Biology subjects

Molari, M.

Publications and source records attributed to Molari, M..

11 recordsLinked to original sources

bioRxiv2026

Competition against the intestinal microbiota selects for pathoadaptive traits in ESBL E. coli

The intestinal tract is a reservoir for Extended-Spectrum {beta}-Lactamase (ESBL)-producing Escherichia coli. Asymptomatic gut colonization by these pathobionts represents a major risk for extraintestinal infections. Despite clinical relevance, the genetic basis of gut colonization by ESBL E. coli remains poorly understood. Here, we determined how the microbiota shapes the fitness landscape of diverse ESBL E. coli strains, defining the functional requirements for intestinal colonization. In microbiota-depleted hosts, colonization relies mostly on metabolic functions. In contrast, in mice harbouring a microbiota, pathoadaptive functions associated with adhesion and biofilm formation are dominant determinants of E. coli fitness, together with accessory virulence functions. Consistent with these observations, experimental evolution in mice reveals convergent adaptation of ESBL E. coli to the presence of a complex microbiota through enhanced adhesion. These findings establish the microbiota as a major ecological driver of pathoadaptation in antibiotic-resistant pathobionts.

microbiology↗

bioRxiv2025

Optimal Maturation Protocols for High-Affinity Antibody Targets: A Path-Integral Approach

Affinity maturation is a stochastic evolutionary process allowing the adaptive immune system to produce B-cells capable of recognizing antigenic molecules. One of the main factors influencing the quality of the maturation outcome, quantified by the affinity of the produced antibodies to the antigen, is the time-course of antigen availability. In this paper, we introduce a stochastic model for affinity maturation, calibrated against in vivo lineage dynamics and deep mutational scanning data, and address the following question: what is the best antigen-concentration protocol maximizing the probability to reach a target affinity value at the end of the process? We introduce a path-integral formalism to identify the maturation trajectories of the rare clones ending at the desired target affinity, estimate their probabilities, and maximize them over the antigen-concentration temporal profile. The theoretical optimal concentration protocol is approximated by a discrete three-injection schedule; we show that such temporal modulation of selection pressure outperforms constant-dosage regimes.

bioinformatics↗

bioRxiv2025

Synteny and linkage decay in bacteriophage pangenomes

Bacteriophages are rich in genetic diversity, due to frequent horizontal transfer and recombination. This makes traditional microbial phylogenetic analyses, often based on the assumption of vertical inheritance, not suitable for interpreting this diversity. Here, inspired by recent work on bacterial pangenomes, we investigate the evolution of a collection of 3425 actinobacteriophage genomes. We find that synteny is strongly conserved: core genes have a well-defined order, and most accessory genes are localized in a few locations along the core genome backbone. Within the core genome alignment, linkage disequilibrium decays rapidly with distance in some groups, while phylogenetic structure in other groups causes long-range linkage. Our quantitative characterizations extend across many groups of phages and indicate widespread homologous recombination restricted by strong gene order conservation.

genomics↗

bioRxiv2025

Automated Bacteriophage Evolution Studies with the Aionostat

Bacteriophages, the viruses that infect bacteria, are the most abundant and diverse biological entities on our planet. They play a critical role in shaping ecosystems and are increasingly recognized for their potential in treating bacterial infections. Yet, our comprehension of their biology and evolutionary dynamics is limited, largely because research has concentrated on a select few well-characterized phages or relies on broad metagenomic studies with limited follow-up analysis of individual phages. This knowledge gap hinders our capacity to exploit their therapeutic and ecological possibilities - and while some studies have attempted to bridge it, such efforts typically require a lot of manual labor, highlighting the need for high-throughput, reproducible methods for in-depth study of phage evolution. To address this gap, we introduce the Aionostat, a novel automated continuous culture device designed to facilitate bacteriophage directed evolution experiments at scale. The Aionostats potential is showcased through two example experiments. In the first, phages from the BASEL collection rapidly adapted to a challenging E. coli strain, acquiring mutations and deletions that improved their infectivity. In the second experiment, we evolved a mixture of these phages on the same E.coli strain, leading to the emergence of recombinant phages with increased fitness. By automating these experiments, the Aionostat enables faster, more reproducible studies of phage evolution that would be impractical to perform by hand, thereby opening new avenues for investigating viral dynamics, engineering phage therapies, and studying evolutionary principles in broader biological contexts.

