Biology subjects

Rowley, P. A.

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

17 recordsLinked to original sources

bioRxiv2025

A Comprehensive Structural and Functional Analysis of Saccharomyces Killer Toxins.

Antifungal killer toxins are cytotoxic proteins that have the potential to combat the growing threat of fungi to human health and agriculture. A lack of empirical tertiary structures has placed limitations on understanding their mechanisms of action and targeting of pathogens. AlphaFold and molecular dynamics simulations were used to create tertiary structure models of all canonical Saccharomyces killer toxins. These models have enabled the prediction of the functional domains of killer toxins and postranslational modifications, including sites of proteolytic cleavage and disulfide bonds. They have also revealed unexpected homology between Saccharomyces killer toxins, suggesting that all but K28 are likely ionophores. Structural homology with the well-studied killer toxins K1 and K2 enabled prior empirical data to predict the antifungal and immunity mechanisms of the K1L, K21, K45, K74, and KHS toxins. The understudied killer toxins Klus, KHR, and K62 were found to have homology to bacterial and plant toxins, including members of the aerolysin family and antifungal lectins. These structural similarities provided clues for the mechanisms of killer toxin carbohydrate binding, oligomerization, and membrane attack. Given the hundreds of sequence homologs of the Saccharomyces killer toxin identified across fungi, modeling studies offer an exciting opportunity to characterize novel toxin-like proteins. This approach is strengthened by the continued use of the model yeast S. cerevisiae to study killer toxins and the wealth of functional data gathered in the decades since their first discovery.

molecular biology↗

bioRxiv2024

Discovery of Novel Targets for Important Human and Plant Fungal Pathogens via Automated Computational Pipeline HitList

Fungi are a major threat to human health and agricultural productivity, causing 1.7 million human deaths and billions of dollars in crop losses and spoilage annually. While various antifungal compounds have been developed to combat these fungi in medical and agricultural settings, but there are concerns that treatment effectiveness is waning due to the emergence of acquired drug resistance and to novel pathogens. Effectiveness is further hampered due to the limited number of modes of action for available antifungal compounds.To develop new strategies for the control and mitigation of fungal disease and spoilage, new antifungals are needed with novel fungal-specific protein targets that can overcome resistance and prevent host toxicity. New antifungals can add new methods of targeting fungi that have no effective control measures. The increasing availability of complete genomes of pathogenic and spoilage fungi has enabled identification of novel protein and RNA targets essential for viability and not found in host plants or humans. In this study, an automated bioinformatics pipeline utilizing BLAST, Clustal{Omega}, and subtractive genomics was created and used to identify potential new targets for any system of hosts and pathogens with available genomic or proteomic data. This pipeline called HitList allows in silico screening of thousands of possible targets. HitList was then used to generate a list of potential antifungal targets for the World Health Organization fungal priority pathogens list and the top 10 agricultural fungal pathogens. Known antifungal targets were found, validating the approach, and an additional eight novel protein targets were discovered that could be used for the rational design of antifungal compounds.

bioinformatics↗

bioRxiv2024

The Prevalence of Killer Yeasts in the Gardens of Fungus-Growing Ants and the Discovery of Novel Killer Toxin named Ksino.

