Paul Andrew Rowley

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 limited understanding of their mechanisms of action and their ability to target pathogens. In this study, AlphaFold and molecular dynamics simulations were used to generate tertiary structure models of all canonical Saccharomyces killer toxins and to place them in the context of historical empirical data. These models enabled the prediction of functional domains and posttranslational modifications, including proteolytic cleavage sites and disulfide bonds. They also revealed unexpected homology between Saccharomyces killer toxins, suggesting that all but K28 are likely ionophores. Structural homology to the well-studied killer toxins K1 and K2 enabled the prediction of the antifungal and immunity mechanisms of K1L, K21, K45, K74, and KHS. 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 provide clues for the mechanisms of killer toxin carbohydrate binding, oligomerization, and membrane attack. This modeling approach will help guide the continued use of the model yeast S. cerevisiae to study killer toxins in the context of the wealth of functional data gathered in the decades since their first discovery.

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 yeast 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 yeasts isolated from other environments, supporting a role for killer yeasts in shaping the community composition. All killer yeasts had genome-encoded killer toxins and 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 the 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 the fungal community composition and the importance of these complex microbial communities in the discovery of novel antifungal molecules. IMPORTANCE: Attine 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 also 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.

K62 is an antifungal killer toxin produced by Saccharomyces paradoxus , encoded by a double-stranded RNA satellite. The toxin exhibits a unique antifungal activity but lacks sequence homology to other killer toxins, and its antifungal mechanism of action remains unknown. To understand the function of K62, its tertiary structure was predicted using AlphaFold, followed by molecular dynamics simulations to create high-confidence molecular models. These analyses revealed that K62 monomers closely resemble the five-beta-strand domain found in pore-forming aerolysin toxins. Models of K62 oligomers yielded a circular complex and beta-barrel with structural and biochemical similarities to aerolysin-family pre-pores and pores. Consistent with the formation of aerolysin-like pores, recombinant K62 assembled into membrane-associated high molecular weight oligomers (>250 kDa) that were heat- and detergent-resistant. K62 has more than 1,000 uncharacterized sequence homologs, which were mostly found in fungi of the Ascomycota, as well as in the Chytridiomycota, Basidiomycota, plants, and bacteria, with evidence of extensive horizontal gene transfer. Homologs were also identified in pathogenic fungal species, including human and plant pathogens from the Candida and Fusarium genera, but unlike aerolysins, K62 appeared to be non-toxic to higher eukaryotes. K62 is the first aerolysin family protein discovered in yeasts, revealing a likely role in fungal niche competition and establishing an entirely new, expansive family of aerolysin-like proteins. IMPORTANCE: Pore-forming toxins are potent biological weapons used across nature, from virulence factors to immune defense proteins. This study identifies K62, a little-known antifungal toxin produced by a wild yeast, as a structural and functional relative of the aerolysin family, which is well-known for forming damaging pores in cell membranes. Using structure prediction, molecular simulations, and biochemical analysis, we show that K62 assembles into large, stable pore-like complexes. Remarkably, K62 is just one member of a large and previously unrecognized family of similar toxin-like proteins found in fungi, plants, and bacteria, including pathogens that affect humans and crops. These findings uncover an unexpected evolutionary link across kingdoms, suggesting that pore-forming toxins may play a widespread role in fungal pathogenesis and microbial warfare. This work lays the foundation for understanding a new group of antifungal molecules and their potential impacts on health, agriculture, and microbial ecology.

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.

Human immunodeficiency virus type-1 (HIV-1) can infect non-dividing cells by passing through the selective permeability barrier of the nuclear pore complex. The viral capsid is essential for successfully delivering the HIV-1 genome into the nucleus. Nucleoporin 153 (NUP153) interacts with the HIV-1 capsid via a C-terminal capsid-binding motif (hereafter named CbM.1) to licence HIV-1 nuclear ingress. Deletion or mutation of CbM.1 in NUP153 causes a reduction in capsid interaction but does not prevent HIV-1 nuclear ingress or completely block capsid interaction. This paper combines molecular modelling with biochemical and HIV infection assays to identify capsid-binding motif 2 (CbM.2) in the C-terminus of NUP153 that is similar in sequence to CbM.1. CbM.2 has an FG dipeptide motif predicted to interact with a hydrophobic pocket in capsid protein (CA) hexamers similar to CbM.1. CA hexamers can interact with CbM.2, and the deletion of both CbM.1 and CbM.2 results in a lower capsid interaction than a single CbM.1 deletion. The loss of CbM.1 is complemented by CbM.2, an interaction dependent on the FG motif. In the context of the nuclear pore complex, a loss-of-function mutation in CbM.1 reduces HIV nuclear ingress as measured by transduction and 2-LTR circles, whereas the mutation of CbM.2 causes a large increase in 2-LTR circles. Our results highlighted a previously unidentified FG dipeptide-containing motif (CbM.2) in NUP153 that binds the HIV-1 capsid at the common hydrophobic pocket on CA hexamers.

