Biology subjects

Melnyk, R. A.

Publications and source records attributed to Melnyk, R. A..

13 recordsLinked to original sources

bioRxiv2026

Balancing receptor engagement and payload catalytic activity broadens the therapeutic window of immunotoxins

Immunotoxins (ITs) are potent targeted cancer therapeutics that couple tumor-selective receptor binding with efficient intracellular delivery and cytotoxic enzymatic activity of bacterial toxins. While ITs have shown impressive efficacy in hematologic malignancies, dose-limiting toxicity remains a major barrier to their broader clinical application, particularly in solid tumors. The exceptional potency of immunotoxin payloads contributes to off-tumor toxicity because even limited uptake by receptor-low cells can be sufficient to cause cell death. Here, we systematically investigated how receptor-binding domain (RBD) affinity and avidity interact with catalytic payload activity to determine IT selectivity. Using CD123-, HER3-, and HER2-targeted ITs, we found that increasing receptor-binding affinity or avidity altered potency but provided limited control over the selectivity between receptor-high and receptor-low cells. In contrast, attenuating catalytic activity produced profound gains in selectivity when payload potency was appropriately matched to receptor engagement. The optimal level of attenuation varied with receptor context and toxin scaffold, demonstrating that maximal selectivity emerges from balancing cellular delivery with catalytic activity rather than maximizing either property alone. In vivo, payload attenuation increased the maximum tolerated dose of a HER2-targeted IT by 400-fold and enabled robust antitumor activity well below its toxicity limit, whereas the WT counterpart showed no detectable efficacy at tolerated doses. Together, these findings uncover and establish payload activity as a tunable determinant of immunotoxin selectivity and provide a framework for matching receptor engagement with catalytic potency to develop safer and more broadly applicable targeted toxins.

pharmacology and toxicology↗

bioRxiv2026

Genome-wide fitness profiling reveals flagellar rotation as an energetic liability during anaerobic maintenance in Pseudomonas aeruginosa

Bacteria in nature and disease frequently spend time in non-growing states, yet the genes that allow cells to survive without growing remain poorly understood. Using Pseudomonas aeruginosa strain PA14 as a model system to study non-growth powered by anaerobic phenazine cycling, we employed randomly barcoded transposon-insertion sequencing (RB-TnSeq) to identify the genes required for this maintenance state using a platform that sustains anaerobic survival via continuous phenazine reoxidation. We found 167 genes whose disruption altered survival, including genes involved in transcription, translation, protein quality control, and cell envelope maintenance. Comparing our results to published TnSeq data from other forms of energy-limited growth arrest (conditions where electron acceptor or carbon availability constrains energy conservation below that required for cell growth/division) in PA14 revealed that while a subset of genes are fitness determinants across distinct growth-arrested states, most are condition-specific. Notably, genes involved in flagellar regulation and assembly were broadly detrimental to survival under this maintenance condition. Leveraging a high-throughput electrochemical system that allows for quantitative and mechanistic dissection of the phenazine cycling-dependent maintenance state, we found that flagellar abundance influences cells survival, metabolic rate, and ATP levels. Moreover, removing the flagellar stator proteins MotAC, which are required for flagellar rotation but not assembly, reversed these defects, indicating that the energetic cost of flagella comes from their rotation rather than construction under these conditions. These results show that in a low-powered maintenance state, limiting energy-dissipating processes, such as proton-motive force loss through flagellar rotation, supports cell survival.

microbiology↗

bioRxiv2024

De novo design of potent inhibitors of Clostridioides difficile toxin B

Clostridioides difficile is a major cause of secondary disease in hospitals. During infection, C. difficile toxin B drives disease pathology. Here we use deep learning and Rosetta-based approaches to de novo design small proteins that block the entry of TcdB into cells. These molecules have binding affinities and neutralization IC50s in the pM range and are compelling candidates for further clinical development. By directly targeting the toxin rather than the pathogen, these molecules have the advantage of immediate cessation of disease and lower selective pressure for escape compared to conventional antibiotics. As C. difficile infects the colon, the protease and pH resistance of the designed proteins opens the door to oral delivery of engineered biologics. Significance statementC. difficile infection (CDI) is a major public health concern with over half a million cases in the United States annually resulting in 30,000 deaths. Current therapies are inadequate and frequently result in cycles of recurrent infection (rCDI). Progress has been made in the development of anti-toxin mAb therapies that can reduce the rate of rCDI, but these remain unaffordable and out of reach for many patients. Using de novo protein design, we developed small protein inhibitors targeting two independent receptor binding sites on the toxin that drives pathology during CDI. These molecules are high affinity, potently neutralizing and stable in simulated intestinal fluid, making them strong candidates for the clinical development of new CDI therapies.

