About

Richard J. Bennett is the Charles A. and Helen B. Stuart Professor of Molecular Microbiology and Immunology and serves as the Chair of the Department of Molecular Microbiology and Immunology at Brown University. His research focuses on the biology of the human fungal pathogen Candida albicans, particularly on mechanisms used to generate phenotypic diversity. His work addresses epigenetic switching between alternative cell types and the genetic changes that occur in natural or recombining populations. Dr. Bennett's academic background includes undergraduate studies at Cambridge University, a PhD at the Imperial Cancer Research Fund in the UK, and postdoctoral studies at Harvard University and the University of California, San Francisco, before establishing his own laboratory at Brown University.

Research topics

• Genetics
• Biology
• Cell biology

Selected publications

• The transcriptional repressor Ssn6 modulates phase separation to regulate fungal gene expression

  mBio · 2026-03-30

  articleOpen accessSenior author

  ABSTRACT The transcriptional regulation of cell fate plays a central role in eukaryotic cell differentiation. In the human fungal pathogen Candida albicans , the white-to-opaque cell fate switch is controlled by an interconnected network of eight transcription factors (TFs). These include Ssn6, a negative regulator of the opaque state that can function with its co-repressor Tup1; together, they form a global repressor complex that is conserved from yeast to mammals. Here, we evaluated the roles of four Ssn6 domains (N, TPR, M, and CTD) in white-opaque switching. Loss of the prion-like N or M domains had limited effects on Ssn6 phenotypes. In contrast, the tetratricopeptide repeat (TPR) domain was critical for function, consistent with this domain mediating interactions with Tup1 and DNA-binding TFs. The intrinsically disordered C-terminal domain (CTD) showed complex roles; deletion of this domain increased Ssn6 activity whereas substitution of acidic residues within this region abolished Ssn6 function. Notably, these phenotypes were linked to the phase separation capacity of Ssn6, as changes to the TPR or CTD altered the properties of Ssn6 condensates in human U2OS cells. Experiments using purified Ssn6 (± Tup1) demonstrated that this protein is recruited into condensates formed by other white-opaque-regulating TFs and alters condensate properties in a TPR- and CTD-dependent manner. Together, these experiments reveal how individual Ssn6 domains can modulate the phase separation properties of DNA-binding TFs and thereby regulate gene expression and cell fate. IMPORTANCE The mechanisms by which transcription factors (TFs) and co-regulators control gene expression remain ill-defined. An interconnected network of TFs regulates an epigenetic switch between white and opaque states in Candida albicans and serves as a model to understand the transcriptional regulation of eukaryotic cell fate. Here, we examine the role of Ssn6, one of the eight core regulators of the white-opaque switch, and reveal key roles for both the structured TPR domain and the disordered C-terminal domain. We demonstrate that Ssn6 is readily incorporated into transcriptional condensates where it disrupts the liquid-like properties of these condensates, both in the presence and absence of the co-repressor Tup1. Together with studies in higher eukaryotes, these results suggest a conserved role for TPR-containing proteins in regulating gene expression via the modulation of the physical properties of transcriptional condensates.

  PublisherDOI

• A CandiChrome toolkit for multicolor labeling of <i>Candida</i> cells

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-05-12

  articleOpen accessSenior authorCorresponding

  Abstract Here, we develop CandiChrome, a multiplex labeling toolkit for Candida albicans , through combined in vitro and in vivo characterization of fluorescent proteins in a standard strain background. To this end, we screened 13 candidate fluorophores across the visible spectrum and assessed their practical performance based on brightness, stability, and usability. This analysis identified a seven-fluorophore set that achieved the most effective balance of signal strength, robustness, and compatibility. We used this optimized panel to build a modular multicolor platform that enables strain labeling, mixed-population imaging, and competition assays in C. albicans . This platform could resolve up to 21 distinct populations by flow cytometry and microscopy. Importantly, CandiChrome supported the resolution of differentially labeled populations both in vitro and in the murine host, supporting the simultaneous tracking of multiple strains in complex settings. Together, these results establish CandiChrome as a flexible platform for multiplex fungal imaging in a pathogenic species where multicolor tools remain underdeveloped.

