The bacillithiol pathway is required for biofilm formation in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that can cause infections that range from superficial skin and mucosal infections to life threatening disseminated infections. S. aureus can attach to medical devices and host tissues and form biofilms that allow the bacteria to evade the host immune system and provide protection from antimicrobial agents. To counter host-generated oxidative and nitrosative stress mechanisms that are part of the normal host responses to invading pathogens, S. aureus utilizes low molecular weight (LMW) thiols, such as bacillithiol (BSH). Additionally, S. aureus synthesizes its own nitric oxide (NO), which combined with its downstream metabolites may also protect the bacteria against specific host responses. We have previously shown that LMW thiols are required for biofilm formation in Mycobacterium smegmatis and Pseudomonas aeruginosa. Here, we show that the S. aureus bshC mutant strain, which is defective in the last step of the BSH pathway and lacks BSH, is impaired in biofilm formation. We also identify a possible S-nitrosobacillithiol reductase (BSNOR), similar in sequence to an S-nitrosomycothiol reductase found in M. smegmatis and show that the putative S. aureus bsnoR mutant strain has reduced levels of BSH and decreased biofilm formation. Our studies also show that NO plays an important role in biofilm formation and that acidified sodium nitrite severely reduces biofilm thickness. These studies provide insight into the roles of oxidative and nitrosative stress mechanisms on biofilm formation and indicate that BSH and NO are key players in normal biofilm formation in S. aureus.

Inferring gene regulatory networks using transcriptional profiles as dynamical attractors.

Genetic regulatory networks (GRNs) regulate the flow of genetic information from the genome to expressed messenger RNAs (mRNAs) and thus are critical to controlling the phenotypic characteristics of cells. Numerous methods exist for profiling mRNA transcript levels and identifying protein-DNA binding interactions at the genome-wide scale. These enable researchers to determine the structure and output of transcriptional regulatory networks, but uncovering the complete structure and regulatory logic of GRNs remains a challenge. The field of GRN inference aims to meet this challenge using computational modeling to derive the structure and logic of GRNs from experimental data and to encode this knowledge in Boolean networks, Bayesian networks, ordinary differential equation (ODE) models, or other modeling frameworks. However, most existing models do not incorporate dynamic transcriptional data since it has historically been less widely available in comparison to "static" transcriptional data. We report the development of an evolutionary algorithm-based ODE modeling approach (named EA) that integrates kinetic transcription data and the theory of attractor matching to infer GRN architecture and regulatory logic. Our method outperformed six leading GRN inference methods, none of which incorporate kinetic transcriptional data, in predicting regulatory connections among TFs when applied to a small-scale engineered synthetic GRN in Saccharomyces cerevisiae. Moreover, we demonstrate the potential of our method to predict unknown transcriptional profiles that would be produced upon genetic perturbation of the GRN governing a two-state cellular phenotypic switch in Candida albicans. We established an iterative refinement strategy to facilitate candidate selection for experimentation; the experimental results in turn provide validation or improvement for the model. In this way, our GRN inference approach can expedite the development of a sophisticated mathematical model that can accurately describe the structure and dynamics of the in vivo GRN.

Triazine-Based Small Molecules: A Potential New Class of Compounds in the Antifungal Toolbox.

Invasive fungal infections caused by Candida species remain a significant public health problem worldwide. The increasing prevalence of drug-resistant infections and a limited arsenal of antifungal drugs underscore the need for novel interventions. Here, we screened several classes of pharmacologically active compounds against mammalian diseases for antifungal activity. We found that the synthetic triazine-based compound melanogenin (Mel) 56 is fungicidal in Candida albicans laboratory and clinical strains with minimal inhibitory concentrations of 8−16 µg/mL. Furthermore, Mel56 has general antifungal activity in several non-albicans Candida species and the non-pathogenic yeast Saccharomyces cerevisiae. Surprisingly, Mel56 inhibited the yeast-to-hyphae transition at sublethal concentrations, revealing a new role for triazine-based compounds in fungi. In human cancer cell lines, Mel56 targets the inner mitochondrial integral membrane prohibitin proteins, PHB1 and PHB2. However, Mel56 treatment did not impact C. albicans mitochondrial activity, and antifungal activity was similar in prohibitin single, double, and triple homozygous mutant strains compared to the wild-type parental strain. These results suggests that Mel56 has a novel mechanism-of-action in C. albicans. Therefore, Mel56 is a promising antifungal candidate warranting further analyses.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow.

