Allosteric inhibition of tRNA synthetase Gln4 by N-pyrimidinyl-ß-thiophenylacrylamides exerts highly selective antifungal activity.

Candida species are among the most prevalent causes of systemic fungal infections, which account for ∼1.5 million annual fatalities. Here, we build on a compound screen that identified the molecule N-pyrimidinyl-ß-thiophenylacrylamide (NP-BTA), which strongly inhibits Candida albicans growth. NP-BTA was hypothesized to target C. albicans glutaminyl-tRNA synthetase, Gln4. Here, we confirmed through in vitro amino-acylation assays NP-BTA is a potent inhibitor of Gln4, and we defined how NP-BTA arrests Gln4's transferase activity using co-crystallography. This analysis also uncovered Met496 as a critical residue for the compound's species-selective target engagement and potency. Structure-activity relationship (SAR) studies demonstrated the NP-BTA scaffold is subject to oxidative and non-oxidative metabolism, making it unsuitable for systemic administration. In a mouse dermatomycosis model, however, topical application of the compound provided significant therapeutic benefit. This work expands the repertoire of antifungal protein synthesis target mechanisms and provides a path to develop Gln4 inhibitors.

Coding Sequence Insertions in Fungal Genomes are Intrinsically Disordered and can Impart Functionally-Important Properties on the Host Protein.

Insertion and deletion mutations (indels) are important mechanisms of generating protein diversity. Indels in coding sequences are under considerable selective pressure to maintain reading frames and to preserve protein function, but once generated, indels provide raw material for the acquisition of new protein properties and functions. We reported recently that coding sequence insertions in the Candida albicans NDU1 protein, a mitochondrial protein involved in the assembly of the NADH:ubiquinone oxidoreductase are imperative for respiration, biofilm formation and pathogenesis. NDU1 inserts are specific to CTG-clade fungi, absent in human ortholog and successfully harnessed as drug targets. Here, we present the first comprehensive report investigating indels and clade-defining insertions (CDIs) in fungal proteomes. We investigated 80 ascomycete proteomes encompassing CTG clade species, the Saccharomycetaceae family, the Aspergillaceae family and the Herpotrichiellaceae (black yeasts) family. We identified over 30,000 insertions, 4,000 CDIs and 2,500 clade-defining deletions (CDDs). Insert sizes range from 1 to over 1,000 residues in length, while maximum deletion length is 19 residues. Inserts are strikingly over-represented in protein kinases, and excluded from structural domains and transmembrane segments. Inserts are predicted to be highly disordered. The amino acid compositions of the inserts are highly depleted in hydrophobic residues and enriched in polar residues. An indel in the Saccharomyces cerevisiae Sth1 protein, the catalytic subunit of the RSC (Remodel the Structure of Chromatin) complex is predicted to be disordered until it forms a ß-strand upon interaction. This interaction performs a vital role in RSC-mediated transcriptional regulation, thereby expanding protein function.

Antifungal activity of alexidine dihydrochloride in a novel diabetic mouse model of dermatophytosis.

Dermatophytosis is one of the most prevalent fungal infections and a major public health problem worldwide. Recent years have seen a change in the epidemiological patterns of infecting fungi, corresponding to an alarming rise in the prevalence of drug-recalcitrant dermatophyte infections. In patients with diabetes mellitus, dermatophytosis is more severe and recurrent. The potency of promising new antifungal drugs in the pipeline must be expanded to include dermatophytosis. To facilitate this effort, we established a clinically pertinent mouse model of dermatophyte infections, in which diabetic mice were infected with Trichophyton mentagrophytes on abraded skin. The diabetic mouse model was optimized as a simple and robust system for simulating dermatophytoses in diabetic patients. The outcome of infection was measured using clinical and mycological parameters. Infected mice with fungal lesions were treated with oral and topical formulations of terbinafine or topical administration of the FDA-approved and repurposed pan-antifungal drug alexidine dihydrochloride (AXD). In this model, AXD was found to be highly effective, with outcomes comparable to those of the standard of care drug terbinafine.

Antibodies targeting Candida albicans Als3 and Hyr1 antigens protect neonatal mice from candidiasis.