  PublisherDOI

• Aneuploidy confers a unique transcriptional and phenotypic profile to Candida albicans

  Nature Communications · 2025-04-06 · 9 citations

  articleOpen access

  Inaccurate chromosome segregation can lead to the formation of aneuploid cells that harbor an imbalanced complement of chromosomes. Several fungal species are not only able to tolerate the detrimental effects of aneuploidy but can use it to adapt to environmental pressures. The fungal pathobiont Candida albicans frequently acquires supernumerary chromosomes that enable growth in the presence of antifungal drugs or in specific host niches, yet the transcriptional changes associated with aneuploidy are not globally defined. Here, a karyotypically diverse set of C. albicans strains revealed that expression generally correlated with gene copy number regardless of the strain karyotype. Unexpectedly, aneuploid strains shared a characteristic transcriptional profile that was distinct from a generalized environmental stress response previously defined in aneuploid yeast cells. This aneuploid transcriptional response led to altered growth and oxidative balances relative to euploid control strains. The increased expression of reactive oxygen species (ROS) mitigating enzymes in aneuploid cells reduced the levels of ROS but caused an acute sensitivity to both internal and external sources of oxidative stress. Taken together, our work demonstrates common transcriptional and phenotypic features of aneuploid C. albicans cells with consequences for infection of different host niches and susceptibility to environmental stimuli.

  PublisherOA PDFDOI

• Steering Clear of the ‘Fix-It’ Trap

  2025-02-25

  book-chapter1st authorCorresponding
  PublisherDOI

• Cross‐species rescue reveals sequence requirements for a rapidly evolving intrinsically disordered region

  PLoS Biology · 2025-09-25 · 2 citations

  articleOpen accessCorresponding

  Many proteins contain intrinsically disordered regions (IDRs) that are essential for their function but do not adopt a stable structure; instead, they exist as an ensemble of conformations. Because these regions lack fixed structural constraints, traditional structure-based and alignment-based approaches are often ineffective for studying their sequence-function relationships. Here, we present an approach that combines molecular evolution with genetic complementation to extract functional sequence features of an IDR. We use the budding yeast RNA-binding protein Rim4 as a model system. Rim4 is required for sporulation, and its IDR facilitates its dual role as a translational activator and repressor. Notably, Rim4's IDR supports assembly into an SDS-resistant amyloid-like form, which is required for its repressor function. We demonstrate that the Rim4 IDR is functionally conserved across orthologous sequences spanning more than 400 million years of evolution, despite extensive sequence divergence. Our results suggest that noncomplementing Rim4 IDRs generally evolve toward higher hydrophobicity, and that reducing hydrophobicity can refunctionalize a nonfunctional IDR sequence that diverged over 200 million years ago. In the refunctionalized IDR, the activator function is restored, whereas assembly into an amyloid-like form remains uncomplemented. Overall, our findings add to evidence that IDRs can perform multiple functions, with each role optimized by distinct biochemical properties, and that evolutionary pressure favoring one function may drive the IDR toward biochemical characteristics that compromise its other functions.

  PublisherDOI

• Sphingolipid Homeostasis, Mitochondrial Activity, and PKA Signaling Drive an Azole-Tolerant State

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-09-12

  preprintOpen access

  ABSTRACT Antifungal therapies frequently fail, resulting in persistent infections caused by the important opportunistic pathogen, Candida albicans , which is highly tolerant to azole drugs. Tolerance arises from a subpopulation of cells that survive prolonged drug exposure, yet the underlying mechanisms remain poorly defined. Here, transcription factor mutant library screens identified Mnl1 as a key repressor of tolerance. Loss of MNL1 elevated cAMP levels and activated the PKA pathway, linking Mnl1 to growth control and stress responses. Additionally, mnl1 cells exhibited altered sphingolipid composition, implicating Mnl1 in regulating membrane permeability, a key determinant of drug efflux and membrane integrity. Increased tolerance in mnl1 cells was also linked to increased mitochondrial function. Thus, Mnl1 regulates tolerance through coordinated control of sphingolipid metabolism, mitochondrial activity, and PKA signaling, highlighting that tolerant cells adopt a distinct physiological state primed to withstand antifungal stress.

