Biology subjects

Muszynski, A.

Publications and source records attributed to Muszynski, A..

4 recordsLinked to original sources

bioRxiv2026

Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways

Single stranded RNA (ssRNA) and single stranded DNA (ssDNA) bacteriophages represent a key component of the global virome, yet the host genetic networks supporting their infection cycles remain poorly understood. Here, we present a comprehensive, genome-wide mapping of the genetic landscape regulating infection cycles for F pilus-dependent ssRNA and ssDNA phages in Escherichia coli. Genetic screens across ssRNA phages spanning all four genogroups of the Leviviricetes revealed a highly conserved network of host dependencies, with the notable exception of the F plasmid gene traD. While primary structural receptor components and dsbA mediated disulfide bond formation are universally required across all lineages to ensure F pilus integrity, traD exhibits a strict genogroup-specific requirement during entry, showing variable essentiality across different viral groups despite sharing an identical primary receptor. Our gene dosage screens revealed that an elevated copy number of the hslU protease or the RNA chaperone stpA restricts infection, identifying clear genetic barriers that can perturb the viral life cycle. Parallel assays with filamentous ssDNA phages produced host factor profiles consistent with published literature, while revealing additional variations in host dependency. These screens confirmed that ssDNA phages strictly rely on the host TolQRA complex for entry downstream of pilus engagement. The assays tracked prominent negative fitness signatures across homeostatic clusters, highlighting how the physiological burden of continuous, non-lytic virion extrusion strains the host envelope. Finally, this comparative approach traced the selectivity of our isolation host (E. coli HSF) to a horizontally acquired capsule architecture from Klebsiella. This surface shield excludes a large panel of double stranded DNA phages isolated on diverse E. coli strains, while allowing virions from ssDNA and ssRNA phages to engage the extended F pilus and bypass the barrier via native pilus retraction. Together, this work provides a systematic, class-wide map of single stranded phage-host interactions, bridging classical genetics with modern viral discovery while establishing a robust host platform to access uncultured viral diversity and a functional blueprint to design next generation diagnostics, protein antibiotics, and biocontrol tools to halt horizontal gene transfer.

microbiology↗

bioRxiv2026

Transposon insertion sequencing of Pseudomonas aeruginosa identifies multiple intersecting pathways essential for extreme colistin resistance

Colistin is used to treat antibiotic resistant gram-negative infections, including those caused by Pseudomonas aeruginosa (Pa). Using a diverse collection of clinical isolates, we identified BWH047, a colistin-resistant isolate with an extremely high minimum inhibitory concentration (MIC, 1280 {micro}g/mL). To characterize the genes conditionally essential for colistin resistance in BWH047, we employed transposon insertion sequencing and identified 20 gene candidates. In-frame deletion validated 75% of the candidates and identified genes in several novel pathways that contribute to colistin resistance, including algU and wapH. We also identified several candidate genes from previously reported colistin resistance pathways (e.g. arn, pmrAB). We further investigated the impact of a colistin resistance-associated inner membrane DedA-family undecaprenyl phosphate flippase, which we named DpcA (DedA of Pseudomonas necessary for colistin resistance A). Deletion of dpcA in BWH047 restored sensitivity to colistin (MIC = 0.5 {micro}g/mL) and resulted in several unique changes to the structure of lipopolysaccharide (LPS), including production of decreased amounts of the colistin resistance-conferring 4-amino-4-deoxy-L-arabinose (L-Ara4N) modification on lipid A. To date, this work represents the most complete analysis of colistin resistance in Pa and identifies novel intersecting pathways that contribute to extreme phenotypic resistance. Author summaryPseudomonas aeruginosa is a bacterium that causes a wide variety of infections. It is especially problematic given its propensity to become resistant to antibiotics. One antibiotic used to treat multidrug-resistant P. aeruginosa infections is colistin. In this study, we investigated colistin resistance mechanisms in a patient-derived, extremely phenotypically resistant P. aeruginosa isolate, BWH047, using transposon insertion sequencing and mass spectrometry. We identified 13 genes conditionally essential for colistin resistance and investigated the role of one of these genes, dpcA, on the composition of the bacterial outer membrane, the target of colistin. Additionally, our study identified novel colistin resistance genes residing in several intersecting pathways that could be targeted to prevent the development of antimicrobial resistance.

