Biology subjects

Mitchell, A. P.

Publications and source records attributed to Mitchell, A. P..

11 recordsLinked to original sources

bioRxiv2026

Two Ume6 target gene classes in the biofilm regulatory network of Candida albicans

Biofilm formation by the fungal pathogen Candida albicans is a major source of infection. The biofilm regulator Ume6 forms complexes with partner transcription factors Efg1, Ndt80, and Upc2 to drive expression of biofilm-related genes. Here we present chromatin immunoprecipitation with sequencing (ChIP-seq) data for Ume6-chromatin association in both wild-type and efg1{Delta}/{Delta} ndt80{Delta}/{Delta} upc2{Delta}/{Delta} backgrounds. Ume6 associates with two promoter region classes. For one class, Ume6 association is significantly decreased in the efg1{Delta}/{Delta} ndt80{Delta}/{Delta} upc2{Delta}/{Delta} background. This class includes 577 genes, many with well-established roles in biofilm formation or the related process of filamentation. For the second class, Ume6 association is unperturbed in the triple mutant background. This class includes 534 genes, some with roles in iron homeostasis (e.g., SFU1) and ergosterol synthesis (e.g., ERG11), which may contribute to the impact of Ume6 on ndt80{Delta}/{Delta} mutant azole drug sensitivity. It also includes FLO9, a putative adhesin gene that is required for the emergent biofilm state produced by efg1{Delta}/{Delta} ndt80{Delta}/{Delta} double mutants. The data show that Ume6 binding to ~half of its targets requires known partners, and Ume6 binding to ~half of its targets does not. Ume6 may bind to the latter set of promoter regions independently of any partner, or perhaps with additional partners that have yet to be discovered.

microbiology↗

bioRxiv2026

A negative role for master regulator Bcr1 in Candida albicans hypha-associated gene expression

Filamentation, a central virulence trait of the fungal pathogen Candida albicans, is required for host cell damage, tissue invasion, and biofilm formation. The transcription factor Bcr1 was first found to be required for biofilm formation but not filamentation. Subsequent studies revealed a negative role for Bcr1 in filamentation in opaque cells, a cell type that is required for mating, under hypoxic conditions, and in a wor2del/del mutant background. Here we characterize a new context in which Bcr1 negatively regulates filamentation and present its associated gene expression impact. We compared the wild-type and bcr1del/del mutant cells in the SC5314 reference strain background at 30 degrees C in glucose media, conditions that do not induce filamentation. The bcr1del/del mutant displayed increased invasive growth in a solid medium, and some filamentation ability in a liquid medium. The bcr1del/del effect on filamentation was augmented by overexpression of hyphal cyclin gene HGC1, which caused pseudohyphal growth in wild-type, efg1del/del, or brg1del/del strains, yet caused hyphal growth in the bcr1del/del strain. RNA-sequencing (RNA-seq) analysis of 30 degrees C cells shows that the bcr1del/del mutant has elevated expression of two genes that drive filamentation: HGC1 and UME6. In published data these same two genes have decreased expression in the bcr1del/del mutant at 37 degrees C, the temperature at which biofilm formation is typically assayed. A growing cadre of biofilm/hyphal regulators can have both positive and negative effects on hypha-associated gene expression, including Bcr1, Efg1, Ndt80, and Nrg1.

microbiology↗

bioRxiv2025

Characterization of ORF19.7608 (PPP1), a Biofilm-induced Gene of Candida albicans