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&RUN data analysis.

A Markerless CRISPR-Mediated System for Genome Editing in Candida auris Reveals a Conserved Role for Cas5 in the Caspofungin Response.

Candida auris is a multidrug-resistant human fungal pathogen that has recently emerged worldwide. It can cause life-threatening disseminated infections in humans, with mortality rates upwards of 50%. The molecular mechanisms underlying its multidrug resistance and pathogenic properties are largely unknown. Few methods exist for genome editing in C. auris, all of which rely on selectable markers that limit the number of modifications that can be made. Here, we present a markerless CRISPR/Cas9-mediated genome editing system in C. auris. Using this system, we successfully deleted genes of interest and subsequently reconstituted them at their native loci in isolates across all five C. auris clades. This system also enabled us to introduce precision genome edits to create translational fusions and single point mutations. Using Cas5 as a test case for this system, we discovered a conserved role for Cas5 in the caspofungin response between Candida albicans and C. auris. Overall, the development of a system for precise and facile genome editing in C. auris that can allow edits to be made in a high-throughput manner is a major step forward in improving our understanding of this important human fungal pathogen. IMPORTANCE Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing life-threatening systemic infections in humans. Few tools are available for genome editing in C. auris. Here, we present a markerless genome editing system for C. auris that relies on CRISPR/Cas9 technology and works to modify the genomes of all known C. auris clades. Using this system, we discovered a conserved role for Cas5 in the caspofungin response between C. albicans and C. auris. Overall, the development of a system for facile genome editing in C. auris is a major step forward in improving our understanding of this important human fungal pathogen.

AddTag, a two-step approach with supporting software package that facilitates CRISPR/Cas-mediated precision genome editing.

CRISPR/Cas-induced genome editing is a powerful tool for genetic engineering, however, targeting constraints limit which loci are editable with this method. Since the length of a DNA sequence impacts the likelihood it overlaps a unique target site, precision editing of small genomic features with CRISPR/Cas remains an obstacle. We introduce a two-step genome editing strategy that virtually eliminates CRISPR/Cas targeting constraints and facilitates precision genome editing of elements as short as a single base-pair at virtually any locus in any organism that supports CRISPR/Cas-induced genome editing. Our two-step approach first replaces the locus of interest with an "AddTag" sequence, which is subsequently replaced with any engineered sequence, and thus circumvents the need for direct overlap with a unique CRISPR/Cas target site. In this study, we demonstrate the feasibility of our approach by editing transcription factor binding sites within Candida albicans that could not be targeted directly using the traditional gene-editing approach. We also demonstrate the utility of the AddTag approach for combinatorial genome editing and gene complementation analysis, and we present a software package that automates the design of AddTag editing.

The Roles of Chromatin Accessibility in Regulating the Candida albicans White-Opaque Phenotypic Switch.

Candida albicans, a diploid polymorphic fungus, has evolved a unique heritable epigenetic program that enables reversible phenotypic switching between two cell types, referred to as "white" and "opaque". These cell types are established and maintained by distinct transcriptional programs that lead to differences in metabolic preferences, mating competencies, cellular morphologies, responses to environmental signals, interactions with the host innate immune system, and expression of approximately 20% of genes in the genome. Transcription factors (defined as sequence specific DNA-binding proteins) that regulate the establishment and heritable maintenance of the white and opaque cell types have been a primary focus of investigation in the field; however, other factors that impact chromatin accessibility, such as histone modifying enzymes, chromatin remodelers, and histone chaperone complexes, also modulate the dynamics of the white-opaque switch and have been much less studied to date. Overall, the white-opaque switch represents an attractive and relatively "simple" model system for understanding the logic and regulatory mechanisms by which heritable cell fate decisions are determined in higher eukaryotes. Here we review recent discoveries on the roles of chromatin accessibility in regulating the C. albicans white-opaque phenotypic switch.

N-Acetylglucosamine (GlcNAc) Sensing, Utilization, and Functions in Candida albicans.