microbiology↗

bioRxiv2025

Persistent trade-offs balance competition and colonization across centuries

Microbial competition drives rapid adaptation, often forcing organisms to specialize in new ecological niches. Adaptations that improve competitive ability can reduce performance in other environments creating trade-offs. Whether such trade-offs persist in nature--or are eroded as lineages adapt through compensatory changes--remains largely unknown. Here we show that a trade-off between competitive ability and host colonization has been stably maintained in natural Pseudomonas populations for centuries. Wild plant-pathogenic Pseudomonas compete using tailocins--phage-derived molecular weapons that bind to specific cell-surface receptors. Genomic surveys and functional assays reveal that the most broadly lethal tailocins remain rare--while the tailocins production increases competitive killing, it also compromises plant colonization. We determine that the polymorphisms behind this trade-off are not transient -- historical genomes spanning two centuries show that the trade-off has been maintained for at least 10-10 generations. Our results demonstrate that, in natural populations, a trade-off between competition and pathogenicity is fundamental and not easily overcome. SignificanceWhen a microbe colonizes a host, it must both establish infection and outcompete other organisms. Short-term experiments show that gains in competitive ability can reduce colonization, creating trade-offs, but whether microbes resolve these conflicts over long evolutionary timescales is unknown. We show that a trade-off between competitive killing and host colonization has been stably maintained for centuries in natural Pseudomonas populations infecting Arabidopsis thaliana. Tailocins--phage-derived weapons--provide strong competitive advantages, yet their production reduces colonization success, explaining why the most broadly lethal variants remain rare. Genomic surveys and historical genomes spanning two centuries reveal that the polymorphisms underlying this trade-off have persisted across 10-10 generations. Understanding such long-lived constraints can inform antimicrobial strategies that exploit evolutionary trade-offs.

evolutionary biology↗

bioRxiv2022

Surface anchoring of the Kingella kingae galactan is dependent on the lipopolysaccharide O-antigen

Kingella kingae is a leading cause of bone and joint infections and other invasive diseases in young children. A key K. kingae virulence determinant is a secreted exopolysaccharide that mediates resistance to serum complement and neutrophils and is required for full pathogenicity. The K. kingae exopolysaccharide is a galactofuranose homopolymer called galactan and is encoded by the pamABC genes in the pamABCDE locus. In this study, we sought to define the mechanism by which galactan is tethered on the bacterial surface, a prerequisite for mediating evasion of host immune mechanisms. We found that the pamD and pamE genes are glycosyltransferases and are required for synthesis of an atypical lipopolysaccharide (LPS) O-antigen. The LPS O-antigen in turn is required for anchoring of galactan, a novel mechanism for association of an exopolysaccharide with the bacterial surface. SignificanceKingella kingae is an emerging pediatric pathogen and produces invasive disease by colonizing the oropharynx, invading the bloodstream, and disseminating to distant sites. This organism produces a uniquely multifunctional exopolysaccharide called galactan that is critical for virulence and promotes intravascular survival by mediating resistance to serum and neutrophils. In this study, we established that at least some galactan is anchored to the bacterial surface via a novel structural interaction with an atypical lipopolysaccharide O-antigen. Additionally, we demonstrated that the atypical O-antigen is synthesized by the pamD and pamE genes, located downstream of the gene cluster responsible for galactan biosynthesis. This work addresses how the K. kingae exopolysaccharide can mediate innate immune resistance and advances understanding of bacterial exopolysaccharides and lipopolysaccharides.

microbiology↗