evolutionary biology↗

bioRxiv2025

Replicative selfish genetic elements are driving rapid pathogenic adaptation of Enterococcus faecium

Understanding how healthcare-associated pathogens adapt in clinical environments can inform strategies to reduce their burden. Here, we investigate the hypothesis that insertion sequences (IS), prokaryotic transposable elements, are a dominant mediator of rapid genomic evolution in healthcare-associated pathogens. Among 28,207 publicly available pathogen genomes, we find high copy numbers of the replicative ISL3 family in healthcare-associated Enterococcus faecium, Streptococcus pneumoniae and Staphylococcus aureus. In E. faecium, the ESKAPE pathogen with the highest IS density, we find that ISL3 proliferation has increased in the last 30 years. To enable better identification of structural variants, we long read-sequenced a new, single hospital collection of 282 Enterococcal infection isolates collected over three years. In these samples, we observed extensive, ongoing structural variation of the E. faecium genome, largely mediated by active replicative ISL3 elements. To determine if ISL3 is actively replicating in clinical timescales in its natural, gut microbiome reservoir, we long read-sequenced a collection of 28 longitudinal stool samples from patients undergoing hematopoietic cell transplantation, whose gut microbiomes were dominated by E. faecium. We found up to six structural variants of a given E. faecium strain within a single stool sample. Examining longitudinal samples from one individual in further detail, we find ISL3 elements can replicate and move to specific positions with profound regulatory effects on neighboring gene expression. In particular, we identify an ISL3 element that upon insertion replaces an imperfect -35 promoter sequence at a folT gene locus with a perfect -35 sequence, which leads to substantial upregulation of expression of folT, driving highly effective folate scavenging. As a known folate auxotroph, E. faecium depends on other members of the microbiota or diet to supply folate. Enhanced folate scavenging may enable E. faecium to thrive in the setting of microbiome collapse that is common in HCT and other critically ill patients. Together, ISL3 expansion has enabled E. faecium to rapidly evolve in healthcare settings, and this likely contributes to its metabolic fitness and may strongly influence its ongoing trajectory of genomic evolution.

microbiology↗

bioRxiv2024

Evolutionary dynamics of genome structure and content among closely related bacteria

Bacterial genomes primarily diversify via gain, loss, and rearrangement of genetic material in their flexible accessory genome. Yet the dynamics of accessory genome evolution are very poorly understood, in contrast to the core genome where diversification is readily described by mutations and homologous recombination. Here, we tackle this problem for the case of very closely related genomes. We comprehensively describe genome evolution within n=222 genomes of E. coli ST131, which likely shared a common ancestor around one hundred years ago. After removing putative recombinant diversity, the total length of the phylogeny is 6000 core genome mutations. Within this diversity, we find 22 modifications to core genome synteny and estimate around 2000 structural changes within the accessory genome, i.e. one structural change for every 3 core genome mutations. 63% of loci with structural diversity could be resolved into individual gain and loss events with ten-fold more gains than losses, demonstrating a dominance of gains due to insertion sequences and prophage integration. Our results suggest the majority of synteny changes and insertions in bacterial genomes are likely deleterious and only persist for a short time before being removed by purifying selection.

evolutionary biology↗

bioRxiv2022

Generative and interpretable machine learning for aptamer design and analysis of in vitro sequence selection

Selection protocols such as SELEX, where molecules are selected over multiple rounds for their ability to bind to a target molecule of interest, are popular methods for obtaining binders for diagnostic and therapeutic purposes. With the increasing amount of such high-throughput experimental data available, machine learning techniques have become increasingly popular for molecular datasets analysis. Here, we show that Restricted Boltzmann Machines (RBMs), a two-layer neural network architecture, can successfully be trained on sequence ensembles from SELEX experiments for thrombin aptamers, and used to estimate the fitness of the sequences obtained through the experimental protocol. As a direct consequence, we show that trained RBMs can be exploited to classify as well as generate novel molecules. To confirm our findings, we experimentally verify the generated sequences from RBM.

