Todd Reynolds

University of Tennessee | UTK · Department of Microbiology

Enzymatic recycling of poly-l-lactic acid (PLLA) plastic has recently become an area of interest; however, investigation of enzymatic mechanisms and engineering strategies to improve activity remains limited. In this study, we have identified a subtilisin from Bacillus pumilus that has the ability to depolymerize high-molecular-weight PLLA. We perf...

Phosphatidylserine (PS) is a key lipid that plays important roles in disease-related biological processes, and therefore, the means to track PS in live cells are invaluable. Herein, we describe the metabolic labeling of PS in Saccharomyces cerevisiae cells using analogues of serine, a PS precursor, derivatized with azide moieties at either the amin...

Shielding the immunogenic cell wall epitope β(1, 3)-glucan under an outer layer of mannosylated glycoproteins is an essential virulence factor deployed by Candida albicans during systemic infection. Accordingly, mutants with increased β(1, 3)-glucan exposure (unmasking) display increased immunostimulatory capabilities in vitro and attenuated virule...

Candida albicans is a common cause of human mucosal yeast infections, and invasive candidiasis can be fatal. Antifungal medications are limited, but those targeting the pathogen cell wall or plasma membrane have been effective. Therefore, virulence factors controlling membrane biogenesis are potential targets for drug development. P4-ATPases contri...

In order to successfully induce disease, the fungal pathogen Candida albicans regulates exposure of antigens like the cell wall polysaccharide ß(1,3)-glucan to the host immune system. C . albicans covers (masks) ß(1,3)-glucan with a layer of mannosylated glycoproteins, which aids in immune system evasion by acting as a barrier to recognition by hos...

Candida albicans is a common cause of human mucosal yeast infections, and invasive candidiasis can be fatal. Antifungal medications are limited, but those targeting the pathogen cell wall or plasma membrane have been effective. Therefore, virulence factors controlling membrane biogenesis are potential targets for drug development. P4-ATPases contri...

Candida spp. are an important source of systemic and mucosal infections in immune compromised populations. However, drug resistance or toxicity has put limits on the efficacy of current antifungals. The C. albicans cell wall is considered a good therapeutic target due to its roles in viability and fungal pathogenicity. One potential method for impr...

The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the CHO1 gene, is a potential drug target for pathogenic fungi, such as Candida albicans . However, both substrate-binding sites of C. albicans Cho1 have not been characterized. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies...

Masking the immunogenic cell wall epitope ß(1,3)-glucan under an outer layer of mannosylated glycoproteins is an important virulence factor deployed by Candida albicans during infection. Consequently, increased ß(1,3)-glucan exposure (unmasking) reveals C . albicans to the host’s immune system and attenuates its virulence. We have previously shown...

Viruses modulate the function(s) of environmentally relevant microbial populations, yet considerations of the metabolic capabilities of individual virus particles themselves are rare. We used shotgun proteomics to quantitatively identify 43 virus-encoded proteins packaged within purified Aureococcus anophagefferens Virus (AaV) particles, normalizin...

The pelagophyte Aureococcus anophagefferens blooms annually in shallow bays around the world, where it is hypothesized to outcompete other phytoplankton in part by using alternative nitrogen sources. The high proportion of natural populations that are infected during the late stages of the bloom suggest viruses cause bloom collapse. We hypothesized...

The pelagophyte Aureococcus anophagefferens has caused recurrent brown tide blooms along the northeast coast of the United States since the mid-1980’s, and more recently spread to other regions of the globe. These blooms, due to the high cell densities, are associated with severe light attenuation that destroys the sea grass beds which provide the...

Candida albicans is a leading cause of systemic bloodstream infections and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the CDP-DAG pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce P...

Poly-lactic acid (PLA) is increasingly used as a biodegradable alternative to traditional petroleum-based plastics. In this study, we identify a novel agricultural soil isolate of Bacillus pumilus (B12) that is capable of degrading high molecular weight PLA films. This degradation can be detected on a short timescale, with significant degradation d...

Candida albicans is an important source of systemic infections in humans. The ability to mask the immunogenic cell wall polymer β (1,3)-glucan from host immune surveillance contributes to fungal virulence. We previously reported that the hyperactivation of the Cek1 MAP kinase cascade promotes cell wall unmasking, thus increasing strain immunogenici...

Candida albicans is an opportunistic human fungal pathogen that causes life-threatening systemic infections, as well as oral mucosal infections. Phospholipids are crucial for pathogenesis in C. albicans, as disruption of phosphatidylserine (PS) and phosphatidylethanolamine (PE) biosynthesis within the cytidine diphosphate diacylglycerol (CDP-DAG) p...