The sensing and efficient utilization of environmental nutrients are critical for the survival of microorganisms in environments where nutrients are limited, such as within mammalian hosts. Candida albicans is a common member of the human microbiota as well as an opportunistic fungal pathogen. The amide derivative sugar N-acetlyglucosamine (GlcNAc) is an important signaling molecule for C. albicans that could be a major nutrient source for this fungus in host settings. In this article, we review progress made over the past two decades on GlcNAc utilization, sensing, and functions in C. albicans and its related fungal species. GlcNAc sensing and catabolic pathways have been intensively studied in C. albicans. The C. albicans protein Ngt1 represents the first identified GlcNAc-specific transporter in eukaryotic organisms. In C. albicans, GlcNAc not only induces morphological transitions including the yeast to hyphal transition and the white to opaque phenotypic switch, but it also promotes fungal cell death. The Ras-cAMP/PKA signaling pathway plays critical roles in regulating these processes. Given the importance of GlcNAc sensing and utilization in C. albicans, targeting GlcNAc associated pathways and key pathway components could be promising in the development of new antifungal strategies.

Epigenetic cell fate in Candida albicans is controlled by transcription factor condensates acting at super-enhancer-like elements.

Cell identity in eukaryotes is controlled by transcriptional regulatory networks that define cell-type-specific gene expression. In the opportunistic fungal pathogen Candida albicans, transcriptional regulatory networks regulate epigenetic switching between two alternative cell states, 'white' and 'opaque', that exhibit distinct host interactions. In the present study, we reveal that the transcription factors (TFs) regulating cell identity contain prion-like domains (PrLDs) that enable liquid-liquid demixing and the formation of phase-separated condensates. Multiple white-opaque TFs can co-assemble into complex condensates as observed on single DNA molecules. Moreover, heterotypic interactions between PrLDs support the assembly of multifactorial condensates at a synthetic locus within live eukaryotic cells. Mutation of the Wor1 TF revealed that substitution of acidic residues in the PrLD blocked its ability to phase separate and co-recruit other TFs in live cells, as well as its function in C. albicans cell fate determination. Together, these studies reveal that PrLDs support the assembly of TF complexes that control fungal cell identity and highlight parallels with the 'super-enhancers' that regulate mammalian cell fate.

Transcriptional control of hyphal morphogenesis in Candida albicans.

Candida albicans is a multimorphic commensal organism and opportunistic fungal pathogen in humans. A morphological switch between unicellular budding yeast and multicellular filamentous hyphal growth forms plays a vital role in the virulence of C. albicans, and this transition is regulated in response to a range of environmental cues that are encountered in distinct host niches. Many unique transcription factors contribute to the transcriptional regulatory network that integrates these distinct environmental cues and determines which phenotypic state will be expressed. These hyphal morphogenesis regulators have been extensively investigated, and represent an increasingly important focus of study, due to their central role in controlling a key C. albicans virulence attribute. This review provides a succinct summary of the transcriptional regulatory factors and environmental signals that control hyphal morphogenesis in C. albicans.

Unraveling How Candida albicans Forms Sexual Biofilms.

Biofilms, structured and densely packed communities of microbial cells attached to surfaces, are considered to be the natural growth state for a vast majority of microorganisms. The ability to form biofilms is an important virulence factor for most pathogens, including the opportunistic human fungal pathogen Candida albicans. C. albicans is one of the most prevalent fungal species of the human microbiota that asymptomatically colonizes healthy individuals. However, C. albicans can also cause severe and life-threatening infections when host conditions permit (e.g., through alterations in the host immune system, pH, and resident microbiota). Like many other pathogens, this ability to cause infections depends, in part, on the ability to form biofilms. Once formed, C. albicans biofilms are often resistant to antifungal agents and the host immune response, and can act as reservoirs to maintain persistent infections as well as to seed new infections in a host. The majority of C. albicans clinical isolates are heterozygous (a/α) at the mating type-like (MTL) locus, which defines Candida mating types, and are capable of forming robust biofilms when cultured in vitro. These "conventional" biofilms, formed by MTL-heterozygous (a/α) cells, have been the primary focus of C. albicans biofilm research to date. Recent work in the field, however, has uncovered novel mechanisms through which biofilms are generated by C. albicans cells that are homozygous or hemizygous (a/a, a/Δ, α/α, or α/Δ) at the MTL locus. In these studies, the addition of pheromones of the opposite mating type can induce the formation of specialized "sexual" biofilms, either through the addition of synthetic peptide pheromones to the culture, or in response to co-culturing of cells of the opposite mating types. Although sexual biofilms are generally less robust than conventional biofilms, they could serve as a protective niche to support genetic exchange between mating-competent cells, and thus may represent an adaptive mechanism to increase population diversity in dynamic environments. Although conventional and sexual biofilms appear functionally distinct, both types of biofilms are structurally similar, containing yeast, pseudohyphal, and hyphal cells surrounded by an extracellular matrix. Despite their structural similarities, conventional and sexual biofilms appear to be governed by distinct transcriptional networks and signaling pathways, suggesting that they may be adapted for, and responsive to, distinct environmental conditions. Here we review sexual biofilms and compare and contrast them to conventional biofilms of C. albicans.