microbiology↗

bioRxiv2021

Commensal Pseudomonas fluorescens protect Arabidopsis from closely-related Pseudomonas pathogens in a colonization-dependent manner

Plants form commensal associations with soil microorganisms, creating a root microbiome that provides benefits to the host including protection against pathogens. While bacteria can inhibit pathogens through production of antimicrobial compounds in vitro, it is largely unknown how microbiota contribute to pathogen protection in planta. We developed a gnotobiotic model system consisting of Arabidopsis thaliana, and an opportunistic pathogen Pseudomonas sp. N2C3, to identify mechanisms that determine the outcome of plant-pathogen-microbiome interactions in the rhizosphere. We screened 25 phylogenetically diverse Pseudomonas strains for their ability to protect against N2C3 and found that commensal strains closely related to N2C3 were more likely to protect against pathogenesis. We used a comparative genomics approach to identify unique genes in the protective strains that revealed no genes that correlate with protection, suggesting that variable regulation of components of the core Pseudomonas genome may contribute to pathogen protection. We found that commensal colonization level was highly predictive of protection and so tested deletions in genes previously shown to be required for Arabidopsis rhizosphere colonization. We identified a response regulator colR that is required for Pseudomonas protection from N2C3 and fitness in competition with N2C3 indicating that competitive exclusion may contribute to pathogen protection. We found that Pseudomonas WCS365 also protects against the agricultural pathogen Pseudomonas fuscovaginae SE-1, the causal agent of bacterial sheath brown rot of rice. This work establishes a gnotobiotic model to uncover mechanisms by which members of the microbiome can protect hosts from pathogens and informs our understanding of the use of beneficial strains for microbiome engineering in dysbiotic soil systems.

microbiology↗

bioRxiv2021

Structure-function analysis of distant diphtheria toxin homologs reveals insights into host adaptation

Diphtheria toxin (DT) is the archetype of bacterial exotoxins implicated in human diseases and has played a central role in defining the field of toxinology since its discovery in 1888. Despite being one of the most extensively characterized bacterial toxins, the origins and molecular evolution of DT host specialization remain unknown. Here, we determined high-resolution structures of two recently discovered distant homologs of DT. These DT-like proteins from non-human associated Streptomyces albireticuli (17% identity to DT) and Seinonella peptonophila (20% identity to DT) display remarkable structural similarity to DT enabling a comparative investigation into DTs unique toxicity toward mammalian cells. We find that the individual domains of DT-like toxins retain two critical features of DTs activity: full catalytic function and ability to translocate across mammalian cell membranes. However, we show that receptor-binding, pH-dependent pore-formation and proteolytic release of the cytotoxic enzyme into the cytosol are not optimized for human cell physiology and thus unable to efficiently deliver the cytotoxic cargo into human hosts. Our work provides structural insights into DTs evolutionary history, and implies key transitions required for the emergence of human-specificity of a major bacterial exotoxin with an important history in human disease.

microbiology↗

bioRxiv2020

Comparative genomics identified a genetic locus in plant-associated Pseudomonas spp. that is necessary for induced systemic susceptibility

