Biology subjects

Brouns, S. J. J.

Publications and source records attributed to Brouns, S. J. J..

14 recordsLinked to original sources

bioRxiv2026

Anti-phage defence systems are enriched in multidrug-resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa encodes diverse defence systems against phages and mobile genetic elements, yet their variation across clinical contexts remains unclear. Here, we present a large-scale comparative analysis of the P. aeruginosa defensome across both public and clinically highly relevant datasets, including from patients with chronic lung disease and multidrug-resistant isolates. Our analysis shows that while influences on defensome composition are minor, multidrug-resistant isolates encode more defence systems and cystic fibrosis-associated isolates have fewer. Across phylogenetic clusters, defensome size correlates with cluster abundance, suggesting that defence-rich lineages persist more successfully across environments. Lastly, comparative analysis with other Pseudomonas species reveals enrichment of anti-plasmid systems in P. aeruginosa. Overall, these findings have important implications for phage therapy: multidrug-resistant infections may be more difficult to treat, while cystic-fibrosis-associated isolates may have higher phage susceptibility. This work provides a framework for understanding defensome variation and guiding the decision-making process of phage-based therapies.

microbiology↗

bioRxiv2025

DepoCatalog: Mapping the Diversity of 105 Recombinant Klebsiella Phage Depolymerases Across Sequence, Structure, and Substrate Specificity

A validated catalog of 105 recombinant depolymerases from Klebsiella phages covers 58 KL-types. 46 novel enzymes from prophages, jumbo phages, and common phages are linked to any known enzymatic activity against 14 classical serotypes and 12 genome-defined KL-types. Using activity-based profiling, structure prediction, and domain dissection, we developed a function-guided classification and a five-class structural catalog. This framework reveals highly specific enzymes active against up to three capsule types. K47 CPS was degraded by three diverse protein groups. Structurally similar depolymerases degrading particular CPS were found in distinct phage taxa, with highly conserved enzymes in Drulisvirus specific to K1-, K2-types. The exclusive depolymerases were found in siphoviruses targeting K2 and K62 serotypes. A case study of five structurally similar enzymes degrading KL22/KL37/KL111 and KL25/KL119 capsules suggested specificity switching via amino acid changes or C-domain modification. Klebsiella phage depolymerases catalog sheds light on their diversity, evolution, and potential application.

microbiology↗

bioRxiv2024

KlebPhaCol: A community-driven resource for Klebsiella research identified a novel gut phage order associated with the human gut

The growing threat of multidrug-resistant Klebsiella pneumoniae, coupled with its role in gut colonisation, has intensified the search for new treatments, including bacteriophage therapy. Despite increasing documentation of Klebsiella-targeting phages, clinical applications remain limited, with key phage-bacteria interactions still poorly understood. A major obstacle is fragmented access to well-characterised phage-bacteria pairings, restricting the collective advancement of therapeutic and mechanistic insights. To address this gap, we created the Klebsiella Phage Collection (KlebPhaCol), an open-source resource comprising 53 phages and 74 Klebsiella isolates, all fully characterised. These phages span five families - including a novel order, Felixvirales, associated with the human gut - and target 27 sequence types (including ST258, ST11, ST14) and 28 capsular-locus types (including KL1 and KL2), across six Klebsiella species. Freely accessible at www.klebphacol.org, KlebPhaCol invites the scientific community to both use and contribute to this resource, fostering collaborative research and a deeper understanding of Klebsiella-phage interactions beyond therapeutic use.

microbiology↗

bioRxiv2024

Reduced prevalence of phage defense systems in Pseudomonas aeruginosa strains from Cystic Fibrosis Patients

