Biology subjects

Mostowy, R. J.

Publications and source records attributed to Mostowy, R. J..

7 recordsLinked to original sources

bioRxiv2026

Structural modularity of receptor-binding proteins underlies host-range strategy diversification in Klebsiella pneumoniae phages

Bacteriophage receptor-binding proteins (RBPs) determine bacterial host recognition and are central to phage host-range evolution, yet the structural principles governing how RBPs diversify and expand host range remain not fully understood. Although phage RBPs are thought to evolve through modular exchange of receptor-binding domains, it is unclear how this modularity is organised across diverse RBP architectures, at what structural scales recombination operates, and how it shapes host-range breadth. Here we combined large-scale AlphaFold3 structural modelling, domain-level annotation, de novo pseudo-domain segmentation, sequence modularity analysis and experimentally determined host-range phenotypes across 192 Klebsiella pneumoniae phages and 382 high-confidence RBPs spanning up to 96 K-types. We generated the first system-wide structural atlas of K. pneumoniae phage RBPs, resolving 39 structurally distinct RBP-classes. Although capsule-degrading beta-helix depolymerases dominated numerically, 161 RBPs across 37 RBP-classes employed non-depolymerase architectures, including 18 novel RBP-classes. Structural and sequence analyses show that this diversity arose through modular reuse of structural domains rather than independent invention. Conserved N-terminal scaffolds linked depolymerase and non-depolymerase RBPs across morphotypes, while receptor-binding regions diversified through recombination operating at and beyond domain boundaries. We found recent modular exchange both within genera, where capsule-specific and capsule-independent RBPs can be swapped to alter host-range strategy, and across morphotypes, where depolymerase modules moved between distant lineages altering capsule specificity. Together, these architectures resolve into six receptor-recognition strategies, establishing multi-scale modularity as the primary organising principle of RBP diversification and a structure-informed framework for guiding phage isolation and engineering against K. pneumoniae. The complete atlas is freely accessible as an interactive community resource at Klebsiella-Phage-RBP-Atlas.

evolutionary biology↗

bioRxiv2025

Capsular specificity in temperate phages of Klebsiella pneumoniae is driven by diverse receptor-binding enzymes

In bacteriophages infecting Klebsiella pneumoniae, capsule specificity is a major determinant of host range due to the presence of capsule-specific depolymerases. Yet for temperate phages, the genetic and functional basis of this specificity remains less well understood. Depolymerases appear unexpectedly rare in prophage genomes, raising unresolved questions about which prophage genes mediate capsule tropism, whether this apparent scarcity reflects biological or ecological differences versus annotation limits, and whether prophage-encoded receptor-binding proteins (RBPs) are functionally active. To address these questions, we analysed 3,900 Klebsiella genomes from diverse ecological niches to identify prophage-encoded proteins mediating capsule tropism. We conducted a genome-wide association study (GWAS) correlating prophage protein clusters (from 8,105 prophages) with confidently assigned bacterial K-loci. GWAS identified high-confidence predictors for 16 out of 35 most diverse K-loci, of which 14 were receptor-binding proteins (RBPs) belonging to classical depolymerases (n = 6), SGNH hydrolases which deacetylate polysaccharides (n = 6), and structurally novel RBPs (n = 2). Overall, we predicted K-locus specificity for 26 putative depolymerases, of which 12 were deemed as strong predictions against 10 K-loci. In parallel, we attempted recombinant production of 50 putative depolymerases selected from 469 candidate proteins identified in prophages from a representative subset of 99 bacterial isolates, together with an additional 10 depolymerases selected based on GWAS predictions. All recombinant proteins were tested against a Klebsiella reference panel of 119 K-types. Of the 50 manually chosen putative depolymerases, 34 failed to yield detectable recombinant expression, a pattern unlikely to be explained by degraded or defective prophages. Of the 14 active enzymes, 5 targeted a K-locus different from that of their bacterial host, and enzyme specificity could not always be reliably inferred from sequence similarity or structural homology. Comparison of GWAS predictions with experimental validation results revealed that 10 of the 12 strongest GWAS predictors were confirmed experimentally, while 2 produced soluble protein but showed no detectable activity against the tested K-types. Together, these results highlight the intrinsic difficulty of predicting activity and capsule specificity of prophage-encoded RBPs from genomic information alone. Finally, analysis of 4,598 high-completeness prophages revealed that SGNH-domain hydrolases are among the most prevalent enzymatic domains in prophage RBPs. Two SGNH-domain RBPs identified by GWAS were experimentally confirmed as active esterases, supporting capsule deacetylation as a widespread alternative to polysaccharide depolymerisation in temperate phages. Our findings reveal that Klebsiella prophages encode structurally diverse RBPs, suggesting temperate phages may rely not only on depolymerisation but also on capsule modification--such as deacetylation--for infection. This also implies that capsule diversity in K. pneumoniae may be substantially underestimated, with implications for phage specificity, competition and vaccine design.

