Christopher G. Pierce

A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.

The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms.

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.

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.

Candidiasis drug discovery and development: New approaches targeting virulence for discovering and identifying new drugs

Introduction: Targeting pathogenetic mechanisms, rather than essential processes, represents a very attractive alternative for the development of new antibiotics. This may be particularly important in the case of antimycotics, due to the urgent need for novel antifungal drugs and the paucity of selective fungal targets. The opportunistic pathogenic fungus Candida albicans is the main etiological agent of candidiasis, the most common human fungal infection. These infections carry unacceptably high mortality rates, a clear reflection of the many shortcomings of current antifungal therapy, including the limited armamentarium of antifungal agents, their toxicity and the emergence of resistance. Moreover, the antifungal pipeline is mostly dry. Areas covered: This review covers some of the most recent progress toward understanding C. albicans pathogenetic processes and how to harness this information for the development of anti-virulence agents. The two principal areas covered are filamentation and biofilm formation, as C. albicans pathogenicity is intimately linked to its ability to undergo morphogenetic conversions between yeast and filamentous morphologies and to its ability to form biofilms. Expert opinion: Filamentation and biofilm formation represent high value targets, yet are clinically unexploited, for the development of novel anti-virulence approaches against candidiasis. Although this has proved a difficult task despite increasing understanding at the molecular level of C. albicans virulence, there are some opportunities and prospects for antifungal drug development targeting these two important biological processes.

The Transcriptional Regulator Nrg1p Controls Candida albicans Biofilm Formation and Dispersion

ABSTRACT 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. albicanstet-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. albicanstet-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. albicanstet-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.

Antifungal therapy with an emphasis on biofilms.

Fungal infections are on the rise as advances in modern medicine prolong the lives of severely ill patients. Fungi are eukaryotic organisms and there are a limited number of targets for antifungal drug development; as a result the antifungal arsenal is exceedingly limited. Azoles, polyenes and echinocandins constitute the mainstay of antifungal therapy for patients with life-threatening mycoses. One of the main factors complicating antifungal therapy is the formation of fungal biofilms, microbial communities displaying resistance to most antifungal agents. A better understanding of fungal biofilms provides for new opportunities for the development of urgently needed novel antifungal agents and strategies.

High-Throughput Screening of a Collection of Known Pharmacologically Active Small Compounds for Identification of Candida albicans Biofilm Inhibitors

ABSTRACT Candida albicans is the most common etiologic agent of systemic fungal infections with unacceptably high mortality rates. The existing arsenal of antifungal drugs is very limited and is particularly ineffective against C. albicans biofilms. To address the unmet need for novel antifungals, particularly those active against biofilms, we have screened a small molecule library consisting of 1,200 off-patent drugs already approved by the Food and Drug Administration (FDA), the Prestwick Chemical Library, to identify inhibitors of C. albicans biofilm formation. According to their pharmacological applications that are currently known, we classified these bioactive compounds as antifungal drugs, as antimicrobials/antiseptics, or as miscellaneous drugs, which we considered to be drugs with no previously characterized antifungal activity. Using a 96-well microtiter plate-based high-content screening assay, we identified 38 pharmacologically active agents that inhibit C. albicans biofilm formation. These drugs were subsequently tested for their potency and efficacy against preformed biofilms, and we identified three drugs with novel antifungal activity. Thus, repurposing FDA-approved drugs opens up a valuable new avenue for identification and potentially rapid development of antifungal agents, which are urgently needed.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