Transcriptional Circuits Regulating Developmental Processes in Candida albicans.

Candida albicans is a commensal member of the human microbiota that colonizes multiple niches in the body including the skin, oral cavity, and gastrointestinal and genitourinary tracts of healthy individuals. It is also the most common human fungal pathogen isolated from patients in clinical settings. C. albicans can cause a number of superficial and invasive infections, especially in immunocompromised individuals. The ability of C. albicans to succeed as both a commensal and a pathogen, and to thrive in a wide range of environmental niches within the host, requires sophisticated transcriptional regulatory programs that can integrate and respond to host specific environmental signals. Identifying and characterizing the transcriptional regulatory networks that control important developmental processes in C. albicans will shed new light on the strategies used by C. albicans to colonize and infect its host. Here, we discuss the transcriptional regulatory circuits controlling three major developmental processes in C. albicans: biofilm formation, the white-opaque phenotypic switch, and the commensal-pathogen transition. Each of these three circuits are tightly knit and, through our analyses, we show that they are integrated together by extensive regulatory crosstalk between the core regulators that comprise each circuit.

Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9.

CRISPR-Cas has proven to be a powerful tool for precision genetic engineering in a variety of difficult genetic systems. In the highly tractable yeast S. cerevisiae, CRISPR-Cas can be used to conduct multiple engineering steps in parallel, allowing for engineering of complex metabolic pathways at multiple genomic loci in as little as 1 week. In addition, CRISPR-Cas can be used to consolidate multiple causal alleles into a single strain, bypassing the laborious traditional methods using marked constructs, or mating. These tools compress the engineering timeline sixfold or more, greatly increasing the productivity of the strain engineer.

Monitoring Phenotypic Switching in Candida albicans and the Use of Next-Gen Fluorescence Reporters.

Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white-opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.

Whole RNA-Sequencing and Transcriptome Assembly of Candida albicans and Candida africana under Chlamydospore-Inducing Conditions.

Candida albicans is the most common cause of life-threatening fungal infections in humans, especially in immunocompromised individuals. Crucial to its success as an opportunistic pathogen is the considerable dynamism of its genome, which readily undergoes genetic changes generating new phenotypes and shaping the evolution of new strains. Candida africana is an intriguing C. albicans biovariant strain that exhibits remarkable genetic and phenotypic differences when compared with standard C. albicans isolates. Candida africana is well-known for its low degree of virulence compared with C. albicans and for its inability to produce chlamydospores that C. albicans, characteristically, produces under certain environmental conditions. Chlamydospores are large, spherical structures, whose biological function is still unknown. For this reason, we have sequenced, assembled, and annotated the whole transcriptomes obtained from an efficient C. albicans chlamydospore-producing clinical strain (GE1), compared with the natural chlamydospore-negative C. africana clinical strain (CBS 11016). The transcriptomes of both C. albicans (GE1) and C. africana (CBS 11016) clinical strains, grown under chlamydospore-inducing conditions, were sequenced and assembled into 7,442 (GE1 strain) and 8,370 (CBS 11016 strain) high quality transcripts, respectively. The release of the first assembly of the C. africana transcriptome will allow future comparative studies to better understand the biology and evolution of this important human fungal pathogen.

An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans.

Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCECandida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

Systematic Genetic Screen for Transcriptional Regulators of the Candida albicans White-Opaque Switch.