microbiology↗

bioRxiv2024

Rapid and Accurate Estimation of Genetic Relatedness Between Millions of Viral Genome Pairs Using MANIAC

Average Nucleotide Identity (ANI) is a widely used metric to estimate genetic relatedness, especially in microbial species delineation. While ANI calculation has been well optimised for bacteria and closely related viral genomes, accurate estimation of ANI below 80%, particularly in large reference datasets, has been challenging due to a lack of accurate and scalable methods. To bridge this gap, here we introduce MANIAC, an efficient computational pipeline optimised for estimating ANI and alignment fraction (AF) in viral genomes with divergence around ANI of 70%. Using a rigorous simulation framework, we demonstrate MANIACs accuracy and scalability compared to existing approaches, even to datasets of hundreds-of-thousands of viral genomes. Applying MANIAC to a curated dataset of complete bacterial dsDNA viruses revealed a multimodal ANI distribution, with a distinct gap around 80%, akin to the bacterial ANI gap ([~]90%) but shifted, likely due to viral-specific evolutionary processes such as recombination dynamics and mosaicism. We then evaluated ANI and AF as predictors of genus-level taxonomy using a logistic regression model. We found that this model has strong predictive power (PR-AUC=0.981), but that it works much better for virulent (PR-AUC=0.997) than temperate (PR-AUC=0.847) bacterial viruses. This highlights the complexity of taxonomic classification in temperate phages, known for their extensive mosaicism, and cautions against over-reliance on ANI in such cases. MANIAC can be accessed under https://github.com/bioinf-mcb/MANIAC. ImportanceWe introduce a novel computational pipeline called MANIAC, designed to accurately assess Average Nucleotide Identity (ANI) and alignment fraction (AF) between diverse viral genomes, scalable to datasets of over 100k genomes. Through the use of computer simulations and real data analyses, we show that MANIAC could ac- curately estimate genetic relatedness between pairs of viral genomes around 60-70% ANI. We applied MANIAC to investigate the question of ANI discontinuity in bacterial dsDNA viruses, finding evidence for an ANI gap, akin to the one seen in bacteria but around ANI of 80%. We then assessed the ability of ANI and AF to predict taxonomic genus boundaries, finding its strong predictive power in virulent, but not in temperate phages. Our results suggest that bacterial dsDNA viruses may exhibit an ANI threshold (on average around 80%) above which recombination helps maintain population cohesiveness, as previously argued in bacteria.

bioinformatics↗

bioRxiv2022

Protein modularity in phages is extensive and associated with functions linked to core replication machinery and host tropism determinants

Biological modularity enhances evolutionary adaptability by allowing rearrangement of functional components. One striking example are bacterial viruses (phages). They exhibit extensive genomic modularity by being built of independent functional modules that evolve separately and combine in various ways, making them astoundingly diverse. While multiple studies have investigated genomic modularity in phages, less attention has been given to protein modularity--proteins having distinct building blocks or domains that can evolve and recombine, enhancing functional and genetic diversity. To better understand the impact of protein modularity on viral evolution, we quantified it by detecting instances of domain mosaicism, defined as a homologous fragment sharing between two otherwise unrelated proteins. We used highly sensitive homology detection to quantify domain mosaicism between pairs of 133,574 representative phage proteins and to understand its relationship with functional diversity in phage genomes. We found that diverse functional classes often shared homologous domains. This phenomenon was often linked to protein modularity, particularly in receptor-binding proteins, endolysins and DNA polymerases. We also identified multiple instances of recent diversification via exchange and gain/loss of domains in receptor-binding proteins, neck passage structures, endolysins and some members of the core replication machinery. Diversification via protein fragment exchange often transcended distant taxonomic and ecological borders. We argue that the ongoing diversification via shuffling of protein domains associated with those functions is reflective of co-evolutionary arms race and the resulting diversifying selection to overcome multiple mechanisms of bacterial resistance against phages.