The opportunistic human pathogen Candida albicans is an important cause of nosocomial infections, in large part because of its propensity to form biofilms on indwelling medical devices such as catheters. The formation of these biofilms is controlled by a complex transcriptional network and involves over a thousand genes, many of which are uncharacterized. We have investigated three genes (ORF19.4654, ORF19.7608, and PBR1), found only in C. albicans and closely related species, that are highly induced under biofilm conditions and encode small proteins with N-terminal signal sequences. Through the construction of fluorescent protein fusions, we have examined the location of the encoded proteins in both planktonic and biofilm cells. Orf19.4654-Scarlet and Pbr1-Scarlet were localized to the vacuole under both conditions. In contrast, the Orf19.7608-GFP fusion generated a punctate pattern only under biofilm conditions and was designated Ppp1 (Punctate Pattern Protein 1). The Ppp1-GFP puncta were similar in location, stability, and size to those formed by the eisosome subunit Sur7, but co-localization studies suggest that Ppp1 and Sur7 define separate elements. The PPP1 mutation does not cause a distinct phenotype under various stress conditions or in the presence of antifungals and does not impact biofilm formation and biomass. These data suggest that while the expression and cellular localization of Ppp1 appear controlled by conditions generating biofilms, and define a unique subcellular localization pattern, Ppp1 protein function is not essential for biofilm formation. IMPORTANCEBiofilm formation is a virulence factor of medical importance in C. albicans. Identifying the biological function and cellular localization of biofilm-related proteins can help in their characterization and understanding of the biofilm network. Microscopy was employed to identify the subcellular localization of putative biofilm proteins, aiming to provide insight into their possible functions. Prb1 and Orf19.4654 localized to the vacuole, while Orf19.7608 (Ppp1) formed puncta. Phenotypic assays to investigate the uniqueness of PPP1 revealed that it does not play a major role in the stress response pathways or antifungal activity. Overall, our study provides insight into the localization of the products of Candida-specific biofilm genes.

microbiology↗

bioRxiv2024

Roles of P-body factors in Candida albicans filamentation and stress response

Hyphal growth is strongly associated with virulence in the human fungal pathogen Candida albicans. While hyphal transcriptional networks have been the subject of intense study, relatively little is known about post-transcriptional regulation. Previous work reported that P-Body (PB) factors Dhh1 and Edc3 were required for C. albicans virulence and filamentation, suggesting an essential role for post-transcriptional regulation of these processes. However, the molecular roles of these factors have not been determined. To further study the function of PB factors in filamentation, we generated homozygous deletions of DHH1 and EDC3 in diverse prototrophic clinical strains using transient CRISPR-Cas9. Homozygous DHH1 deletion strongly impaired growth, altered filamentation, and exhibited unusual colony morphology in response to heat stress in five strain backgrounds. Using RNA-seq, we found DHH1 deletion disrupts the regulation of thousands of genes under both yeast and hyphal growth conditions in SC5314 and P57055. This included upregulation of many stress response genes in the absence of external stress, similar to deletion of the S. cerevisiae DHH1 homolog. In contrast, we found EDC3 was not required for heat tolerance or filamentation in diverse strains. These results support a model in which DHH1, but not EDC3, represses hyphal stress response transcripts in yeast and remodels the transcriptome during filamentation. Our work supports distinct requirements for specific mRNA decay factors, bolstering evidence for post-transcriptional regulation of filamentation in C. albicans. Author summaryIn the dimorphic fungal pathogen C. albicans, the hyphal phenotype strongly correlates with pathogenicity. While transcriptional control of hyphal growth has been extensively studied, comparatively little is known about post-transcriptional regulation of this significant morphological shift. PB factors are associated with mRNA decay and translational repression. Here we investigate the roles of two PB factors in growth, filamentation, and gene expression. Although deletion of PB factor EDC3 did not impact growth or filamentation, dhh1{Delta}/{Delta} had greatly impaired growth and heat tolerance as well as unusual hyphal phenotypes in multiple clinical isolate strains. Additionally, we found that the transcriptomes of SC5314 and P57055 dhh1{Delta}/{Delta} yeast and hyphae were highly dysregulated. The extensive transcriptomic impacts of the absence of Dhh1 correlated with our phenotypic findings. Stress-associated genes were induced under non-stress conditions and the filamentation response was blunted under physiologically relevant in vitro conditions. We demonstrate that mRNA decay factors play distinct roles in regulating C. albicans morphology and that Dhh1 contributes to environmentally appropriate expression of the stress response and hyphal growth.

genetics↗

bioRxiv2023

Candida albicans stimulates the formation of a multi-receptor complex that mediates epithelial cell invasion during oropharyngeal infection