Plant root-associated microbes promote plant growth and induce systemic resistance (ISR) to foliar pathogens. In an attempt to find novel growth-promoting and ISR-inducing strains, we previously identified strains of root-associated Pseudomonas spp. that promote plant growth but unexpectedly induced systemic susceptibility (ISS) rather than ISR to foliar pathogens. Here we demonstrate that the ISS-inducing phenotype is common among root-associated Pseudomonas spp. Using comparative genomics, we identified a single P. fluorescens locus that is unique to ISS strains. We generated a clean deletion of the 11-gene ISS locus and found that it is necessary for the ISS phenotype. Although the functions of the predicted genes in the locus are not apparent based on similarity to genes of known function, the ISS locus is present in diverse bacteria and a subset of the genes have previously been implicated in pathogenesis in animals. Collectively these data show that a single bacterial locus contributes to modulation of systemic plant immunity. ImportanceMicrobiome-associated bacteria can have diverse effects on health of their hosts, yet the genetic and molecular basis of these effects have largely remained elusive. This work demonstrates that a novel bacterial locus can modulate systemic plant immunity. Additionally, this work demonstrates that growth promoting strains may have unanticipated consequences on plant immunity and this is critical to consider when engineering the plant microbiome for agronomic improvement.

microbiology↗

bioRxiv2020

Exploiting toxin internalization receptors to enhance delivery of proteins to lysosomes for enzyme replacement therapy

Lysosomal storage diseases are a group of over 70 inherited genetic diseases caused by a defect or deficiency in a lysosomal protein. Enzyme replacement therapy, in which a functional copy of the defective enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases; however, the inefficient uptake of recombinant enzymes into cells and tissues via the low-affinity mannose-6-phosphate receptor prohibits broader utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endo-lysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that the targeted TPP1 chimera binds with high affinity to target cells and is delivered into lysosomes with much greater efficiency than TPP1 alone. Further, we demonstrate efficient and durable uptake of the chimera in vivo following intracerebroventricular injection in mice lacking TPP1. Targeting the highly efficient diphtheria toxin internalization pathway represents a novel approach for improving the efficacy and utility of enzyme replacement therapy for treating lysosomal storage diseases.

pharmacology and toxicology↗

bioRxiv2019

Inhibition of tumor growth by a novel engineered chimeric toxin that cleaves activated mutant and wild-type RAS

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small molecule approaches has proven to be a major challenge. Here we describe the development of a novel pan-RAS biologic inhibitor comprised of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Further, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS. Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising new approach to developing RAS therapeutics against a broad array of cancers. SignificanceRAS oncoproteins have long been considered among the most elusive drug targets in cancer research. At issue is the lack of accessible drug binding sites and the extreme affinity for its GTP substrate. Covalent inhibitors against the KRAS G12C mutant have shown early clinical promise, however, targeting the other oncogenic RAS mutants across three RAS isoforms has proven challenging. Inhibition of activated wild-type RAS in the absence of canonical RAS mutations is also highly desirable in certain tumors. Here, we demonstrate delivery of an extremely potent pan-RAS and RAP1 cleaving enzyme in therapeutic quantities to specific receptor-bearing cells in vitro and in vivo. We aim to advance this approach to engineer the first targeted pan-RAS inhibitor for cancer therapy. One Sentence SummaryEngineered chimeric toxin halts tumor growth in vivo via RAS cleavage

cancer biology↗

bioRxiv2019

bioPROTACs as versatile modulators of intracellular therapeutic targets: Application to proliferating cell nuclear antigen (PCNA)

Targeted degradation approaches have recently generated much excitement as a paradigm shift to address human disease in unprecedented ways. Amongst these, small molecule based approaches such as Proteolysis targeting chimeras (PROTACs) have attracted the lions share of attention due to their potential to tackle historically intractable targets and achieve greater potency, efficacy, and specificity over traditional small molecule inhibitors. Despite their promise, the identification of high-affinity ligands that can serve as starting points for PROTAC strategies remains challenging. As a complementary approach, we describe herein a class of intracellular biologics termed bioPROTACs. The substrate binding component of these fusion proteins consists of a peptide or an antibody-mimetic which allows for an unprecedented diversity of protein targets that can be addressed. The high-affinity binder is linked directly to an E3 ubiquitin ligase to harness the power of targeted degradation. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. Indeed, 9 out of 16 binder-E3 combinations tested resulted in greater than 70% target clearance. Through a systematic approach, we then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA), a sliding DNA clamp with critical roles in DNA replication and repair. The bioPROTAC, termed Con1-SPOP, elicited rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Compared to RNAi-based approaches which typically take days to manifest, PCNA knockdown using Con1-SPOP was evident within 4 h. The advantage of degradation versus stoichiometric inhibition was also clearly demonstrated with bioPROTAC strategies. Combining superior pharmacological inhibition and relative ease of development, bioPROTACs are powerful tools for interrogating the degradability of a substrate, for guiding the identification of the fittest E3 ligase, for studying the functional consequences associated with target protein down-regulation, and potentially for making therapeutic impacts.