The human fungal pathogen Candida albicans can reversibly switch between two cell types named "white" and "opaque," each of which is stable through many cell divisions. These two cell types differ in their ability to mate, their metabolic preferences and their interactions with the mammalian innate immune system. A highly interconnected network of eight transcriptional regulators has been shown to control switching between these two cell types. To identify additional regulators of the switch, we systematically and quantitatively measured white-opaque switching rates of 196 strains, each deleted for a specific transcriptional regulator. We identified 19 new regulators with at least a 10-fold effect on switching rates and an additional 14 new regulators with more subtle effects. To investigate how these regulators affect switching rates, we examined several criteria, including the binding of the eight known regulators of switching to the control region of each new regulatory gene, differential expression of the newly found genes between cell types, and the growth rate of each mutant strain. This study highlights the complexity of the transcriptional network that regulates the white-opaque switch and the extent to which switching is linked to a variety of metabolic processes, including respiration and carbon utilization. In addition to revealing specific insights, the information reported here provides a foundation to understand the highly complex coupling of white-opaque switching to cellular physiology.

Ssn6 Defines a New Level of Regulation of White-Opaque Switching in Candida albicans and Is Required For the Stochasticity of the Switch.

UNLABELLED: The human commensal and opportunistic pathogen Candida albicans can switch between two distinct, heritable cell types, named "white" and "opaque," which differ in morphology, mating abilities, and metabolic preferences and in their interactions with the host immune system. Previous studies revealed a highly interconnected group of transcriptional regulators that control switching between the two cell types. Here, we identify Ssn6, the C. albicans functional homolog of the Saccharomyces cerevisiae transcriptional corepressor Cyc8, as a new regulator of white-opaque switching. In A: or α mating type strains, deletion of SSN6 results in mass switching from the white to the opaque cell type. Transcriptional profiling of ssn6 deletion mutant strains reveals that Ssn6 represses part of the opaque cell transcriptional program in white cells and the majority of the white cell transcriptional program in opaque cells. Genome-wide chromatin immunoprecipitation experiments demonstrate that Ssn6 is tightly integrated into the opaque cell regulatory circuit and that the positions to which it is bound across the genome strongly overlap those bound by Wor1 and Wor2, previously identified regulators of white-opaque switching. This work reveals the next layer in the white-opaque transcriptional circuitry by integrating a transcriptional regulator that does not bind DNA directly but instead associates with specific combinations of DNA-bound transcriptional regulators. IMPORTANCE: The most common fungal pathogen of humans, C. albicans, undergoes several distinct morphological transitions during interactions with its host. One such transition, between cell types named "white" and "opaque," is regulated in an epigenetic manner, in the sense that changes in gene expression are heritably maintained without any modification of the primary genomic DNA sequence. Prior studies revealed a highly interconnected network of sequence-specific DNA-binding proteins that control this switch. We report the identification of Ssn6, which defines an additional layer of transcriptional regulation that is critical for this heritable switch. Ssn6 is necessary to maintain the white cell type and to properly express the opaque cell transcriptional program. Ssn6 does not bind DNA directly but rather associates with specific combinations of DNA-bound transcriptional regulators to control the switch. This work is significant because it reveals a new level of regulation of an important epigenetic switch in the predominant fungal pathogen of humans.

Genome-Wide Chromatin Immunoprecipitation in Candida albicans and Other Yeasts.

Chromatin immunoprecipitation experiments are critical to investigating the interactions between DNA and a wide range of nuclear proteins within a cell or biological sample. In this chapter we outline an optimized protocol for genome-wide chromatin immunoprecipitation that has been used successfully for several distinct morphological forms of numerous yeast species, and include an optimized method for amplification of chromatin immunoprecipitated DNA samples and hybridization to a high-density oligonucleotide tiling microarray. We also provide detailed suggestions on how to analyze the complex data obtained from these experiments.

Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas.

CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs. The gRNA cassettes can be easily made by PCR and delivered in any combination. We employed this method to rapidly phenotype up to five specific allele combinations and identify synergistic effects. To prototype a pathway for the production of muconic acid, we integrated six DNA fragments totaling 24 kb across three loci in naive Saccharomyces cerevisiae in a single transformation. With minor modifications, we integrated a similar pathway in Kluyveromyces lactis. The flexibility afforded by combinatorial gRNA delivery dramatically accelerates complex strain engineering for basic research and industrial fermentation.