Killer toxins are proteinaceous antifungal molecules produced by yeasts, with activity against a wide range of human and plant pathogenic fungi. Fungus gardens of attine ants in Brazil were surveyed to determine the presence of killer toxin-producing yeasts and to define their antifungal activities and ecological importance. Our results indicate that 10 out of 59 yeasts species isolated from fungal gardens are killer yeasts. Killer yeasts were less likely to inhibit the growth of yeasts isolated from the same environment but more effective at inhibiting yeast isolated from other environments, supporting a role for killer yeasts in shaping community composition. All killer yeasts harbored genome-encoded killer toxins lacked cytoplasmic toxin-encoding elements (i.e., double-stranded RNA satellites and linear double-stranded DNAs). Of all the killer yeasts associated with attine ants, Candida sinolaborantium (strain LESF 1467) showed a broad spectrum of antifungal activities against 39 out of 69 57% of yeast strains tested for toxin susceptibility. The complete genome sequence of C. sinolaborantium LESF 1467 identified a new killer toxin, Ksino, with similarities in primary sequence and tertiary structure to the Saccharomyces cerevisiae killer toxin named Klus. Surveys of publicly available genome databases identified homologs of Ksino in the genomes of yeast strains of Saccharomycetes and Pichiomycetes, as well as other species of Ascomycota and Basidiomycota filamentous fungi. This demonstrates that killer yeasts can be widespread in attine ant fungus gardens, possibly influencing fungal community composition and the importance of these complex microbial communities for discovering novel antifungal molecules. ImportanceAttine ants perform essential ecosystem services through the harvesting of substrates for fungiculture. The cultured fungi are a food source for attine ants. Characterizing antifungal toxin-producing yeasts (killer yeasts) is vital to understanding how they might protect gardens from invasion by unwanted fungal species. This study describes a new toxin named Ksino from the yeast Candida sinolaborantium, a member of a new group of toxins found across many different species of fungi. This work supports the role of killer yeasts in the ecology of fungicultures and competition between fungi. The observed high prevalence of killer yeasts in fungal gardens also enables the discovery of novel antifungal molecules with the potential to be applied against disease-causing fungi.

microbiology↗

bioRxiv2024

Defining the alpha domains of K1-like killer toxins

Antifungal killer toxins produced by yeasts undergo post-translational processing to enable the formation of mature and active toxins. The preprocessed, immature K1 killer toxin from Saccharomyces cerevisiae has four different domains (delta, alpha, gamma, and beta) that are cleaved to produce a mature toxin consisting of a disulfide-linked alpha and beta heterodimer. K1 homologs from various yeast species of the Saccharomycotina have been previously identified by primary sequence homology. Primary sequence analysis has identified putative cleavage sites across all homologs, with some conservation with cleavage sites identified in K1 and the likely importance of the conserved carboxypeptidases Kex1 and Kex2. The identification of sites of proteolytic processing has enabled an accurate definition of the domain boundaries of delta, alpha, gamma, and beta domains in all K1 homologs. Functional assays revealed that many K1 homologs contain cytotoxic alpha domains. Interestingly, several killer toxins that lacked measurable antifungal activity still exhibited toxic alpha domains when expressed independently. These findings suggest that alpha domain toxicity is conserved across K1 homologs, and that variations in post-translational processing and domain interactions may influence full-length toxin functionality. This work provides insights into the evolutionary conservation and diversity of yeast killer toxins.

biochemistry↗

bioRxiv2024

Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost

Plasmids play a major role in rapid adaptation of bacteria by facilitating horizontal transfer of diverse genes, most notably those conferring antibiotic resistance. While most plasmids that replicate in a broad range of bacteria also persist well in diverse hosts, there are exceptions that are poorly understood. We investigated why a broad-host range plasmid, pBP136, originally found in clinical Bordetella pertussis isolates, quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution we found that inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid maintenance in E. coli. This gene inactivation resulted in decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. It also caused transcriptional changes in many chromosomal genes primarily related to metabolism. In silico analyses suggested that the change in plasmid transcriptome may be initiated by Upf31 interacting with the plasmid regulator KorB. Expression of upf31 in trans negatively affected persistence of pBP136{Delta}upf31 as well as the closely related archetypal IncP-1{beta} plasmid R751, which is stable in E. coli and natively encodes a truncated upf31 allele. Our results demonstrate that while the upf31 allele in pBP136 might advantageously modulate gene expression in its original host, B. pertussis, it has harmful effects in E. coli. Thus, evolution of a single plasmid gene can change the range of hosts in which that plasmid persists, due to effects on the regulation of plasmid gene transcription.

microbiology↗

bioRxiv2023

An apparent lack of synergy between degradative enzymes against Staphylococcus aureus biofilms

The use of enzymes represents an approach to combat bacterial infections by degrading extracellular biomolecules to disperse Staphylococcus aureus biofilms. Commercial enzyme preparations, including cellulase, amylase, pectinase, zymolyase, and pepsin, exhibit concentration-dependent dispersion of S. aureus biofilms. Here, we report that low concentrations of these enzymes generally lack synergy when combined or added together sequentially to biofilms. Only the addition of a protease (pepsin) followed by a commercial mixture of degradative enzymes from Arthrobacter luteus (zymolyase 20T), demonstrated synergy and was effective at dispersing S. aureus biofilms. A more purified mixture of Arthrobacter luteus enzymes (zymolyase 100T) showed improved dispersal of S. aureus biofilms compared to zymolyase 20T but lacked synergy with pepsin. This study emphasizes the complexity of enzymatic biofilm dispersal and the need for tailored approaches based on the properties of degradative enzymes and biofilm composition.