cell biology↗

bioRxiv2019

Bacterially produced spermidine induces plant systemic susceptibility to pathogens

plant biology↗

bioRxiv2018

A genome-wide screen identifies genes in rhizosphere-associated Pseudomonas required to evade plant defenses

Pseudomonas fluorescens and related plant root- (\"rhizosphere\") associated species contribute to plant health by modulating defenses and facilitating nutrient uptake. To identify bacterial fitness determinants in the rhizosphere of the model plant Arabidopsis thaliana, we performed a Tn-Seq screen using the biocontrol and growth-promoting strain Pseudomonas sp. WCS365. The screen, which was performed in parallel on wild-type and an immunocompromised Arabidopsis, identified 231 genes that positively affect fitness in the rhizosphere of wild-type plants. A subset of these genes negatively affect fitness in the rhizosphere of immunocompromised plants. We postulated that these genes might be involved in avoiding plant defenses and verified 7 Pseudomonas sp. WCS365 candidate genes by generating clean deletions. We found that two of these deletion strains, {Delta}morA (encodes a putative diguanylate cyclase/phosphodiesterase) and {Delta}spuC (encodes a putrescine aminotransferase) formed enhanced biofilms and inhibited plant growth. Inhibition of plant growth by {Delta}spuC and {Delta}morA was the result of pattern triggered immunity (PTI) as measured by induction of an Arabidopsis PTI reporter and FLS2/BAK1-dependent inhibition of plant growth. We found that MorA acts as a phosphodiesterase to inhibit biofilm formation suggesting a possible role in biofilm dispersal. We found that both putrescine and its precursor arginine promote biofilm formation that is enhanced in the {Delta}spuC mutant, which cannot break down putrescine suggesting that putrescine might serve as a signaling molecule in the rhizosphere. Collectively, this work identified novel bacterial factors required to evade plant defenses in the rhizosphere.\n\nImportanceWhile rhizosphere bacteria hold the potential to improve plant health and fitness, little is known about the bacterial genes required to evade host immunity. Using a model system consisting of Arabidopsis and a beneficial Pseudomonas sp. isolate, we identified bacterial genes required for both rhizosphere fitness and for evading host immune responses. This work advances our understanding of how evasion of host defenses contributes to survival in the rhizosphere.

microbiology↗

bioRxiv2018

Convergent gain and loss of genomic islands drives lifestyle changes in plant-associated bacteria

Host-associated bacteria can have both beneficial and detrimental effects on host health. While some of the molecular mechanisms that determine these outcomes are known, little is known about the evolutionary histories of pathogenic or mutualistic lifestyles. Using the model plant Arabidopsis, we found that closely related strains within the Pseudomonas fluorescens species complex promote plant growth and occasionally cause disease. To elucidate the genetic basis of the transition between commensalism and pathogenesis, we developed a computational pipeline and identified genomic islands that correlate with outcomes for plant health. One island containing genes for lipopeptide biosynthesis and quorum sensing is required for pathogenesis. Conservation of the quorum sensing machinery in this island allows pathogenic strains to eavesdrop on quorum signals in the environment and coordinate pathogenic behavior. We found that genomic loci associated with both pathogenic and commensal lifestyles were convergently gained and lost in multiple lineages through homologous recombination, possibly constituting an early step in the differentiation of pathogenic and commensal lifestyles. Collectively this work provides novel insights into the evolution of commensal and pathogenic lifestyles within a single clade of host-associated bacteria.

microbiology↗

bioRxiv2017

Filling Gaps in Bacterial Amino Acid Biosynthesis Pathways with High-throughput Genetics

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

microbiology↗