evolutionary biology↗

bioRxiv2019

Shigella sonnei infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence

Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.\n\nAuthor SummaryShigella sonnei is predominantly responsible for dysentery in developed countries, and is replacing Shigella flexneri in areas undergoing economic development and improvements in water quality. Using Shigella infection of zebrafish (in vivo) and human neutrophils (in vitro), we discover that S. sonnei is more virulent than S. flexneri because of neutrophil tolerance mediated by its O-antigen oligosaccharide acquired from the environmental bacteria Plesiomonas shigelloides. To inspire new approaches for S. sonnei control, we show that increased phagolysosomal acidification or innate immune training can promote S. sonnei clearance by neutrophils in vivo. These findings have major implications for our evolutionary understanding of Shigella, and may explain why exposure to P. shigelloides in low and middle-income countries (LMICs) can protect against dysentery incidence.

microbiology↗

bioRxiv2019

Diversity and evolution of surface polysaccharide synthesis loci in Enterobacteriales

Bacterial capsules and lipopolysaccharides are diverse surface polysaccharides (SPs) that serve as the frontline for interactions with the outside world. While SPs can evolve rapidly, their diversity and evolutionary dynamics across different taxonomic scales has not been investigated in detail. Here, we focused on the bacterial order Enterobacteriales (including the medically-relevant Enterobacteriaceae), to carry out comparative genomics of two SP locus synthesis regions, cps and kps, using 27,334 genomes from 45 genera. We identified high-quality cps loci in 22 genera and kps in 11 genera, around 4% of which were detected in multiple species. We found SP loci to be highly dynamic genetic entities: their evolution was driven by high rates of horizontal gene transfer (HGT), both of whole loci and component genes, and relaxed purifying selection, yielding large repertoires of SP diversity. In spite of that, we found the presence of (near-)identical locus structures in distant taxonomic backgrounds that could not be explained by recent exchange, pointing to long-term selective preservation of locus structures in some populations. Our results reveal differences in evolutionary dynamics driving SP diversity within different bacterial species, with lineages of Escherichia coli, Enterobacter hormachei and Klebsiella aerogenes most likely to share SP loci via recent exchange; and lineages of Salmonella enterica, Citrobacter sakazakii and Serratia marcescens most likely to share SP loci via other mechanisms such as long-term preservation. Overall, the evolution of SP loci in Enterobacteriales is driven by a range of evolutionary forces and their dynamics and relative importance varies between different species.

evolutionary biology↗

bioRxiv2019

Horizontal gene transfer rate is not the primary determinant of observed antibiotic resistance frequencies in Streptococcus pneumoniae

The extent to which evolution is constrained by the rate at which horizontal gene transfer (HGT) allows DNA to move between genetic lineages is an open question, which we address in the context of antibiotic resistance in Streptococcus pneumoniae. We analyze microbiological, genomic and epidemiological data from the largest-to-date sequenced pneumococcal carriage study in 955 infants from a refugee camp on the Thailand-Myanmar border. Using a unified framework, we simultaneously test prior hypotheses on rates of HGT and a key evolutionary covariate (duration of carriage) as determinants of resistance frequencies. We conclude that in this setting, there is only weak evidence for the rate of HGT playing a role in the evolutionary dynamics of resistance. Instead, observed resistance frequencies are best explained as the outcome of selection acting on a pool of variants, irrespective of the rate at which resistance determinants move between genetic lineages.

evolutionary biology↗