Pre-term infants in neonatal intensive care units are vulnerable to fungal sepsis. In this patient population, Candida albicans remains the predominant fungal pathogen causing high morbidity and mortality, despite antifungal therapy. Thus, new preventative/therapeutic strategies against neonatal candidiasis are needed. Previously, we have reported that vaccination with recombinant forms of the C. albicans N-termini of the cell wall proteins Als3 (rAls3p-N) and Hyr1 (rHyr1p-N) protected adult mice from disseminated candidiasis. Further, in a Phase 1b/2a NDV-3A (an rAls3p-N formulated with alum) protected women from recurrent vulvovaginal candidiasis, with anti-Als3p IgG2 isotype being a biomarker for efficacy. Here, we performed a proof of concept study to evaluate if anti-Als3p or anti-Hyr1p antibodies are important for prevention of disseminated candidiasis in neonates. Als3 and Hyr1 antigens when adjuvanted with complete Freund's adjuvant (CFA)/incomplete Freund's adjuvant (IFA) induced a robust antibody response with a ten-fold higher titer of IgG2, than attained by either antigen formulated with alum. Transplacental transfer of these antibodies significantly reduced fungal burden in the kidneys of mice pups, and adoptive transfer of vaccinated mothers' sera into pups displayed similar levels of protection. Neutrophils were found important for this efficacy. Finally, anti-Hyr1 antisera potentiated the activity of fluconazole in protecting from C. albicans infection. Our current studies are the first in the field to emphasize the importance of anti-Als3 and anti-Hyr1 antibodies in preventing neonatal candidiasis. Considering that Candida infections in low birthweight infants is a lethal infection, active and passive vaccination strategies using these antigens could have profound clinical relevance.

A Simple Method for Growth of Candida albicans Biofilms Under Continuous Media Flow and for Recovery of Biofilm Dispersed Cells.

Majority of nosocomial infections are associated with biofilms growing on indwelling medical devices. These biofilms are nourished by a continuous flow of body fluids and subjected to shear stress forces. Cells dispersed from C. albicans biofilms are highly virulent and developmentally distinct from their parent biofilms. To study biofilm dispersed cells, it becomes imperative to isolate newly dispersed cells using a flow biofilm model. In this chapter, we detail the methods underlying assembly and workings of a simple flow biofilm model using materials commonly available in most microbiological laboratories. Biofilms developed using this system are robust and particularly suitable for studies requiring large amounts of biofilm (dispersed) cells for downstream analyses. Importantly, this apparatus mimics in vivo flow conditions, thereby making it a physiologically relevant model.

A Simple 96-Well Plate-Based Method for Development of Candida Biofilms Under Static Conditions.

We describe a rapid and simple in vitro method for development of Candida biofilms under static growth conditions. This 96-well microtiter-based method measures metabolic activity of sessile cells and can also be easily adapted for antifungal susceptibility testing. The entire procedure takes 2-3 days to complete, reliably quantifies biofilms, and provides reproducible results that are imperative toward the standardization of antifungal susceptibility testing of biofilms.

Niclosamide-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis.

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.

An evolutionarily diverged mitochondrial protein controls biofilm growth and virulence in Candida albicans.

A forward genetic screening approach identified orf19.2500 as a gene controlling Candida albicans biofilm dispersal and biofilm detachment. Three-dimensional (3D) protein modeling and bioinformatics revealed that orf19.2500 is a conserved mitochondrial protein, structurally similar to, but functionally diverged from, the squalene/phytoene synthases family. The C. albicans orf19.2500 is distinguished by 3 evolutionarily acquired stretches of amino acid inserts, absent from all other eukaryotes except a small number of ascomycete fungi. Biochemical assays showed that orf19.2500 is required for the assembly and activity of the NADH ubiquinone oxidoreductase Complex I (CI) of the respiratory electron transport chain (ETC) and was thereby named NDU1. NDU1 is essential for respiration and growth on alternative carbon sources, important for immune evasion, required for virulence in a mouse model of hematogenously disseminated candidiasis, and for potentiating resistance to antifungal drugs. Our study is the first report on a protein that sets the Candida-like fungi phylogenetically apart from all other eukaryotes, based solely on evolutionary "gain" of new amino acid inserts that are also the functional hub of the protein.

GRP78 and Integrins Play Different Roles in Host Cell Invasion during Mucormycosis.