Candida albicans biofilm formation and its clinical consequences

In the last several years there has been increasing recognition in the Microbiology field that biofilms constitute the predominant mode of growth for most microorganisms in their natural habitats [1, 2]. Different from planktonic (free floating) organisms, biofilms can be defined as structured microbial communities, attached to a biotic or abiotic surface, and most frequently encapsulated within a matrix of self-produced exopolymeric material. Most importantly, the same is also true for the relative minority of microorganisms that are pathogenic to humans, and according to the CDC today it is estimated that about 65% of all treated infections are associated with microbial biofilm formation on the surface of tissues, organs or medical devices. Biofilm formation carries important clinical implications, as sessile cells typically display increased levels of resistance to most antibiotics and also to host defence mechanisms. In addition, the protective structure of a biofilm provides cells with a safe sanctuary in which they are able to withstand adverse environmental conditions. In essence, biofilms act as reservoirs for persistent sources of infection. Thus, the net effect is that microbial biofilms negatively impact the health of a growing number of patients, that ultimately translates to a soaring financial burden to our health care system [3]. Candida albicans is no exception to this rule, and this often benign commensal of humans is now the fungal species most frequently associated with formation of biofilms affecting different types of immunosuppressed patients [4]. There is little doubt that different manifestations of candidiasis, including oropharyngeal candidiasis, denture stomatitis, endocarditis and catheter-related candidemia and candiduria, among others, are intimately associated with the formation of biofilms on host surfaces and/or implantable medical devices [5, 6]. A variety of biomaterials used in clinical practice are able to support biofilm formation by Candida and, ironically, the increase in candidiasis in recent years (now the third to fourth most common nosocomial infection in US hospitals and abroad) has been virtually concomitant with the increase in use of a broad range of medical implant devices, mostly in immunocompromised patients [7, 8]. The increasing awareness of the importance of Candida biofilms is reflected by the number of publications on this topic: a simple PubMed search using the terms “Candida” and “biofilm” returned 549 articles, with over 90% of these articles having been published in the last ten years. But, how does C. albicans form a biofilm and what are the most important characteristics linked to biofilm formation? In order to answer these fundamental questions multiple groups of investigators have developed different models of C. albicans biofilm formation, with varying degrees of sophistication, both in vitro and most recently in vivo. Using these models and now armed with state of the art analytical techniques (including advanced microscopy techniques, genomics and proteomics, etc.) researchers have been able to shed some light on the structural characteristics and molecular mechanisms governing biofilm formation by this opportunistic pathogenic fungus [4]. From these studies it is now clear that these biofilms are not a simple accumulation of cells, but rather highly structured microbial communities, postulated to represent an optimal spatial arrangement to facilitate the influx of nutrients and disposal of waste products. In general, most investigators in the field agree that C. albicans biofilm development encompasses different phases, including initial adherence, colonization, proliferation, maturation and ultimately dispersion so that the “biofilm life-cycle” can be repeated all over again [4, 9, 10]. Mature C. albicans biofilms typically consist of an intricate network of yeasts, hyphae and pseudohyphae within ramifying water channels and are encased within exopolymeric material. They exhibit a rather complex three-dimensional architecture, likely indicative of a high degree of specialization reminiscent of what is found in primitive tissue. Our current understanding at the molecular level of the mechanisms controlling C. albicans biofilm formation is still somewhat limited; however, in recent years studies by multiple groups of investigators have begun to unravel some of the major driving forces behind the transition to the biofilm life style [4, 11]. Besides some insights into biofilm metabolism, these studies have revealed a pivotal role for morphogenetic conversions (the ability to reversibly switch between yeast and filamentous forms in response to different environmental stimuli), adhesive interactions and quorum sensing mechanisms in C. albicans biofilm development, with also some very important implications in mating. Two very interesting reviews published in this very issue of this Journal provide readers with up to date information on the Ras/cAMP/PKA signaling pathway that regulates filamentation and virulence in C. albicans and on the role of farnesol as a quorum sensing molecule: both of these phenomena are critical for the C. albicans biofilm mode of growth. For example, the C. albicansΔefg1 mutant strain is locked in the yeast form and forms only a rudimentary (monolayer) biofilm, thus pointing to the Efg1p regulator protein (a main component of the Ras/cAMP/PKA signaling pathway) as a key factor in C. albicans biofilm formation [12]. Moreover, HWP1 and ALS3, whose expression is under the control of Efg1p, encode hypha-specific cell wall proteins that play complementary adhesive functions critical in C. albicans biofilm development [13]. Likewise, farnesol, and other autoregulatory molecules also control C. albicans biofilm formation via quorum sensing mechanisms [14, 15]. As mentioned before, C. albicans biofilm formation carries important negative clinical implications, mostly due to the fact that cells in biofilms are recalcitrant to antifungal therapy. As such, biofilm formation is now widely considered one of the major virulence attributes of C. albicans and a key contributing factor to the unacceptably high mortality rates associated with candidiasis. A plethora of articles have now been published reporting that fungal biofilms show intrinsic resistance to azole derivatives and display high levels of resistance against polyenes, two of the most common classes of antifungal agents. Contrary to these observations echinocandins, a new class of antifungal agents targeting cell wall glucan, seem to display excellent anti-biofilm activity at therapeutic concentrations [4, 16]. Antifungal drug resistance of C. albicans cells within biofilms is likely multifactorial and, among other mechanisms, may be due to i) metabolic and physiological state of sessile fungal cells, ii) elevated cellular density within the biofilm; iii) the protective effect of the biofilm matrix, including presence of glucans which may bind to molecules of certain antifungal agents (azoles) ; iv) differential expression of genes linked to resistance, including those encoding efflux pumps; v) differences in sterol composition of the cell wall membrane; and vi) presence of a subpopulation of “persister” cells [4]. In conclusion, sessile C. albicans cells within biofilms possess distinct developmental properties and phenotypic characteristics that are in stark contrast to planktonic cells. Because of this, infections associated with C. albicans biofilm formation represent an escalating problem in health care and negatively impact the health of an increasing number of individuals as progress in modern medicine prolong the lives of severely ill patients. The increased recognition by the research and medical community of the role that biofilms play during infection should, without any doubt, lead to major advances in the diagnosis, prevention and treatment of biofilm-associated candidiasis in the near future.

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.