biophysics↗

bioRxiv2022

PanGraph: scalable bacterial pan-genome graph construction

The genomic diversity of microbes is commonly parameterized as single nucleotide polymorphisms relative to a reference genome of a well-characterized, but arbitrary, isolate. However, any reference genome contains only a fraction of the microbial pangenome, the total set of genes observed in a given species. Reference-based approaches are thus blind to the dynamics of the accessory genome, as well as variation within gene order and copy number. With the wide-spread usage of long-read sequencing, the number of high-quality, complete genome assemblies has increased dramatically. Traditional computational approaches towards whole-genome analysis either scale poorly with the number of genomes, or treat genomes as dissociated "bags of genes", and thus are not suited for this new era. Here, we present PanGraph, a Julia-based library and command line interface for aligning whole genomes into a graph. Each genome is represented as an undirected path along vertices, which in turn, encapsulate homologous multiple sequence alignments. The resultant data structure succinctly summarizes population-level nucleotide and structural polymorphisms and can be exported into a several common formats for either downstream analysis or immediate visualization.

bioinformatics↗

bioRxiv2021

FAIR enough? A perspective on the status of nucleotide sequence data and metadata on public archives

Knowledge derived from nucleotide sequence data is increasing in importance in the life sciences, as well as decision making (mainly in biodiversity policy). Metadata standards have been established to facilitate sustainable sequence data management according to the FAIR principles (Findability, Accessibility, Interoperability, Reusability). Here, we review the status of metadata available for raw read Illumina amplicon and whole genome shotgun sequencing data derived from ecological metagenomic material that are accessible at the European Nucleotide Archive (ENA), as well as the compliance of the primary sequence data (fastq files) with data submission requirements. While overall basic metadata, such as geographic coordinates, were retrievable in 98% of the cases for this type of sequence data, interoperability was not always ensured and other (mainly conditionally) mandatory parameters were often not provided at all. Metadata standards, such as the Minimum Information about any(x) Sequence (MIxS), were only infrequently used despite a demonstrated positive impact on metadata quality. Furthermore, the sequence data itself did not meet the prescribed requirements in 31 out of 39 studies that were manually inspected. To tackle the most immediate needs to improve FAIR sequence data management, we provide a list of minimal suggestions to researchers, research institutions, funding agencies, reviewers, publishers, and databases, that we believe might have a potentially large positive impact on sequence data and metadata FAIRness, which is crucial for further research and its derived applications.

molecular biology↗

bioRxiv2020

The hidden pangenome: comparative genomics reveals pervasive diversity in symbiotic and free-living sulfur-oxidizing bacteria

Sulfur-oxidizing Thioglobaceae, often referred to as SUP05 and Arctic96BD clades, are widespread and common to hydrothermal vents and oxygen minimum zones. They impact global biogeochemical cycles and exhibit a variety of host-associated and free-living lifestyles. The evolutionary driving forces that led to the versatility, adoption of multiple lifestyles and global success of this family are largely unknown. Here, we perform an in-depth comparative genomic analysis using all available and newly generated Thioglobaceae genomes. Gene content variation was common, throughout taxonomic ranks and lifestyles. We uncovered a pool of variable genes within most Thioglobaceae populations in single environmental samples and we referred to this as the hidden pangenome. The hidden pangenome is often overlooked in comparative genomic studies and our results indicate a much higher intra-specific diversity within environmental bacterial populations than previously thought. Our results show that core-community functions are different from species core genomes suggesting that core functions across populations are divided among the intra-specific members within a population. Defense mechanisms against foreign DNA and phages were enriched in symbiotic lineages, indicating an increased exchange of genetic material in symbioses. Our study suggests that genomic plasticity and frequent exchange of genetic material drives the global success of this family by increasing its evolvability in a heterogeneous environment.

microbiology↗

bioRxiv2020

Quantitative modeling of the effect of antigen dosage on B-cell affinity distributions in maturating germinal centers

Affinity maturation is a complex dynamical process allowing the immune system to generate antibodies capable of recognizing antigens. We introduce a model for the evolution of the distribution of affinities across the antibody population in germinal centers. The model is amenable to detailed mathematical analysis, and gives insight on the mechanisms through which antigen availability controls the rate of maturation and the expansion of the antibody population. It is also capable, upon maximum-likelihood inference of the parameters, to reproduce accurately the distributions of affinities of IgG-secreting cells we measure in mice immunized against Tetanus Toxoid under largely varying conditions (antigen dosage, delay between injections). Both model and experiments show that the average population affinity depends non-monotonically on the antigen dosage. We show that combining quantitative modelling and statistical inference is a concrete way to investigate biological processes underlying affinity maturation (such as selection permissiveness), hardly accessible through measurements.

immunology↗