Mucormycosis, caused by Rhizopus species, is a life-threatening fungal infection that occurs in patients immunocompromised by diabetic ketoacidosis (DKA), cytotoxic chemotherapy, immunosuppressive therapy, hematologic malignancies, or severe trauma. Inhaled Rhizopus spores cause pulmonary infections in patients with hematologic malignancies, while patients with DKA are much more prone to rhinoorbital/cerebral mucormycosis. Here, we show that Rhizopus delemar interacts with glucose-regulated protein 78 (GRP78) on nasal epithelial cells via its spore coat protein CotH3 to invade and damage the nasal epithelial cells. Expression of the two proteins is significantly enhanced by high glucose, iron, and ketone body levels (hallmark features of DKA), potentially leading to frequently lethal rhinoorbital/cerebral mucormycosis. In contrast, R. delemar CotH7 recognizes integrin ß1 as a receptor on alveolar epithelial cells, causing the activation of epidermal growth factor receptor (EGFR) and leading to host cell invasion. Anti-integrin ß1 antibodies inhibit R. delemar invasion of alveolar epithelial cells and protect mice from pulmonary mucormycosis. Our results show that R. delemar interacts with different mammalian receptors depending on the host cell type. Susceptibility of patients with DKA primarily to rhinoorbital/cerebral disease can be explained by host factors typically present in DKA and known to upregulate CotH3 and nasal GRP78, thereby trapping the fungal cells within the rhinoorbital milieu, leading to subsequent invasion and damage. Our studies highlight that mucormycosis pathogenesis can potentially be overcome by the development of novel customized therapies targeting niche-specific host receptors or their respective fungal ligands.IMPORTANCE Mucormycosis caused by Rhizopus species is a fungal infection with often fatal prognosis. Inhalation of spores is the major route of entry, with nasal and alveolar epithelial cells among the first cells that encounter the fungi. In patients with hematologic malignancies or those undergoing cytotoxic chemotherapy, Rhizopus causes pulmonary infections. On the other hand, DKA patients predominantly suffer from rhinoorbital/cerebral mucormycosis. The reason for such disparity in disease types by the same fungus is not known. Here, we show that the unique susceptibility of DKA subjects to rhinoorbital/cerebral mucormycosis is likely due to specific interaction between nasal epithelial cell GRP78 and fungal CotH3, the expression of which increases in the presence of host factors present in DKA. In contrast, pulmonary mucormycosis is initiated via interaction of inhaled spores expressing CotH7 with integrin ß1 receptor, which activates EGFR to induce fungal invasion of host cells. These results introduce a plausible explanation for disparate disease manifestations in DKA versus those in hematologic malignancy patients and provide a foundation for development of therapeutic interventions against these lethal forms of mucormycosis.

Monoclonal IgM Antibodies Targeting Candida albicans Hyr1 Provide Cross-Kingdom Protection Against Gram-Negative Bacteria.

Recent years have seen an unprecedented rise in the incidence of multidrug-resistant (MDR) Gram-negative bacteria (GNBs) such as Acinetobacter and Klebsiella species. In view of the shortage of novel drugs in the pipeline, alternative strategies to prevent, and treat infections by GNBs are urgently needed. Previously, we have reported that the Candida albicans hypha-regulated protein Hyr1 shares striking three-dimensional structural homology with cell surface proteins of Acinetobacter baumannii. Moreover, active vaccination with rHyr1p-N or passive immunization with anti-Hyr1p polyclonal antibody protects mice from Acinetobacter infection. In the present study, we use molecular modeling to guide design of monoclonal antibodies (mAbs) generated against Hyr1p and show them to bind to priority surface antigens of Acinetobacter and Klebsiella pneumoniae. The anti-Hyr1 mAbs block damage to primary endothelial cells induced by the bacteria and protect mice from lethal pulmonary infections mediated by A. baumannii or K. pneumoniae. Our current studies emphasize the potential of harnessing Hyr1p mAbs as a cross-kingdom immunotherapeutic strategy against MDR GNBs.

Candida auris Biofilm Colonization on Skin Niche Conditions.