microbiology↗

bioRxiv2022

Killer Toxins for the Control of Diastatic Yeasts in Brewing

Secondary fermentation in craft beers by diastatic yeasts can result in undesirable consequences, such as off-flavors, increased alcohol content, gushing, and the explosion of packaging. Currently, there are no strategies for inhibiting diastatic yeasts once they have invaded a commercial brewing process. Many strains of yeasts can naturally produce "killer" toxins that inhibit the growth of competing yeasts. Testing the effectiveness of canonical toxins against diastatic yeasts revealed that most (90%) strains are susceptible to the K1 killer toxin. Only two diastatic strains resistance to killer toxins was due to toxin production and associated immunity. Four diastatic strains resistant to K1 and K2 toxins were screened against a library of 192 novel killer yeasts to discover novel antifungal activities. This identified novel K2 killer yeasts that were more potent at inhibiting diastatic yeasts than the canonical killer yeast. As proof-of-principle for the control of diastatic yeasts during fermentation, killer yeasts were found to be effective at inhibiting hyperattenuation after a simulated diastatic contamination in a 1,000-liter industrial-style fermentation.

microbiology↗

bioRxiv2022

yEvo: a modular eukaryotic genetics and evolution research experience for high school students

Microbial experimental evolution paired with whole-genome sequencing allows researchers to observe evolutionary processes in real-time. The resources for carrying out and analyzing microbial evolution experiments have become more accessible. It is now possible to expand these studies beyond the research laboratory and into the classroom. We have developed a series of five connected and standards-aligned yeast evolution laboratory modules, called "yEvo," for high school biology students. The modules have been designed to enable students to take agency in answering open-ended research questions. In Module 1, students evolve the bakers yeast Saccharomyces cerevisiae to tolerate an over-the-counter antifungal drug, and in subsequent modules, investigate how evolved yeasts adapted to this stressful condition at both the phenotype and genotype levels. Pre- and post-surveys from 72 students at two different schools and one-on-one interviews with students and teachers were used to assess our program goals to iteratively improve these modules over three years. We also measured changes in student conceptions of mutation and evolution, confidence in scientific practices, and interest in STEM and biology careers. Students who participated in our experimental evolution module showed improvements in activity-specific concepts, including the importance of variation in evolution and the random nature of mutation. They additionally reported increased confidence in their ability to design a valid biology experiment. Student experimental data replicated literature findings on mechanisms of clotrimazole resistance and has led to new insights into this phenomenon. This collaborative endeavor will serve as a model for other university researchers and K-16 classrooms interested in engaging in open-ended research questions using yeast as a model system.

scientific communication and education↗

bioRxiv2022

Defining the HIV Capsid Binding Site of Nucleoporin 153

The interaction between the HIV-1 capsid (CA) and human nucleoporin 153 (NUP153) is vital for delivering the HIV-1 preintegration complex into the nucleus via the nuclear pore complex. The interaction with CA requires a phenylalanine/glycine-containing motif in the C-terminus of NUP153. This study used molecular modeling and biochemical assays to determine the amino acids of NUP153 that are essential for its interactions with CA. Molecular dynamics, FoldX, and PyRosetta simulations delineated the minimal CA binding motif of NUP153 based on the known structure of NUP153 bound to the HIV-1 CA hexamer. Computational predictions were experimentally validated by testing the interaction of NUP153 with CA using an in vitro binding assay and a cell-based TRIM-NUP153C restriction assay. This multidisciplinary approach identified eight amino acids from P1411 to G1418 that stably engage with CA, with significant correlations between molecular models and empirical experiments. Specifically, P1411, V1414, F1415, T1416, F1417, and G1418 were confirmed as critical amino acids required to interact NUP153 with CA. IMPORTANCEHuman immunodeficiency virus (HIV) can infect non-dividing cells by interacting with host nuclear pores. The host nuclear pore protein NUP153 directly interacts with the HIV capsid to promote viral nuclear entry. This study used a multidisciplinary approach combining computational and experimental techniques to map the essential amino acids of NUP153 required for HIV capsid interaction. This approach revealed that the HIV capsid interacts specifically with only six amino acids of NUP153, suggesting other FG-containing motifs could also interact with the capsid. Based on molecular modeling, naturally occurring polymorphisms in human and non-human primates would be predicted to prevent NUP153 interaction with capsid, potentially protecting from HIV infection.