Cystic fibrosis is a genetic disorder that affects mucus clearance, particularly of the lungs. As a result, cystic fibrosis patients often experience infections from bacteria, which contribute to the disease progression. Pseudomonas aeruginosa is one of the most common opportunistic pathogens associated with cystic fibrosis. The presence of P. aeruginosa complicates the treatment due to its high antibiotic resistance. Thus, research is ongoing to treat these infections with bacterial viruses instead, known as bacteriophages. Notably, P. aeruginosa clinical strains possess a variety of phage defense mechanisms that may limit the effectiveness of phage therapy. In this study, we compared the defense system repertoire of P. aeruginosa strains isolated from cystic fibrosis patients with those from non-cystic fibrosis patients. Our findings reveal that P. aeruginosa strains isolated from cystic fibrosis patients have fewer phage defense mechanisms per strain than from non-cystic fibrosis patients, suggesting altered phage selection pressures in strains colonizing CF patient lungs. ImportanceCystic fibrosis patients often experience chronic Pseudomonas aeruginosa lung infections, which are challenging to treat with antibiotics and contribute to disease progression and eventual respiratory failure. Phage therapy is being explored as an alternative treatment strategy for these infections. However, assessing strain susceptibility to phage treatment is essential for ensuring efficacy. To address this, we investigated whether CF-associated clinical P. aeruginosa strains have a distinct phage defense repertoire compared to those isolated from other lung patients. We observedthat CF-associated P. aeruginosa strains have significantly fewer phage defenses, possibly affecting the susceptibility of these strains to phage infection.

microbiology↗

bioRxiv2023

De novo peptide sequencing with InstaNovo: Accurate, database-free peptide identification for large scale proteomics experiments

Bottom-up mass spectrometry-based proteomics is challenged by the task of identifying the peptide that generates a tandem mass spectrum. Traditional methods that rely on known peptide sequence databases are limited and may not be applicable in certain contexts. De novo peptide sequencing, which assigns peptide sequences to the spectra without prior information, is valuable for various biological applications; yet, due to a lack of accuracy, it remains challenging to apply this approach in many situations. Here, we introduce InstaNovo, a transformer neural network with the ability to translate fragment ion peaks into the sequence of amino acids that make up the studied peptide(s). The model was trained on 28 million labelled spectra matched to 742k human peptides from the ProteomeTools project. We demonstrate that InstaNovo outperforms current state-of-the-art methods on benchmark datasets and showcase its utility in several applications. Building upon human intuition, we also introduce InstaNovo+, a multinomial diffusion model that further improves performance by iterative refinement of predicted sequences. Using these models, we could de novo sequence antibody-based therapeutics with unprecedented coverage, discover novel peptides, and detect unreported organisms in different datasets, thereby expanding the scope and detection rate of proteomics searches. Finally, we could experimentally validate tryptic and non-tryptic peptides with targeted proteomics, demonstrating the fidelity of our predictions. Our models unlock a plethora of opportunities across different scientific domains, such as direct protein sequencing, immunopeptidomics, and exploration of the dark proteome. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/555055v3_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@20063org.highwire.dtl.DTLVardef@1679c01org.highwire.dtl.DTLVardef@1332940org.highwire.dtl.DTLVardef@1d9339f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

High resolution reconstruction of a Jumbo bacteriophage infecting capsulated bacteria using hyperbranched tail fibers

The Klebsiella jumbo myophage {phi}Kp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combined cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, and machine learning approaches to explore the capsid, tail, and tail fibers of this phage at high resolution. We determined the structure of the capsid and tail at 4.3[A] and 4.1[A] resolution. We observed that the tail fibers were highly branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide {phi}Kp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Taken together, our study provides structural and functional insight into how {phi}Kp24 adapts to the highly variable surfaces of capsulated bacterial pathogens, which will be useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.

microbiology↗

bioRxiv2022

Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

molecular biology↗

bioRxiv2021

Single Cell Variability of CRISPR-Cas Interference and Adaptation

CRISPR-Cas defence is a combination of adaptation to new invaders by spacer acquisition, and interference by targeted nuclease activity. While these processes have been studied on a population level, the individual cellular variability has remained unknown. Here, using a microfluidic device combined with time-lapse microscopy, we monitor invader clearance in a population of Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we show large cell-to-cell variability of clearance times, which originates from primed CRISPR adaptation. Faster growth and cell division, as well as higher levels of Cascade, increase the chance of clearance by interference. In contrast, faster growth is associated with decreased chances of clearance by priming. A mathematical model explains the experimental findings, and identifies Cascade binding to the mutated invader DNA, rather than spacer integration, as the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond to invading threats in a timely manner. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.

microbiology↗

bioRxiv2021

SCOPE: Flexible targeting and stringent CARF activation enables type III CRISPR-Cas diagnostics