Candida auris, an emerging multidrug-resistant yeast, has recently been associated with outbreaks of invasive infections in health care facilities worldwide. Its success as a nosocomial pathogen lies in its capability to sustain for prolonged periods in the intensive care unit (ICU), adeptly colonize skin, and spread among patients. Little is known of the mechanism behind the predilection of C. auris for skin or the extent of its resilience on it. Now, M. V. Horton, C. J. Johnson, J. F. Kernien, T. D. Patel, et al. (mSphere 5:e00910-19, 2020, https://doi.org/10.1128/mSphere.00910-19) demonstrate that in synthetic sweat medium designed to mimic axillary skin conditions, C. auris can grow into multilayers of cells called biofilms that can resist desiccation. C. auris' propensity to form biofilms was further elaborated using a novel ex vivo porcine skin model of skin colonization. These studies provide early evidence that C. auris biofilm cells persisting on skin could serve as source of continuing outbreaks in health care facilities. Interventions blocking C. auris biofilm growth on skin will help control the spread of this pathogen.

A Study of In vitro Antifungal Susceptibility Patterns of Dermatophytic Fungi at a Tertiary Care Center in Western India.

BACKGROUND: Recent years have seen an alarming rise in the prevalence of recalcitrant and relapsing dermatophyte infections in India associated with lack of clinical response to standard antifungal regimens. AIMS AND OBJECTIVES: A study was undertaken to identify the antifungal susceptibility patterns of dermatophyte species isolated from lesions of dermatophytoses in patients examined at our center. MATERIALS AND METHODS: A total of 85 patients with clinically diagnosed dermatophytoses were subjected to skin scrapings for potassium hydroxide mount (microscopic examination) and culture using Sabouraud's agar medium containing chloramphenicol and cycloheximide (incubated at 30°C). Antifungal susceptibilities [minimum inhibitory concentration-90 (MIC-90)] of the identified dermatophytes were tested for seven systemic and topical antifungal agents (terbinafine, griseofulvin, itraconazole, fluconazole, sertaconazole, ketoconazole, and clotrimazole) using Clinical and Laboratory Standards Institute broth microdilution method (M38-A). RESULTS: Trichophyton rubrum (50%) and Trichophyton mentagrophytes complex (47.2%) were the two major species isolated. Isolates of both showed downy and granular forms (61.11%, 38.89% and 32.35%, 67.65%, respectively). The overall in-vitro susceptibility profiles (MIC-90 ranges in µg/mL) of the seven drugs for T. rubrum and T. mentagrophytes complex respectively were as follows: terbinafine (0.008-0256, 0.016-0.256), griseofulvin (0.03-1, 0.06-1), itraconazole (0.125-2, 0.25-2), fluconazole (0.125-1, 0.25-32), sertaconazole (0.03-1, 0.03-1), ketoconazole (0.06-1, 0.125-1), and clotrimazole (0.03-2, 0.06-1). CONCLUSIONS: This study indicates a rising proportion of T. mentagrophytes complex with increased proportion of granular form (T. mentagrophytes var. mentagrophytes). This study represents the current antifungal susceptibility profile of dermatophytic infections in a tertiary care medical center in western India with rising MICs to terbinafine and itraconazole.

The NDV-3A vaccine protects mice from multidrug resistant Candida auris infection.

Candida auris is an emerging, multi-drug resistant, health care-associated fungal pathogen. Its predominant prevalence in hospitals and nursing homes indicates its ability to adhere to and colonize the skin, or persist in an environment outside the host-a trait unique from other Candida species. Besides being associated globally with life-threatening disseminated infections, C. auris also poses significant clinical challenges due to its ability to adhere to polymeric surfaces and form highly drug-resistant biofilms. Here, we performed bioinformatic studies to identify the presence of adhesin proteins in C. auris, with sequence as well as 3-D structural homologies to the major adhesin/invasin of C. albicans, Als3. Anti-Als3p antibodies generated by vaccinating mice with NDV-3A (a vaccine based on the N-terminus of Als3 protein formulated with alum) recognized C. auris in vitro, blocked its ability to form biofilms and enhanced macrophage-mediated killing of the fungus. Furthermore, NDV-3A vaccination induced significant levels of C. auris cross-reactive humoral and cellular immune responses, and protected immunosuppressed mice from lethal C. auris disseminated infection, compared to the control alum-vaccinated mice. The mechanism of protection is attributed to anti-Als3p antibodies and CD4+ T helper cells activating tissue macrophages. Finally, NDV-3A potentiated the protective efficacy of the antifungal drug micafungin, against C. auris candidemia. Identification of Als3-like adhesins in C. auris makes it a target for immunotherapeutic strategies using NDV-3A, a vaccine with known efficacy against other Candida species and safety as well as efficacy in clinical trials. Considering that C. auris can be resistant to almost all classes of antifungal drugs, such an approach has profound clinical relevance.

