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Virtually all Candida species linked to clinical candidiasis are capable of forming highly resistant biofilms on different types of surfaces, which poses an additional significant threat and further complicates therapy of these infections. There is a scarcity of antifungal agents, and their effectiveness, particularly against biofilms, is limited. Here we provide a historical perspective on antifungal agents and therapy of Candida biofilms. As we reflect upon the past, consider the present, and look towards the future of antifungal therapy of Candida biofilms, we believe that there are reasons to remain optimistic, and that the major challenges of Candida biofilm therapy can be conquered within a reasonable timeframe.

Automated imaging techniques have been in increasing demand for the more advanced analysis and efficient characterization of cellular phenotypes. The success of the image-based profiling method hinges on assays that can rapidly and simultaneously capture a wide range of phenotypic features. We have developed an automated image acquisition method for fungal cytological profiling (FCP) using an imaging flow cytometer that can objectively measure over 250 features of a single fungal cell. Fungal cells were labeled with calcofluor white and FM4-64FX, which bind to the cell wall and lipophilic membrane, respectively. Images of single cells were analyzed using IDEAS® software. We first acquired FCPs of fungal cells treated with fluconazole, amphotericin B, and caspofungin, each with a distinct mode of action, to establish FCP databases of profiles associated with specific antifungal treatment. Once fully established, we investigated the potential application of this technique as a screening methodology to identify compounds with novel antifungal activity against Candida albicans and Cryptococcus neoformans. Altogether, we have developed a rapid, powerful, and novel image-profiling method for the phenotypic characterization of fungal cells, also with potential applications in antifungal drug development.

ABSTRACT 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.

ABSTRACT Candidiasis affects a wide variety of immunocompromised and medically compromised patients. Candida albicans, a major human fungal pathogen, accounts for about 50% of all cases, while the remainder are caused by the less pathogenic non-albicans Candida species (NACS). These species are believed to be less pathogenic, in part, because they do not filament as readily or robustly as C. albicans, although definitive evidence is lacking. To address this question, we used strains for two NACS, Candida tropicalis and Candida parapsilosis, which were genetically engineered to constitutively express the key transcriptional regulator UME6 and drive strong filamentation both in vitro and during infection in vivo. Unexpectedly, both strains showed a dramatic reduction in organ fungal burden in response to UME6 expression. Consistent with these findings, we observed that a C. tropicalis hyperfilamentous mutant was significantly reduced and a filamentation-defective mutant was slightly increased for organ fungal burden. Comprehensive immune profiling generally did not reveal any significant changes in the host response to UME6 expression in the NACS that could explain the increased clearance of infection. Interestingly, whole-genome transcriptional profiling indicated that while genes important for filamentation were induced by UME6 expression in C. tropicalis and C. parapsilosis, other genes involved in a variety of processes important for pathogenesis were strongly downregulated. These findings suggest that there are fundamental evolutionary differences in the relationship between morphology and pathogenicity among Candida species and that NACS do not necessarily possess the same virulence properties as C. albicans. IMPORTANCE Many immunocompromised individuals, including HIV/AIDS and cancer patients, are susceptible to candidiasis. About half of all cases are caused by the major fungal pathogen Candida albicans, whereas the remainder are due to less pathogenic non-albicans Candida species (NACS). Generation of filamentous cells represents a major virulence property of C. albicans, and the NACS are believed to be less pathogenic, in part, because they do not filament as well as C. albicans does. To address this question, we determined the pathogenicity of two NACS strains that have been genetically engineered to promote filamentation during infection. Surprisingly, these strains showed a dramatic reduction in pathogenicity. The host immune response did not appear to be affected. However, unlike C. albicans, filamentation of the NACS was associated with downregulation of several genes important for pathogenicity processes. Our results suggest that there are fundamental evolutionary differences in the relationship between filamentation and pathogenesis in NACS compared to C. albicans.

Fungal infections are an important and increasing global threat, carrying not only high morbidity and mortality rates, but also extraordinary healthcare costs [...]

Prior antibiotic treatment is a risk factor for Clostridioides difficile infection (CDI); the commensal gut microbiota plays a key role in determining host susceptibility to the disease. Previous studies demonstrate that the pre-colonization of mice with a commensal fungus, Candida albicans, protects against a lethal challenge with C. difficile spores. The results reported here demonstrate that the cecum contents of antibiotic-treated mice with C. albicans colonization contained different levels of several lipid species, including non-esterified, unsaturated long-chain fatty acids compared to non-C. albicans-colonized mice. Mice fed olive oil for one week and challenged with C. difficile spores showed enhanced survival compared to PBS-fed mice. The amount of olive oil administered was not sufficient to cause weight gain or to result in significant changes to the bacterial microbiota, in contrast to the effects of a high-fat diet. Furthermore, the direct exposure of C. difficile bacteria in laboratory culture to the unsaturated fatty acid oleic acid, the major fatty acid found in olive oil, reduced the transcription of genes encoding the toxins and reduced the survival of bacteria in the post-exponential phase. Therefore, the effects of C. albicans on the metabolite milieu contributed to the attenuation of C. difficile virulence.