  PublisherOA PDFDOI

• Mss2 shapes the virulence of <i>Candida albicans</i> through reactive oxygen species (ROS) and calcium signaling, independent of direct transcriptional control

  Virulence · 2025-11-20 · 1 citations

  articleOpen access

  . These findings refine our understanding of fungal invasion and virulence and reveal that targeting mitochondrial signaling could be an important area for antifungal therapeutic interventions.

  PublisherDOI

• Transcriptional control of <i>C. albicans</i> white-opaque switching and modulation by environmental cues and strain background

  mBio · 2025-04-09 · 1 citations

  articleOpen accessSenior author

  ABSTRACT The opportunistic fungal pathogen Candida albicans can undergo cellular transitions in response to environmental cues that impact its lifestyle and its interactions with the human host. This is exemplified by the white-opaque switch, which is a heritable transition between two phenotypic states that is regulated by a highly interconnected network of transcription factors (TFs). To obtain greater understanding of the transcriptional regulation of the switch, we generated a genome-wide, tetracycline-inducible TF library in the WO-1 strain background and identified those TFs whose forced expression induces white cells to switch to the opaque state. This set of opaque-inducing TFs was also evaluated for their ability to induce switching in a second strain background, that of the standard reference strain SC5314, as well as during growth on different laboratory media. These experiments identify 14 TFs that can drive white-to-opaque switching when overexpressed but that do so in a highly strain- and media-specific manner. In particular, changes in pH, amino acids, and zinc concentrations had marked effects on the ability of TFs to drive phenotypic switching. These results provide insights into the complex transcriptional regulation of switching in C. albicans and reveal that an interplay between genetic and environmental factors determines TF function and cell fate. IMPORTANCE The white-opaque switch in Candida albicans represents a model system for understanding an epigenetic switch in a eukaryotic pathogen. Here, we generated an inducible library of the set of transcription factors (TFs) present in C. albicans and identify 14 TFs that can drive the white-to-opaque transition when ectopically expressed. We demonstrate that several of these TFs induce the switch in a highly strain- and media-specific manner. This highlights that both strain background and changes in experimental conditions (including different water sources) can profoundly impact the phenotypic consequences of TF overexpression. Moreover, the inducible TF library provides an invaluable tool for the further analysis of TF function in this important human pathogen.

  PublisherDOI

• The <i>Candida albicans</i> transcription factor Efg1 governs hyphal morphogenesis independently of the cAMP-protein kinase A pathway