Anti-CotH3 antibodies protect mice from mucormycosis by prevention of invasion and augmenting opsonophagocytosis.

Mucorales are fungal pathogens that cause mucormycosis, a lethal angioinvasive disease. Previously, we demonstrated that Rhizopus, the most common cause of mucormycosis, invades endothelial cells by binding of its CotH proteins to the host receptor GRP78. Loss of CotH3 renders the fungus noninvasive and attenuates Rhizopus virulence in mice. Here, we demonstrate that polyclonal antibodies raised against peptides of CotH3 protected diabetic ketoacidotic (DKA) and neutropenic mice from mucormycosis compared to mice treated with control preimmune serum. Passive immunization with anti-CotH3 antibodies enhanced neutrophil inlfux and triggered Fc receptor-mediated enhanced opsonophagocytosis killing of Rhizopus delemar. Monoclonal antibodies raised against the CotH3 peptide also protected immunosuppressed mice from mucormycosis caused by R. delemar and other Mucorales and acted synergistically with antifungal drugs in protecting DKA mice from R. delemar infection. These data identify anti-CotH3 antibodies as a promising adjunctive immunotherapeutic option against a deadly disease that often poses a therapeutic challenge.

Candida albicans biofilm growth and dispersal: contributions to pathogenesis.

The fungal species Candida albicans is most frequently associated with biofilm formation in immune-compromised and medically compromised patients, and it is now firmly established that biofilm formation represents a major virulence factor during candidiasis. A growing body of evidence has demonstrated that C. albicans biofilm development is a highly regulated and coordinated process, where adhesive interactions, morphogenetic conversions, and consortial behavior play significant roles. Cells within the biofilms are protected from environmental stresses including host immune defenses and antifungal treatment, which carries important clinical consequences for the treatment of biofilm-associated infections. Dispersal of cells from biofilms represents one of the hallmarks of the biofilm life-style, and in the case of C. albicans dispersed cells are responsible for candidemia and dissemination leading to the establishment of invasive disease.

NDV-3A vaccination prevents C. albicans colonization of jugular vein catheters in mice.

NDV-3A, a novel fungal vaccine undergoing clinical trials, contains a recombinant version of the Candida albicans rAls3 N-terminus protein (rAls3p-N) in aluminum hydroxide. In a Phase 1b/2a clinical trial, NDV-3A protected women from recurrent vulvovaginal candidiasis. Here, we reveal that active immunization in mice with NDV-3A induces high titers of anti-rAls3p-N antibodies that interfere with C. albicans ability to adhere to and invade endothelial cells, and form biofilm in vitro. Anti-rAls3p-N antibodies also significantly inhibit yeast dispersal from the hyphal layers of biofilms. Compared to placebo, NDV-3A vaccination inhibited C. albicans dissemination to kidneys and prevented colonization of central venous catheters in mice. Overall, these preclinical studies suggest that NDV-3A may serve as an immunotherapeutic strategy for prevention of infections on indwelling medical devices.

Metabolic Profiling of Candida auris, a Newly-Emerging Multi-Drug Resistant Candida Species, by GC-MS.

Candida auris, a newly-emerging Candida species, is a serious global health threat due to its multi-drug resistant pattern, difficulty to diagnose, and the high mortality associated with its invasive and bloodstream infections. Unlike C. albicans, and C. dubliniensis which can form true hyphae, C. auris grows as yeast or pseudohyphae and is capable of developing biofilms. The reasons for the inability of C. auris to form true hyphae are currently unknown. Metabolites secreted by microorganisms, including Candida, are known as important factors in controlling morphogenesis and pathogenesis. Metabolic profiling of C. auris and C. albicans cultures was performed using gas chromatography⁻mass spectrometry (GC⁻MS). Compared to C. albicans, C. auris secreted several hyphae-inhibiting metabolites, including phenylethyl, benzyl and isoamyl alcohols. Furthermore, a biofilm-forming metabolite-tyrosol-was identified. On the other hand, several other biomarkers identified from C. auris but not from C. albicans cultures may be produced by the organism to overcome the host immune system or control fungal adaptations, and hence ease its invasion and infections. The results from this study are considered as the first identification of C. auris metabolic activities as a step forward to understand its virulence mechanisms.