molecular biology↗

bioRxiv2022

A Novel Virus Discovered in the Yeast Pichia membranifaciens.

Mycoviruses are widely distributed across fungi, including yeasts of the Saccharomycotina subphylum. It was recently discovered that the yeast species Pichia membranifaciens contained double stranded RNAs (dsRNAs) that were predicted to be of viral origin. The fully sequenced dsRNA is 4,578 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses of the family Totiviridae. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is related to other totiviruses previously described within the Saccharomycotina yeasts. PmV-L-A is part of a monophyletic subgroup within the I-A totiviruses, implying a common ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the previously solved structure of the Saccharomyces cerevisiae virus L-A (ScV-L-A) Gag protein. The predicted tertiary structure of the PmV-L-A Pol and its homologs provide details of the potential mechanism of totivirus RNA-dependent RNA polymerases (RdRps) because of structural similarities to the RdRps of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts is important because of their parallels with mammalian viruses and the emerging role of totiviruses in animal disease.

molecular biology↗

bioRxiv2021

yEvo: Experimental evolution in high school classrooms selects for novel mutations and epistatic interactions that impact clotrimazole resistance in S. cerevisiae.

Antifungal resistance in pathogenic fungi is a growing global health concern. Non-pathogenic laboratory strains of Saccharomyces cerevisiae are a useful model for studying mechanisms of antifungal resistance that are relevant to understanding the same processes in pathogenic fungi. We developed a series of lab modules in which high school students used experimental evolution to study antifungal resistance by isolating azole-resistant S. cerevisiae and examining the genetic basis of resistance. All 99 sequenced clones from these experiments possessed mutations previously shown to impact azole resistance, demonstrating the efficacy of our protocols. We additionally found recurrent mutations in an mRNA degradation pathway and an uncharacterized mitochondrial protein (Csf1) that have possible mechanistic connections to azole resistance. The scale of replication in this high school-led initiative allowed us to identify epistatic interactions, as evidenced by pairs of mutations that occur in the same clone more frequently than expected by chance (positive epistasis) or less frequently (negative epistasis). We validated one of these pairs, a negative epistatic interaction between gain-of-function mutations in the multidrug resistance transcription factors Pdr1 and Pdr3. This high school-university collaboration can serve as a model for involving members of the broader public in the scientific process to make meaningful discoveries in biomedical research.

genetics↗

bioRxiv2020

Nuku, a family of primate retrogenes derived from KU70

The ubiquitous DNA repair protein, Ku70p, has undergone extensive copy number expansion during primate evolution. Gene duplications of KU70 have the hallmark of long interspersed element-1 (LINE-1) mediated retrotransposition with evidence of target-site duplications, the poly-A tails, and the absence of introns. Evolutionary analysis of this expanded family of KU70-derived "NUKU" retrogenes reveals that these genes are both ancient and also actively being created in extant primate species. NUKU retrogenes show evidence of functional divergence away from KU70, as evinced by their altered pattern of tissue expression and possible translation in the human testes. Molecular modeling predicted that mutations in Nuku2p at the interaction interface with Ku80p would prevent the assembly of the Ku heterodimer. The lack of Nuku2p-Ku80p interaction was confirmed by yeast two-hybrid assay, which contrasts the robust interaction of Ku70p-Ku80p. While several NUKU retrogenes appear to have been degraded by mutation, NUKU2 shows evidence of positive natural selection, suggesting that this retrogene is undergoing neofunctionalization. Although Nuku proteins do not appear to antagonize retroviruses in cell culture, the observed expansion and rapid evolution of NUKUs could be being driven by alternative selective pressures related to infectious disease or an undefined role in primate physiology.