  mBio · 2025-10-31 · 1 citations

  articleOpen access

  ABSTRACT Candida albicans is one of the most common causes of human fungal disease. An intensively studied C. albicans virulence traits is its ability to adopt both yeast and filamentous morphologies. Accordingly, the regulation of the yeast-to-filament transition has been an area of intense study in medical mycology. A long-standing mechanistic paradigm in this field is that the cAMP-protein kinase A pathway phosphorylates a master transcriptional regulator of C. albicans filamentation, Efg1, to drive filamentation. This model has been generalized over the years despite the fact that the initial work showed that it applied to only specific in vitro filamentation conditions. Here, we reinvestigated the protein kinase A-Efg1 paradigm by generating new strains containing alleles of EFG1 with the putative protein kinase A phosphorylation site (T208) mutated to either Ala or Glu which blocks or mimics phosphorylation, respectively. We integrated these alleles into efg1 ∆∆ mutants so that they are expressed from the endogenous promoter. We assayed the effect of these Efg1 mutations on (i) in vitro filamentation under a wide range of inducing conditions, (ii) biofilm formation, (iii) in vivo filamentation, (iv) virulence in a model of disseminated candidiasis, and (v) gene expression during in vitro filamentation. Using updated genetic approaches, we found no evidence that blocking or mimicking protein kinase phosphorylation of Efg1 affected its function during filamentation, biofilm formation, or infection. Therefore, additional studies will be required to identify the mechanisms by which Efg1 is regulated during hyphal morphogenesis. IMPORTANCE Candida albicans is a common human fungal pathogen causing both superficial mucosal and life-threatening invasive disease. The virulence of C. albicans is associated with its ability to form filamentous hyphae and pseudohyphae. The regulation of this process is widely attributed to the phosphorylation of a transcription factor Efg1 by the protein kinase A signaling pathway. Since its initial description 25 years ago, this model has informed the design and interpretation of many studies of C. albicans . Through the use of updated genetic methods, we have found that protein kinase A-mediated phosphorylation of Efg1 is dispensable for filamentation, biofilm formation, and virulence in an experimental model of candidiasis. Although these data are negative in nature, the centrality of the protein kinase A-Efg1 paradigm to C. albicans pathogenesis studies increases their impact and should catalyze new studies to understand how this master regulator of C. albicans biology is itself regulated.

  PublisherDOI

• Gene dosage and protein valency impact phase separation and fungal cell fate

  PLoS Genetics · 2025-08-08 · 1 citations

  articleOpen accessSenior authorCorresponding

  Cell fate decisions in eukaryotes are regulated by interconnected networks of transcription factors (TFs) that drive heritable changes in identity. However, much is unknown about how TFs act together to control cell fate, despite links to cellular dysfunction and disease when TF function is aberrant. Here, we addressed the interplay between TFs that control heritable switching in the diploid fungal pathogen Candida albicans. This species can propagate in two distinct cell states, white and opaque, with epigenetic transitions between states regulated by a core network of eight TFs plus >100 auxiliary TFs. The role of these TFs was dissected using simple and complex haploinsufficiency (CHI) analyses to examine the impact of gene dosage on cell fate. Among single heterozygotes, loss of one allele of WOR1 had the greatest impact on white-opaque switching, consistent with its role as the master opaque regulator, while CHI analysis revealed strong genetic interactions between other core TFs including WOR3 and WOR4. Wor1 function was also highly sensitive to its interaction valency, a measure of the number of inter-molecular interactions it can undergo. Engineered strains with increased Wor1 valency, either via the addition of extra prion-like domains (PrLDs) or by forced dimerization, increased switching frequencies by up to two orders of magnitude. Increasing Wor1 valency increased its propensity to form phase-separated condensates both in vitro and in mammalian cells. Together, these experiments establish that changes to TF gene dosage and TF valency can alter cell fate determination, with these changes linked to the propensity of TFs to undergo condensate formation.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R21AI112363

  NIH · $447k · 2017

• NIH Grant R21AI122011

  NIH · $450k · 2018

• Characterization of the Secretome of P. Destructans, the Causative Agent of White-Nose Syndrome in Bats

  NSF · $500k · 2015–2019

• NIH Grant R21AI081560

  NIH · $442k · 2012

• NIH Grant R21AI139592

  NIH · $447k · 2021

Frequent coauthors

• Iuliana V. Ene

  Institut Pasteur

  69 shared

• Matthew P. Hirakawa

  Sandia National Laboratories California

  59 shared

• Matthew Z. Anderson

  University of Wisconsin–Madison

  40 shared

• Corey Frazer

  Providence College

  39 shared

• Shen-Huan Liang

  Brown University

  32 shared

• Pallavi Kakade

  Brown University

  28 shared

• Stephen K. Jones

  Vilnius University

  22 shared

• Grégory Thomson

  Institut Curie

  21 shared

Labs

• Bennett LabPI

Education

• B.A.

  Cambridge University

• Ph.D.

  Imperial Cancer Research Fund