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA (rather than DNA) and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of a target RNA and production of cyclic oligoadenylate (cOA) second messenger molecules that may trigger dormancy or cell death. In this study, we discovered that a largely exposed seed region at the 3 end of the crRNA is essential for target RNA binding and cleavage, whereas base pairing at a unique region at the 5 end of the guide is required to trigger cOA production. Moreover, we uncovered that the natural variation in the composition of type III complexes within a single host results in different guide lengths, and hence variable seed regions. This shifting seed may prevent escape by invading genetic elements, while controlling cOA production very tightly to prevent unnecessary damage to the host. Lastly, we used these findings to develop a new diagnostic tool, named SCOPE, which was used for the specific detection of SARS-CoV-2 from human nasal swab samples, showing sensitivities in the atto-molar range.

biochemistry↗

bioRxiv2020

Extracting transition rates in single-particle tracking using analytical diffusion distribution analysis

Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. Observed diffusion coefficients in vivo, for example, enable researchers to determine whether biomolecules are moving alone, as part of a larger complex or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models especially for molecules that rapidly interchange between different diffusional states. Here, we present analytic diffusion distribution analysis (anaDDA), a framework that allows extracting transition rates from distributions of observed diffusion coefficients. We show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics especially in the fast regime of 0.1-10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times, to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalysed experimental data of DNA polymerase I diffusing in live E. coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expanded to extract detailed information about the behaviour of biomolecules in living cells.

biophysics↗

bioRxiv2020

Prophages are associated with extensive, tolerated CRISPR-Cas auto-immunity

CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of CRISPR-Cas defense, or a regulatory mechanism for gene expression. Here we investigated the incidence, distribution, and evasion of STS in over 100,000 bacterial genomes. We found STS in all CRISPR-Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR-Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR-Cas systems appears to be rare, suggesting a level of escape from the potentially deleterious effects of STS by other mechanisms such as anti-CRISPR proteins and CRISPR target mutations. We propose a scenario where it is common to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects. The mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages, and contribute both to viral dissemination and bacterial diversification.

bioinformatics↗

bioRxiv2018

CRISPR-based DNA and RNA detection with liquid phase separation

The ability to detect specific nucleic acid sequences allows for a wide range of applications including identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence both enzymes activate a unique, indiscriminate nucleic acid cleavage activity, which has been exploited for detection of sequence specific nucleotides using labelled reporter molecules. We here present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Turbidity arises from coacervates of positively charged polyelectrolytes with long poly(dT) or poly(U) oligonucleotides. In the presence of a target sequence, long oligonucleotides are progressively shortened, changing the solution from turbid to transparent. We explain how oligonucleotide cleavage resolves LLPS by using a mathematical model which we validate with poly(dT) phase separation experiments. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.

biophysics↗

bioRxiv2018

An open microscopy framework suited for tracking dCas9 in live bacteria

Super-resolution microscopy is frequently employed in the life sciences, but the number of freely accessible and affordable microscopy frameworks, especially for single particle tracking photo-activation localization microscopy (sptPALM), remains limited. To this end, we designed the miCube: a versatile super-resolution capable fluorescence microscope, which combines high spatiotemporal resolution, good adaptability, low price, and easy installation. By providing all details, we hope to enable interested researchers to build an identical or derivative instrument. The capabilities of the miCube are assessed with a novel sptPALM assay relying on the heterogeneous expression of target genes. Here, we elucidate mechanistic details of catalytically inactive Cas9 (dead Cas9) in live Lactococcus lactis. We show that, lacking specific DNA target sites, the binding and unbinding of dCas9 to DNA can be described using simplified rate constants of kbound[->]free = 30-80 s-1 and kfree[->]bound = 15-40 s-1. Moreover, after providing specific DNA target sites via DNA plasmids, the plasmid-bound dCas9 population size decreases with increasing dCas9 copy number via a mono-exponential decay, indicative of simple disassociation kinetics.

biophysics↗

bioRxiv2017

Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

CRISPR-Cas loci provide an RNA-guided adaptive immune system against invading genetic elements. Interference in type I systems relies on the RNA-guided surveillance complex Cascade for target DNA recognition and the trans-acting Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here we employed single-molecule FRET to probe the helicase activity with a high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling in the target DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of three base pairs, that each underlie three successive 1-nt unwinding events. Reeling is highly repetitive, compensating for inefficient nicking activity of the nuclease domain. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure well controlled degradation of target DNA only.

biophysics↗