evolutionary biology↗

bioRxiv2020

Vaginal isolates of Candida glabrata are Uniquely Susceptible to Killer Toxins Produced by Saccharomyces Yeasts

Compared to other species of Candida yeasts, the growth of Candida glabrata was inhibited by many different strains of Saccharomyces killer yeasts. The ionophoric K1 and K2 killer toxins were broadly inhibitory to all clinical isolates of C. glabrata from patients with recurrent vulvovaginal candidiasis, despite high levels of resistance to clinically relevant antifungal therapeutics.

microbiology↗

bioRxiv2020

The Species-specific Acquisition and Diversification of a Novel Family of Killer Toxins in Budding Yeasts of the Saccharomycotina.

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of the se genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this novel killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the yeast genome. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification, with evidence of gene amplification and positive natural selection. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a new family of biologically active killer toxins. The phylogenetic relationship between K1L and its homologs suggests gene flow via dsRNAs and DNAs across taxonomic divisions to enable the acquisition of a diverse arsenal of killer toxins for use in niche competition.

genetics↗

bioRxiv2020

The design and implementation of restraint devices for the injection of pathogenic microorganisms into Galleria mellonella.

The injection of laboratory animals with pathogenic microorganisms poses a significant safety risk because of the potential for injury by accidental needlestick. This is especially true for researchers using invertebrate models of disease due to the small size of the animals and the required precision and accuracy of the injection. The immobilization of the greater wax moth larvae (Galleria mellonella) is often achieved by grasping a larva firmly between finger and thumb. Needle resistant gloves or forceps can be used to reduce the risk of a needlestick but can result in animal injury, a loss of throughput, and inconsistencies in experimental data. Immobilization devices are commonly used for the manipulation of small mammals, and in this manuscript, we describe the construction of injection chambers that can be used to entrap and restrain G. mellonella larvae prior to injection with pathogenic microbes. These devices significantly reduce the manual handling of larvae and provide an engineering control to protection against accidental needlestick injury, while maintaining a high rate of injection.

microbiology↗

bioRxiv2018

HIV-1 Vpr accessory protein interacts with REAF and mitigates its associated anti-viral activity.

The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and in cycling T cells to a lesser extent. Virion packaged Vpr is released in target cells shortly after entry, suggesting its requirement in the early phase of infection. Previously, we described REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without intact vpr is more highly restricted by REAF and, using delivery by VLPs, that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells and we detect, by co-immunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts very quickly during the early phase of replication and induces the degradation of REAF within 30 minutes of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localisation and interaction with cullin4A-DBB1 (DCAF1) E3 ubiquitin ligase is required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, which impedes HIV infection in macrophages.\n\nImportanceFor at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo. A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages, or to explain why Vpr is carried in the virus particle. Here we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one which inhibits virus replication early during reverse transcription. REAF is degraded by Vpr within 30 minutes of virus entry, in a manner dependent on the nuclear localization of Vpr and its interaction with the cells protein degradation machinery.

microbiology↗

bioRxiv2017

Control of Genetic Parasites Mediated Through Nucleoporin Evolution

Yeasts serve as long-term hosts to several types of genetic parasites. Few studies have addressed the evolutionary trajectory of yeast genes that control the stable co-existence of these parasites with their host cell. In Saccharomyces yeasts, the retrovirus-like Ty retrotransposons must access the nucleus. We show that several genes encoding components of the yeast nuclear pore complex have experienced natural selection for substitutions that change the encoded protein sequence. By replacing these S. cerevisiae genes with orthologs from other Saccharomyces species, we discovered that natural sequence changes have affected the control of Ty retrotransposons. Specifically, changing the genetic sequence of NUP84 or NUP82 to what is found in other Saccharomyces species alters the retrotransposition of S. cerevisiae Ty1 and Ty3, respectively. Importantly, all tested housekeeping functions of NUP84 and NUP82 remained equivalent across species. The nuclear pore complex is the gatekeeper of the nucleus. It appears that nucleoporins are adapting to modulate the control of genetic parasites which access the nucleus, which is achieved despite the strict constraints imposed by host nuclear pore complex function.

evolutionary biology↗