Alexidine Dihydrochloride Has Broad-Spectrum Activities against Diverse Fungal Pathogens.

Invasive fungal infections due to Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans constitute a substantial threat to hospitalized immunocompromised patients. Further, the presence of drug-recalcitrant biofilms on medical devices and emergence of drug-resistant fungi, such as Candida auris, introduce treatment challenges with current antifungal drugs. Worse, currently there is no approved drug capable of obviating preformed biofilms, which increase the chance of infection relapses. Here, we screened a small-molecule New Prestwick Chemical Library, consisting of 1,200 FDA-approved off-patent drugs against C. albicans, C. auris, and A. fumigatus, to identify those that inhibit growth of all three pathogens. Inhibitors were further prioritized for their potency against other fungal pathogens and their ability to kill preformed biofilms. Our studies identified the bis-biguanide alexidine dihydrochloride (AXD) as a drug with the highest antifungal and antibiofilm activity against a diverse range of fungal pathogens. Finally, AXD significantly potentiated the efficacy of fluconazole against biofilms, displayed low mammalian cell toxicity, and eradicated biofilms growing in mouse central venous catheters in vivo, highlighting its potential as a pan-antifungal drug.IMPORTANCE The prevalence of fungal infections has seen a rise in the past decades due to advances in modern medicine leading to an expanding population of device-associated and immunocompromised patients. Furthermore, the spectrum of pathogenic fungi has changed, with the emergence of multidrug-resistant strains such as C. auris High mortality related to fungal infections points to major limitations of current antifungal therapy and an unmet need for new antifungal drugs. We screened a library of repurposed FDA-approved inhibitors to identify compounds with activities against a diverse range of fungi in varied phases of growth. The assays identified alexidine dihydrochloride (AXD) to have pronounced antifungal activity, including against preformed biofilms, at concentrations lower than mammalian cell toxicity. AXD potentiated the activity of fluconazole and amphotericin B against Candida biofilms in vitro and prevented biofilm growth in vivo Thus, AXD has the potential to be developed as a pan-antifungal, antibiofilm drug.

Candida albicans Dispersed Cells Are Developmentally Distinct from Biofilm and Planktonic Cells.

Candida albicans surface-attached biofilms such as those formed on intravenous catheters with direct access to the bloodstream often serve as a nidus for continuous release of cells capable of initiating new infectious foci. We previously reported that cells dispersed from a biofilm are yeast cells that originate from the top-most hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm dispersal yeast cells displayed enhanced virulence-associated characteristics and drug resistance. However, little is known about their molecular properties. To address that issue, in this study we aimed to define the molecular characteristics of these biofilm dispersal cells. We found that the inducer of dispersal, PES1, genetically interacts with the repressor of filamentation, NRG1, in a manner consistent with the definition of dispersed cells as yeast cells. Further, using a flow biofilm model, we performed comprehensive comparative RNA sequencing on freshly dispersed cells in order to identify unique transcriptomic characteristics. Gene expression analysis demonstrated that dispersed cells largely inherit a biofilm-like mRNA profile. Strikingly, however, dispersed cells seemed transcriptionally reprogrammed to acquire nutrients such as zinc and amino acids and to metabolize alternative carbon sources, while their biofilm-associated parent cells did not induce the same high-affinity transporters or express gluconeogenetic genes, despite exposure to the same nutritional signals. Collectively, the findings from this study characterize cell dispersal as an intrinsic step of biofilm development which generates propagules more adept at colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before the cells experience the associated external signals.IMPORTANCECandida albicans surface-attached biofilms serve as a reservoir of cells to perpetuate and expand an infection; cells released from biofilms on catheters have direct access to the bloodstream. Biofilm dispersal yeast cells exhibit enhanced adhesion, invasion, and biofilm formation compared to their planktonic counterparts. Here, we show using transcriptome sequencing (RNA-seq) that dispersed yeast cells are developmentally distinct from the cells in their parent biofilms as well as from planktonic yeast cells. Dispersal cells possess an anticipatory expression pattern that primes them to infect new sites in the host, to survive in nutrient-starved niches, and to invade new sites. These studies identified dispersal cells as a unique proliferative cell type of the biofilm and showed that they could serve as targets for antibiofilm drug development in the future.