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.

ABSTRACT 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.

Candida species are both opportunistic fungal pathogens and common members of the human mycobiome. Over the years, the main focus of the fungal field has been on understanding the pathogenic potential and disease manifestation of these organisms. Therefore, understanding of their commensal lifestyle, interactions with host epithelial barriers, and initial transition into pathogenesis is less developed. In this review, we will describe the current knowledge on the commensal lifestyle of these fungi, how they are able to adhere to and colonize host epithelial surfaces, compete with other members of the microbiota, and interact with the host immune response, as well as their transition into opportunistic pathogens by invading the gastrointestinal epithelium.

A majority of infections caused by Candida albicans—the most frequent fungal pathogen—are associated with biofilm formation. A salient feature of C. albicans biofilms is the presence of the biofilm matrix. This matrix is composed of exopolymeric materials secreted by sessile cells within the biofilm, in which all classes of macromolecules are represented, and provides protection against environmental challenges. In this review, we summarize the knowledge accumulated during the last two decades on the composition, structure, and function of the C. albicans biofilm matrix. Knowledge of the matrix components, its structure, and function will help pave the way to novel strategies to combat C. albicans biofilm infections.

ABSTRACT The contributions of commensal fungi to human health and disease are not well understood. Candida species such as C. albicans and C. glabrata are opportunistic pathogenic fungi and common colonizers of the human intestinal tract. They have been shown to affect the host immune system and interact with the gut microbiome and pathogenic microorganisms. Therefore, Candida species could be expected to play important ecological roles in the host gastrointestinal tract. Previously, our group demonstrated that pre-colonization of mice with C. albicans protected them against lethal C. difficile infection (CDI). Here, we show that mice pre-colonized with C. glabrata succumbed to CDI more rapidly than mice that were not pre-colonized suggesting an enhancement in C. difficile pathogenesis. Further, when C. difficile was added to pre-formed C. glabrata biofilms, an increase in matrix and overall biomass was observed. These effects were also shown with C. glabrata clinical isolates. Interestingly, the presence of C. difficile increased C. glabrata biofilm susceptibility to caspofungin, indicating potential effects on the fungal cell wall. Defining this intricate and intimate relationship will lead to an understanding of the role of Candida species in the context of CDI and novel aspects of Candida biology. IMPORTANCE Most microbiome studies have only considered the bacterial populations while ignoring other members of the microbiome such as fungi, other eukaryotic microorganisms, and viruses. Therefore, the role of fungi in human health and disease has been significantly understudied compared to their bacterial counterparts. This has generated a significant gap in knowledge that has negatively impacted disease diagnosis, understanding, and the development of therapeutics. With the development of novel technologies, we now have an understanding of mycobiome composition, but we do not understand the roles of fungi in the host. Here, we present findings showing that Candida glabrata, an opportunistic pathogenic yeast that colonizes the mammalian gastrointestinal tract, can impact the severity and outcome of a Clostridioides difficile infection (CDI) in a murine model. These findings bring attention to fungal colonizers during CDI, a bacterial infection of the gastrointestinal tract.

ABSTRACT Scientific contributions by members from historically marginalized communities (HMCs) have been largely ignored, uncredited, and in some cases erased from history. This has contributed to science, technology, engineering, and math (STEM) curricula lacking diversity. In this study, we present an Honors seminar course aimed to highlight the discoveries of scientists from HMCs, centered around reading primary literature in a way that builds our students' research skills. The course provides students with opportunities for active learning, skill building, and mentorship that are key for persistence of students in the STEM “leaky pipeline.” Students also read biographies of scientists from HMCs, interact with guest speakers, and choose scientists to highlight (in final papers and presentations) and publicize (through the creation of Wikipedia pages). Additionally, students use community-building methodologies to build a safe classroom and gain tools to have conversations about diversity, inequities, and intersectionality in STEM. In self-reporting surveys, 93.7% of students strongly agreed that their appreciation for marginalized scientists increased and 92.6% reported that the course met very well the goal of refining their research skills. These findings support the effectiveness of this novel course. We provide two lists (one of 137 scientists and one of 57 scientist biographies) that will allow faculty teaching a wide range of science classes to select examples of scientists and discoveries to highlight in their courses. This course represents a novel platform to diversify STEM curricula while engaging and empowering students from historically marginalized communities.