Candida albicans is among the most common causes of human fungal infections and is an important source of mortality. C. albicans is able to diminish its detection by innate immune cells through masking of β (1,3)-glucan in the inner cell wall with an outer layer of heavily glycosylated mannoproteins (mannan). However, mutations or drugs that disrup...

Growth curves were measured to determine the growth rate of strains in YPD vs YPM. Cells were grown overnight in YPD, diluted back to 0.1 OD600 and transferred to fresh YPD or YPM. A growth curve was performed with three replicates per condition, and plotted based on the growth rate of different strains measured in 48 hrs. The growth at each time-p...

Deleting one PKC1 allele in cho1Δ/Δ did not rescue β (1,3)-glucan exposure. One PKC1 allele was deleted by the SAT1-flipper method. Cells were then stained with anti-β (1,3)-glucan primary antibody and phycoerythrin (PE)-conjugated secondary antibody. The statistical analysis was carried out by doing One-way ANOVA. (TIF)

Plasmids used in this study. (DOCX)

The STE11ΔN467 strain exhibits significantly increased β (1,3)-glucan exposure compared to wild-type. Overnight cultures of Candida cells was incubated with anti-β (1,3)-glucan primary antibody and PE-conjugated secondary antibody, followed by flow cytometry to quantify the fluorescence intensity. Data represent three biological replicates. The sta...

MKC1 was knocked out in C. albicans via CRISPR-Cas9. Western blotting was performed using anti-Mkc1 antibody to confirm the absence of Mkc1 in the MKC1 knockout mutants compared to wild-type (WT) and other strains. Tubulin was probed with anti-tubulin antibody as a loading control. (TIF)

C. albicans strains used in this study. (DOCX)

The exposed β (1,3)-glucan in STE11ΔN467 YPM cells was not restricted to bud scars. Overnight cultures of wild-type and STE11ΔN467 grown in YPM were co-stained with anti-β(1,3)-glucan antibody and Cy3 secondary to visualize exposed β(1,3)-glucan and calcofluor white to visualize chitin. (TIF)

CDC42G12V increases β (1,3)-glucan exposure, but also reduces the viable cell population. (A) Propidium iodide staining was performed to quantify the live cells in Candida strains. (B) β (1,3)-glucan exposure in live (gated for propidim iodide negative cells) wild-type and CDC42G12V populations was measured by flow cytometry. (TIF)

Primers used in this study. (XLSX)

Plasmid and Strain Construction. (DOCX)

Candida albicans mutants for phosphatidylserine (PS) synthase ( cho1 ΔΔ) and PS decarboxylase ( psd1 ΔΔ psd2 ΔΔ) are compromised for virulence in mouse models of systemic infection and oropharyngeal Candidiasis (OPC). Both of these enzymes are necessary to synthesize phosphatidylethanolamine (PE) by the de novo pathway, but these mutants are still...

The phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) play important roles in the virulence of Candida albicans and loss of PS synthesis or synthesis of PE from PS (PS decarboxylase) severely compromises virulence in C. albicans in a mouse model of systemic candidiasis. This review discusses synthesis of PE and PS in C. albica...

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characte...

Candida albicans is the most common human fungal pathogen. The ability to undergo the morphological transition from yeast to hyphal growth is critical for its pathogenesis. Farnesol, a precursor in the isoprenoid/sterol pathway, is a quorum-sensing molecule produced by C. albicans that inhibits hyphal growth in this polymorphic fungus. Interestingl...

Phosphatidylserine (PS) synthase (Cho1p) and the PS decarboxylase enzymes (Psd1p and Psd2p), which synthesize PS and phosphatidylethanolamine (PE), respectively, are crucial for Candida albicans virulence. Mutations that disrupt these enzymes compromise virulence. These enzymes are part of the cytidyldiphosphate-diacylglycerol (CDP-DAG) pathway (i....

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections including life threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner...

In order to expand the repertoire of antifungal compounds a novel, high-throughput phenotypic drug screen targeting fungal phosphatidylserine (PS) synthase (Cho1p) was developed based on antagonism of the toxin papuamide A (Pap-A). Pap-A is a cyclic depsipeptide that binds to PS in the membrane of wild-type Candida albicans, and permeabilizes its p...

Synthesis of SB-224289 fragments. Detailed scheme for synthesis of fragments of SB-224289. (DOCX)

Microbial secretion is integral for regulating cell homeostasis as well as releasing virulence factors during infection. Phosphatidylserine synthase (CHO1) and phosphatidylserine decarboxylase (PSD1 and PSD2) are Candida albicans genes involved in phospholipid biosynthesis, and mutations in these genes affect mitochondrial function, cell wall thick...