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans. Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 were required for C. albicans stimulation of c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorated OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/529756v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1448372org.highwire.dtl.DTLVardef@111b411org.highwire.dtl.DTLVardef@144f3b8org.highwire.dtl.DTLVardef@1dcf548_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIc-Met is an oral epithelial cell receptor for Candida albicans C_LIO_LIC. albicans infection causes c-Met and the epidermal growth factor receptor (EGFR) to form a complex with E-cadherin, which is required for c-Met and EGFR function C_LIO_LIC. albicans Hyr1 and Als3 interact with c-Met and EGFR, inducing oral epithelial cell endocytosis and virulence during oropharyngeal candidiasis C_LIO_LIDual blockade of c-Met and EGFR ameliorates oropharyngeal candidiasis C_LI

microbiology↗

bioRxiv2021

Identification of Serum Bridging Molecules that Mediate Human Endothelial Cell Invasion by Candida species

During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although Candida albicans, which forms hyphae, readily invades endothelial cells, other medically important species of Candida are poorly invasive in standard in vitro assays. Here, we show that Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei can bind to vitronectin and high molecular weight kininogen present in human serum. Acting as bridging molecules, vitronectin and kininogen bind to v integrins and the globular C1q receptor (gC1qR), inducing human endothelial cells to endocytose the fungus. This mechanism of endothelial cell invasion is poorly supported by mouse endothelial cells, but can be restored when mouse endothelial cells are engineered to express human gC1qR or v integrin. Overall, these data indicate that bridging molecule-mediated endocytosis is a common pathogenic strategy used by many medically important Candida spp. to invade human vascular endothelial cells. SignificanceThe invasion of vascular endothelial cells is a key step in the pathogenesis of hematogenously disseminated candidiasis. How species of Candida other than C. albicans invade endothelial cells is poorly understood because these fungi are weakly invasive in serum-free media. Here, we demonstrate that C. glabrata and other Candida spp. bind to the serum proteins kininogen and vitronectin, which act as bridging molecules and mediate the adherence and endocytosis of the organisms by endothelial cells. These serum proteins induce endocytosis when they interact with the globular C1q receptor and v integrins on human, but not mouse endothelial cells. Thus, bridging molecule-mediated endocytosis is a common mechanism by which medically important Candida spp. invade human endothelial cells.

microbiology↗

bioRxiv2021

Inferring Mycobacterium bovis transmission between cattle and badgers using isolates from the Randomised Badger Culling Trial

Mycobacterium bovis (M. bovis) is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial, M. bovis isolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission of M. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger.

genomics↗

bioRxiv2021

In vivo complex haploinsufficiency-based genetic analysis identifies a transcription factor circuit regulating Candida albicans oropharyngeal infection and epithelial cell endocytosis

Oropharyngeal candidiasis (OPC) is a common infection that complicates a wide range of medical conditions which can cause either mild or severe disease depending on the patient. The pathobiology of OPC shares many features with candidal biofilms of abiotic surfaces. The transcriptional regulation of C. albicans formation of biofilms on abiotic surfaces has been extensively characterized and involves six key transcription factors (Efg1, Ndt80, Rob1, Bcr1, Brg1, and Tec1). To determine whether this same in vitro biofilm transcriptional regulatory network played a role in OPC, we have carried out the first systematic genetic interaction analysis in a mouse model of C. albicans infection. Whereas all six transcription factors are required for in vitro biofilm formation, only three homozygous deletion mutants (tec1{Delta}{Delta}, bcr1{Delta}{Delta}, and rob1{Delta}{Delta}) and one heterozygous mutant (tec1{Delta}/TEC1) have reduced infectivity in a mouse model of OPC, indicating the network is more robust in vivo than in vitro. Although single mutants (heterozygous or homozygous) of BRG1 and EFG1 have no effect on fungal burden, the double heterozygous and homozygous mutants have dramatically reduced infectivity, indicating a critical genetic interaction between these two transcription factors. Using epistasis analysis, we have formulated a genetic circuit [EFG1+BRG1][->]TEC1[->]BCR1 that is required for OPC infectivity and oral epithelial cell endocytosis. Surprisingly, we also found transcription factor mutants with in vitro defects in filamentation such as efg1{Delta}{Delta} and brg1{Delta}{Delta} filament during oral infection and that decreased filamentation did not correlate with decreased infectivity. Taken together, these data indicate that key in vitro biofilm transcription factors are involved in OPC but that the network characteristics and functional connections are remodeled significantly during interactions with tissues. Author SummaryThe pathology of oral candidiasis has features of biofilm formation, a well-studied process in vitro. Based on that analogy, we hypothesized that network of transcription factors that regulates in vitro biofilm formation might have similarities and differences in during oral infection. To test this, we employed the first systematic genetic interaction analysis of C. albicans in a mouse model of oropharyngeal infection. This revealed that the six regulators involved in in vitro biofilm formation played roles in vivo but that the functional connections between factors were quite distinct. Surprisingly, we also found that, while many of the factors are required for filamentation in vitro, none of the transcription factor deletion mutants was deficient for this key virulence trait in vivo. These observations clearly demonstrate that C. albicans regulates key aspects of its biology differently in vitro and in vivo.