Plasmids promote adaptation of bacteria by facilitating horizontal transfer of diverse genes, notably those conferring antibiotic resistance. Some plasmids, like those of the incompatibility group IncP-1, are known to replicate and persist in a broad range of bacteria. We investigated a poorly understood exception, the IncP-1β plasmid pBP136 from a clinical Bordetella pertussis isolate, which quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution, we found that the inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid persistence in E. coli. The gene inactivation caused alterations in the plasmid regulatory system, including decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. This is consistent with our findings that Upf31 represses its own transcription. It also caused secondary transcriptional changes in many chromosomal genes. In silico analyses predicted that Upf31 interacts with the plasmid regulator KorB at its C-terminal dimerization domain (CTD). We showed experimentally that adding the CTD of upf31/pBP136 to the naturally truncated upf31 allele of the stable IncP-1β archetype R751 results in plasmid destabilization in E. coli. Moreover, mutagenesis showed that upf31 alleles encoded on nearly half of the sequenced IncP-1β plasmids also possess this destabilization phenotype. While Upf31 might be beneficial in many hosts, we show that in E. coli some alleles have harmful effects that can be rapidly alleviated with a single mutation. Thus, broad-host-range plasmid adaptation to new hosts can involve fine-tuning their transcriptional circuitry through evolutionary changes in a single gene.

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, ClustalΩ, 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.

Secondary fermentation in beer can result in undesirable consequences, such as off-flavors, increased alcohol content, hyperattenuation, gushing, and the spontaneous explosion of packaging. Strains of Saccharomyces cerevisiae var. diastaticus are a major contributor to such spoilage due to their production of extracellular glucoamylase enzyme encoded by the STA1 gene. Saccharomyces yeasts can naturally produce antifungal proteins named “killer” toxins that inhibit the growth of competing yeasts. Challenging diastatic yeasts with killer toxins revealed that 91% of strains are susceptible to the K1 killer toxin produced by S. cerevisiae . Screening of 192 killer yeasts identified novel K2 toxins that could inhibit all K1-resistant diastatic yeasts. Variant K2 killer toxins were more potent than the K1 and K2 toxins, inhibiting 95% of diastatic yeast strains tested. Brewing trials demonstrated that adding killer yeast during a simulated diastatic contamination event could prevent hyperattenuation. Currently, most craft breweries can only safeguard against diastatic yeast contamination by good hygiene and monitoring for the presence of diastatic yeasts. The detection of diastatic yeasts will often lead to the destruction of contaminated products and the aggressive decontamination of brewing facilities. Using killer yeasts in brewing offers an approach to safeguard against product loss and potentially remediate contaminated beer. IMPORTANCE: The rise of craft brewing means that more domestic beer in the marketplace is being produced in facilities lacking the means for pasteurization, which increases the risk of microbial spoilage. The most damaging spoilage yeasts are “diastatic” strains of Saccharomyces cerevisiae that cause increased fermentation (hyperattenuation), resulting in unpalatable flavors such as phenolic off-flavor, as well as over-carbonation that can cause exploding packaging. In the absence of a pasteurizer, there are no methods available that would avert the loss of beer due to contamination by diastatic yeasts. This manuscript has found that diastatic yeasts are sensitive to antifungal proteins named “killer toxins” produced by Saccharomyces yeasts, and in industrial-scale fermentation trials, killer yeasts can remediate diastatic yeast contamination. Using killer toxins to prevent diastatic contamination is a unique and innovative approach that could prevent lost revenue to yeast spoilage and save many breweries the time and cost of purchasing and installing a pasteurizer.

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. Importance: Attine 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.