ScOpi1p is a well-characterized transcriptional repressor and master regulator of inositol and phospholipid biosynthetic genes in the baker's yeast Saccharomyces cerevisiae. An ortholog has been shown to perform a similar function in the pathogenic fungus Candida glabrata, but with the distinction that CgOpi1p is essential for growth in this organi...

The virulence of Candida albicans in a mouse model of invasive candidiasis is dependent on the phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE). Disruption of the PS synthase gene CHO1 (i.e., cho1Δ/Δ) eliminates PS and blocks the de novo pathway for PE biosynthesis. In addition, the cho1Δ/Δ mutant's ability to cause invasive...

Saccharomyces cerevisiae strains of the ∑1278b background generate biofilms, referred to as mats, on low density agar (0.3%) plates made with rich media (YPD). Mat formation involves adhesion of yeast cells to the surface of the agar substrate and each other as the biofilm matures, resulting in elaborate water channels that create filigreed pattern...

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in...

The dimorphic switch from a single-cell budding yeast to a filamentous form enables Saccharomyces cerevisiae to forage for nutrients and the opportunistic pathogen Candida albicans to invade human tissues and evade the immune system. We constructed a genome-wide set of targeted deletion alleles and introduced them into a filamentous S. cerevisiae s...

Saccharomyces cerevisiae generates complex biofilms called mats on low-density (0.3%) agar plates. The mats can be morphologically divided into two regions: (i) hub, the interior region characterized by the presence of wrinkles and channels, and (ii) rim, the smooth periphery. Formation of mats depends on the adhesin Flo11p, which is also required...

Development of high-throughput functional assays for the screening of novel cellulases from insects is crucial to the identification of novel enzymes with applications for biofuel production. Our goal was to develop a functional assay to identify and characterize insect cellulases by expressing them in S. cerevisae cultures. We cloned the full-leng...

Phospholipid biosynthetic pathways play crucial roles in the virulence of several pathogens; however, little is known about how phospholipid synthesis affects pathogenesis in fungi such as Candida albicans. A C. albicans phosphatidylserine (PS) synthase mutant, cho1 Delta/Delta, lacks PS, has decreased phosphatidylethanolamine (PE), and is avirulen...

Inositol is essential in eukaryotes, and must be imported or synthesized. Inositol biosynthesis in Saccharomyces cerevisiae is controlled by three non-essential genes that make up the inositol regulon: ScINO2 and ScINO4, which together encode a heterodimeric transcriptional activator, and ScOPI1, which encodes a transcriptional repressor. ScOpi1p i...

myo-Inositol (inositol) is an essential nutrient that is used for building phosphatidylinositol and its derivatives in eukaryotes and even in some eubacteria such as the mycobacteria. As a consequence, fungal, protozoan and mycobacterial pathogens must be able to acquire inositol in order to proliferate and cause infection in their hosts. There are...

Candida albicans is an important cause of life-threatening systemic bloodstream infections in immunocompromised patients. In order to cause infections, C. albicans must be able to synthesize the essential metabolite inositol or acquire it from the host. Based on the similarity of C. albicans to Saccharomyces cerevisiae, it was predicted that C. alb...

The ability of Saccharomyces cerevisiae to form morphologically complex colony-like structures called mats requires expression of the cell surface glycoprotein Flo11p and growth on a semisolid surface. As the mat grows, it forms two visually distinct populations called the rim (edge of the mat) and the hub (interior of the mat), which can be physic...

Mat formation in the bakers' yeast Saccharomyces cerevisiae is a surface-associated phenomenon in which yeast cells spread over the surface of a low-density agar petri plate as a complex film. This spreading growth occurs by sliding motility and is dependent on the adhesion protein (adhesin) Flo11p. In order to identify molecular pathways that gove...

The increase in hospital-acquired fungal infections has been attributed to the ability of fungi to adhere not only to human tissues, but also to the plastic prostheses and invasive devices that are used to treat disease. These properties are conferred by a family of fungal cell-surface proteins, called adhesins. Adhesins might also have a central r...

Biofilms are formed by the aggregation of microorganisms into multicellular structures that adhere to surfaces. Here we show that bakers' yeast Saccharomyces cerevisiae can initiate biofilm formation. When grown in low-glucose medium, the yeast cells adhered avidly to a number of plastic surfaces. On semi-solid (0.3% agar) medium they formed "mats"...