Biology subjects

Csorgo, B.

Publications and source records attributed to Csorgo, B..

4 recordsLinked to original sources

bioRxiv2026

A phiKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases

Bacteria employ diverse DNA-targeting systems, including restriction-modification (R-M) and CRISPR-Cas, to cleave invading bacteriophage genomes. In response, phages encode counter-defense strategies that block or mitigate DNA damage. Here, we screened a panel of Pseudomonas aeruginosa phages against native and heterologous DNA-targeting systems and identified the Phikmvvirus phage genus as broadly resistant to multiple CRISPR-Cas and R-M systems. Following CRISPR-Cas12a exposure, most protospacer sequences remained genetically unchanged. However, at an intergenic protospacer, mutations accumulated with high frequency at the Cas12a cleavage site rather than within PAM or seed sequences, resembling repair-associated indels observed after genome editing in eukaryotic cells. Genetic screens to isolate Cas12a- and EcoRI-sensitized phage mutants revealed perturbations to the phage DNA ligase. A Cas12a-sensitive mutant phage was rescued by DNA ligase expression in trans, which was also sufficient to reverse CRISPR targeting of an unrelated phage. Together, our results support a model in which phiKMV-like phages tolerate certain nucleases through ligase-dependent repair of nuclease-induced double-stranded breaks.

microbiology↗

bioRxiv2026

A preclinical resistance framework discovers the virulence risks of antibiotics in development

Several new antibiotics target multidrug-resistant pathogens, yet resistance is still evaluated mainly by drug-susceptibility, leaving consequences for bacterial pathogenicity poorly understood. Here, we develop a framework integrating resistance evolution, genomic surveillance and host-pathogen phenotyping to classify antibiotics by resistance potential and pathogenic consequences. Applying this framework to Klebsiella pneumoniae identified functionally distinct antibiotic candidates associated with elevated virulence risk. Resistance evolution rapidly increased virulence through clinically-relevant mutations, without direct selection for pathogenicity. Despite distinct genetic routes, resistance converged on cell-envelope rewiring. A single resistance mutation increased epithelial adhesion, intracellular colonization, macrophage immune-evasion, and tissue persistence in murine infection models, transforming K. pneumoniae into a more invasive and cytotoxic pathogen. Risk-profile analysis revealed partial decoupling of resistance and pathogenicity, with some low-resistance antibiotics yielding highly-virulent populations. These findings establish resistance-driven virulence as an underappreciated translational hazard and call for incorporating host-pathogen interactions into resistance surveillance and preclinical antibiotic development.

microbiology↗

bioRxiv2025

END nucleases: Antiphage defense systems targeting multiple hypermodified phage genomes

Prokaryotes carry clusters of phage defense systems in "defense islands" that have been extensively exploited bioinformatically and experimentally for discovery of immune functions. However, little effort has been dedicated to determining which specific system(s) within defense islands limit lytic phage reproduction in clinical bacterial strains. Here, we employed the CRISPR-based Cascade-Cas3 system to delete defense islands in a Pseudomonas aeruginosa clinical isolate to identify mechanisms of lytic phage antagonism. Deletion of one island in a cystic fibrosis-derived clinical isolate sensitized the strain to phages from the Pbunavirus family, which are commonly used as therapeutics. The causal defense system is a Type IIS restriction endonuclease-like protein (ENDPaCF1), common in Pseudomonads, however it lacks an associated methyltransferase typical Type IIS R-M systems. ENDPaCF1 protects bacteria against phages with hypermodified DNA and is surprisingly agnostic to the specific structure of the modification, which is unlike typical type IV restriction endonucleases. In ENDPaCF1, the endonuclease domain is fused to a catalytically inactive Endonuclease III (iEndoIII), a domain that recognizes non-canonical bases to repair DNA in prokaryotes and eukaryotes. We therefore propose that nucleases containing an iEndoIII domain (END nucleases) can sense diverse DNA hypermodifications. Our findings reveal modularity of the sensing and cleavage domains, as expected of a modification-dependent endonucleases. We further show that some hypermodified phages, including Pbunavirus family members and Wrowclawvirus family (Pa5oct-like) of jumbo phages, encode END nuclease inhibitors that directly bind to the nuclease, likely via the iEndoIII domain. These inhibitors are necessary for Pbunavirus to plaque on clinical isolates and sufficient to enable other hypermodified phages to plaque in the presence of this defense system.

microbiology↗

bioRxiv2019

A minimal CRISPR-Cas3 system for genome engineering

CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an "all-in-one" vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an "anti-anti-CRISPR" strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.

molecular biology↗