microbiology↗

bioRxiv2021

Intravital imaging of Candida albicans identifies differential in vitro and in vivo filamentation phenotypes for transcription factor deletion mutants

Candida albicans is an important cause of human fungal infections. A widely studied virulence trait of C. albicans is its ability to undergo filamentation to hyphae and pseudohyphae. Although yeast, pseudohyphae and hyphae are present in pathological samples of infected mammalian tissue, it has been challenging to characterize the role of regulatory networks and specific genes during in vivo filamentation. In addition, the phenotypic heterogeneity of C. albicans clinical isolates is becoming increasingly recognized and correlating this heterogeneity with pathogenesis remains an important goal. Here, we describe the use of an intravital imaging approach to characterize C. albicans filamentation in a mammalian model of infection by taking advantage of the translucence of mouse pinna (ears). Using this model, we have found that the in vitro and in vivo filamentation phenotypes of different C. albicans isolates can vary significantly, particularly when in vivo filamentation is compared to solid agar-based assays. We also show that the well-characterized transcriptional regulators Efg1 and Brg1 appear to play important roles both in vivo and in vitro. In contrast, Ume6 is much more important in vitro than in vivo. Finally, strains that are dependent on Bcr1 for in vitro filamentation are able to form filaments in vivo. This intravital imaging approach provides a new approach to the systematic characterization of this important virulence trait during mammalian infection. Our initial studies provide support for the notion that the regulation and initiation of C. albicans filamentation in vivo is distinct from in vitro induction. ImportanceCandida albicans is one of the most common causes of fungal infections in humans. C. albicans undergoes a transition from a round yeast form to a filamentous form during infection which is critical for its ability to cause disease. Although this transition has been studied in the laboratory for years, methods to do so in an animal model of infection have been limited. We have developed a microscopy method to visualize fluorescently labeled C. albicans undergoing this transition in the subcutaneous tissue of mice. Our studies indicate that the regulation of C. albicans filamentation during infection is distinct from that observed in laboratory conditions.

microbiology↗

bioRxiv2020

Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which governs the ability of the organism to proliferate in the lung. The other was ace1, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the {Delta}ace1 mutant is due to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators. Author summaryAlthough A. fumigatus causes the majority of cases of invasive aspergillosis, the function of most of the genes in its genome remains unknown. To identify genes encoding transcription factors that may be important for virulence, we used a NanoString nCounter to measure the mRNA levels of A. fumigatus transcription factor genes in the lungs of mice with invasive aspergillosis. The transcriptional profiling data indicate that the organism is exposed to nutrient limitation and stress during growth in the lungs, and that it responds by up-regulating genes that encode mycotoxins and secondary metabolites. In vitro, this response was most closely mimicked by growth in medium that was deficient in nitrogen, iron and/or zinc. Using the transcriptional profiling data, we identified two transcription factors that govern A. fumigatus virulence. These were RlmA, which is governs proliferation in the lung and Ace1, which controls the production of mycotoxins and secondary metabolites.

microbiology↗

bioRxiv2018

The Streptococcus pneumoniae Competence-Induced BriC Peptide Promotes Nasopharyngeal Colonization and Impacts Biofilm Development

Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

microbiology↗