form filaments has been strongly linked to its capacity to cause disease in humans. We previously described the construction of a strain in which filamentation can be modulated both in vitro and in vivo by placing one copy of the NRG1 gene under the control of a tetracycline-regulatable promoter. To further characterize the role of NRG1 in controlling filamentous growth, and in an attempt to determine whether NRG1 downregulation is a requirement for filamentation per se, or is only necessary under certain environmental conditions, we have conducted an analysis of the growth of the tet-NRG1 strain under a variety of in vitro conditions. Through overexpression of NRG1, we were able to block filamentation of C. albicans in both liquid media and on solid media. Filamentation in response to the low-oxygen environment of embedded growth was also inhibited. In all of these conditions, normal filamentation could be restored by down regulating expression from the tet-NRG1 allele. Interestingly, although elevated NRG1 levels were able to inhibit the formation of true hyphae in response to a wide range of environmental stimuli, elevated NRG1 expression did not affect the formation of pseudohyphae on nitrogen-limiting synthetic low ammonia dextrose (SLAD) medium. This work further illustrates the key role played by NRG1 in the control of filamentation and suggests that, although NRG1 repression plays a key role in regulating true hyphal growth, it apparently does not regulate pseudohyphal growth in the same fashion.

Adhesion of Candida albicans to endothelial cells under physiological conditions of flow.

Candida albicans is a commensal organism that under certain circumstances can become pathogenic. During systemic infection C. albicans is disseminated via the circulation to distant organs, where it causes multiple organ failure. Despite the severity of systemic C. albicans infection, little is known about the mechanisms involved in the adhesion of this organism to the endothelium lining blood vessels. Previous studies have used static assays to examine adhesion. However, these do not realistically model blood vessels, where circulating C. albicans cells must adhere to the endothelium under conditions of flow and shear stress. Furthermore, there is conflicting evidence concerning the role played by yeast, pseudohyphal, and hyphal forms of C. albicans in adhesion to endothelium. To test the hypothesis that there may be differences in the abilities of these three morphogenic forms of C. albicans to adhere to endothelium under conditions of flow, we developed an in vitro flow adhesion assay. We found that all three forms of C. albicans rapidly bound to confluent endothelial cells under conditions of flow. Maximum adhesion was found at low shear stress levels similar to that found in postcapillary venules. Moreover, yeast forms bound in significantly greater numbers than did pseudohyphal and hyphal forms, respectively, contrasting with previous findings from static assays. These findings are consistent with recent in vivo data suggesting that yeast forms may be capable of adhering to the endothelium and migrating into the tissues before undergoing morphogenic change to cause tissue damage.

Efficacy of a genetically engineered Candida albicans tet-NRG1 strain as an experimental live attenuated vaccine against hematogenously disseminated candidiasis.

We report on the efficacy of the genetically engineered Candida albicans tet-NRG1 strain as an experimental live, attenuated vaccine against disseminated candidiasis in both immunocompetent and immunodeficient mice mostly dependent on T-cell immunity. This experimental vaccination model may represent an important tool to unravel the mechanisms of protective immunity during candidiasis.