Anna L. Lazzell

Expression levels of a filament-specific transcriptional regulator are sufficient to determine Candida albicans morphology and virulence

Candida albicans, the major human fungal pathogen, undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments (elongated cells attached end-to-end). Because typical C. albicans infections contain a mixture of these morphologies it has, for many years, been difficult to assess the relative contribution of each form to virulence. In addition, the regulatory mechanisms that determine growth in pseudohyphal and hyphal morphologies are largely unknown. To address these questions we have generated a C. albicans strain that can be genetically manipulated to grow completely in the hyphal form under non-filament-inducing conditions in vitro. This was achieved by inducing high-level constitutive expression of UME6, a recently identified filament-specific transcriptional regulator of C. albicans hyphal extension. We show that high-level UME6 expression significantly increases hyphal formation and promotes virulence in a mouse model of systemic candidiasis. Our results strongly suggest that shifting the morphology of a C. albicans population toward the hyphal form, and/or increasing hyphal-specific gene expression, during the course of infection is sufficient to improve virulence potential. We also demonstrate that lower levels of UME6 expression specify growth largely in the pseudohyphal form and that increasing UME6 levels is sufficient to cause cells to gradually shift from pseudohyphal to hyphal morphology. In addition, we show that UME6 levels differentially induce the expression of several known filament-specific transcripts. These findings suggest that a common transcriptional regulatory mechanism functions to specify both pseudohyphal and hyphal morphologies in a dosage-dependent manner.

UME6, a Novel Filament-specific Regulator of Candida albicans Hyphal Extension and Virulence

The specific ability of the major human fungal pathogen Candida albicans, as well as many other pathogenic fungi, to extend initial short filaments (germ tubes) into elongated hyphal filaments is important for a variety of virulence-related processes. However, the molecular mechanisms that control hyphal extension have remained poorly understood for many years. We report the identification of a novel C. albicans transcriptional regulator, UME6, which is induced in response to multiple host environmental cues and is specifically important for hyphal extension. Although capable of forming germ tubes, the ume6Delta/ume6Delta mutant exhibits a clear defect in hyphal extension both in vitro and during infection in vivo and is attenuated for virulence in a mouse model of systemic candidiasis. We also show that UME6 is an important downstream component of both the RFG1-TUP1 and NRG1-TUP1 filamentous growth regulatory pathways, and we provide evidence to suggest that Nrg1 and Ume6 function together by a negative feedback loop to control the level and duration of filament-specific gene expression in response to inducing conditions. Our results suggest that hyphal extension is controlled by a specific transcriptional regulatory mechanism and is correlated with the maintenance of high-level expression of genes in the C. albicans filamentous growth program.

Inhibition of Filamentation Can Be Used To Treat Disseminated Candidiasis

ABSTRACT Candida albicans remains the leading causative agent of invasive fungal infection. Although the importance of filamentation in C. albicans pathogenesis has been extensively investigated, in vivo studies to date have been unable to dissect the role of this developmental process in the establishment of infection versus the development of active disease as characterized by damage to the host leading to mortality. To address this issue, we genetically engineered a C. albicans tet-NRG1 strain in which filamentation and virulence can be modulated both in vitro and in vivo simply by the presence or absence of doxycycline (DOX): this strain enabled us, in a prior study, to demonstrate that yeast-form cells were able to infect the deep organs but caused no disease unless filamentation (induced by the addition of DOX) was allowed to occur. In the present study, we examined whether inhibiting filamentation (by withdrawing the DOX) at 24 or 48 h postinfection could serve as an effective therapeutic intervention against candidiasis. The results obtained indicate that DOX removal led to an alteration in the morphology of the infecting fungal cells and a dramatic increase in survival, but as with conventional antifungal drug therapy regimens, mortality rates increased markedly the longer this intervention was delayed. These observations reinforce the importance of invasive filamentous growth in causing the damage to the host and the lethality associated with active disease and suggest this process could be fruitfully targeted for the development of new antifungal agents.

Treatment and prevention of Candida albicans biofilms with caspofungin in a novel central venous catheter murine model of candidiasis

OBJECTIVES We sought to develop a novel model of central venous catheter (CVC)-associated candidiasis in mice and to use this model to examine the efficacy of caspofungin to treat and prevent Candida albicans biofilms in vivo. METHODS We used catheterized mice, commercially available from the National Cancer Institute, to form C. albicans biofilms inside CVCs. Once the model was developed, we examined the efficacy of caspofungin for the treatment of preformed biofilms and for the prevention of C. albicans biofilm formation. RESULTS We developed a relatively simple murine model of CVC-associated candidiasis that minimized the number of manipulations necessary for in vivo biofilm formation. C. albicans biofilms formed in vivo display structural features similar to those observed for models of in vitro- and other in vivo-formed biofilms. Following model development, 0.25 microg/mL of caspofungin was instilled in the catheter to treat preformed biofilms. The results indicated that caspofungin treatment significantly reduced biofilm fungal load in the catheters and dissemination to kidneys compared with untreated controls. In a second set of experiments catheters were pre-treated by filling with 60 microg/mL of caspofungin before challenge with C. albicans via the CVC. Again, the results indicated a significant reduction in biofilm fungal load and dissemination to kidneys compared with untreated controls. CONCLUSIONS We have developed a novel model of CVC-associated candidiasis in mice. Using this model we demonstrate the efficacy of caspofungin for the treatment and prevention of C. albicans biofilms in vivo.

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.

Use of a Genetically Engineered Strain To Evaluate the Pathogenic Potential of Yeast Cell and Filamentous Forms during Candida albicans Systemic Infection in Immunodeficient Mice

ABSTRACT The pathogenesis of Candida albicans systemic infection is complex and results from the balance between its intrinsic virulence attributes and the host immune responses. Morphogenetic transitions between yeast cell and filamentous forms are considered one of the main virulence attributes in C. albicans. We have examined the pathogenesis of a genetically engineered C. albicans strain in which morphogenetic conversions can be externally manipulated in immunodeficient mice; these included B-cell deficient, nude (T cell deficient), SCID (lacking both functional T and B cells), and DBA/2N (C5 deficient with impaired neutrophil activity) mice. We also tested mice severely immunosuppressed by cyclophosphamide-cortisone acetate treatment. Mice with specific immune defects were able to survive an infection by yeast cells but not filamentous forms. However, yeast cells displayed a pathogenic effect leading to lethality in the severely immunosuppressed mice.

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 that 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 antivirulence approaches against C. albicans and other fungal infections that would be less likely to foster the emergence of resistance.

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.

Efficacy of a Genetically Engineered Candida albicans tet-NRG1 Strain as an Experimental Live Attenuated Vaccine against Hematogenously Disseminated Candidiasis

ABSTRACT 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.

Pseudohyphal Regulation by the Transcription Factor Rfg1p in Candida albicans

ABSTRACT 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.