Biology subjects

Aylward, F.

Publications and source records attributed to Aylward, F..

13 recordsLinked to original sources

bioRxiv2026

Gentamicin Treatment in a Murine Infection Model Suggests Extracellular Orientia tsutsugamushi Contributes to Systemic Dissemination

Orientia tsutsugamushi, an obligate intracellular bacterial pathogen, infects multiple cell types and disseminates via the blood and lymphatic system to affect various organs in human and other mammalian hosts. This bacterium replicates exclusively within living cells; however, it also exists transiently in a metabolically inactive extracellular form. While infected leukocytes are known contributors to the systemic spread of the bacterium, the role of the inactive extracellular form in dissemination has been unclear. In this study, we demonstrate that extracellular bacteria are present in the cell-free plasma fraction of infected human and murine blood. Furthermore, treatment with gentamicin, a membrane-impermeable antibiotic, significantly reduces systemic bacterial growth in a murine infection model. These findings suggest a potential role for the extracellular form of the bacterium in the spread of infection and raise the possibility of exploring the use of gentamicin to augment conventional antibiotic therapy during scrub typhus infection.

microbiology↗

bioRxiv2026

Multi-level mosaicism shapes the evolution of small eukaryotic DNA viruses

Eukaryote-infecting small dsDNA viruses, including those referred to as virophages, polintons, and their relatives, are major constituents of ecosystems across the globe as both agents of mortality and as integrated elements that shape host genome evolution. As the ubiquity of these viruses has become apparent, there have been efforts to organize their diversity into major lineages in order to better understand their biogeography, ecological impact, and evolution. Virophages, polintons, and polinton-like viruses were recently placed in the sub-phylum Polisuviricotina, though efforts to resolve relationships within this sub-phylum have been complicated by the vast diversity of these viruses and the existence of horizontal gene transfer between their genomes. To address these complications, we have used a gene profile occurrence based method of phylogenetic tree construction to aid in comparative genomic analysis of Polisuviricotina. We find that mosaicism is common within Polisuviricotina, in particular, reassociation of replication modules and morphogenic modules occurs throughout major Polisuviricotina groups. In addition to mosaic exchanges of full replication modules, we also observe extensive domain swapping within some replicases, indicating that mosaic recombination occurs on both the genome and subgenic level in these viruses. Analysis of individual replication modules suggests that they may be capable of some level of autonomous mobility, partially decoupling their evolution from the morphogenic module. This model helps explain the high levels of mosaicism in these viruses and has implications for the mechanisms through which small dsDNA viruses evolve.

microbiology↗

bioRxiv2026

A chimeric infection program and epigenetic conflict underpin endogenous giant virus latency

Giant endogenous viral elements (GEVEs) are ubiquitous in eukaryotic genomes, but how the virus adapts to the host and is capable of reactivation despite host defense mechanisms remains unclear. Here we examined this process in the green alga Chlamydomonas reinhardtii and its latent giant virus, punuivirus, which is integrated into the host genome. We reveal a chimeric genomic architecture in which punuivirus latency genes are enriched in spliceosomal introns and 6-methyladenine marks, mirroring host transcriptional regulation, while virulence genes are predominantly controlled by a conserved viral promoter. Using single-cell RNA-seq, we show that nearly 70% of cells express viral transcripts, but only a small subpopulation commits to viral production, expressing virulence genes with host transcriptional shutoff. Moreover, we find that the intact punuivirus GEVE remains refractory to host 5-methylcytosine (5mC) silencing, whereas a naturally occurring deletion variant is associated with widespread 5mC accumulation across the GEVE and elsewhere in the host genome. The deleted region encodes a highly expressed ten-eleven translocation dioxygenase (TET), and epigenetic profiling of intact and deletion strains implicates this GEVE gene in removing 5mC marks, thereby maintaining the virus in a transcriptionally permissive latent state. Our study provides novel insights into the molecular underpinnings of host-virus co-existence in a giant virus-alga interaction.

microbiology↗

bioRxiv2026

Mimicin, an antimicrobial protein encoded by mimivirus

Antimicrobial peptides (AMPs) are innate defense molecules found in all domains of life. Giant viruses of amoeba are known to thrive among complex microbial relationships within its hosts cells, hinting at the existence of virus-derived antimicrobial strategies. Here we show that viruses belonging to the Mimiviridae and Marseilleviridae families contain a higher density of in silico predicted AMPs per genome size than other viruses of amoeba. The investigation of potential AMPs led to the description of Mimicin, a taxonomically restricted 74 amino acid long protein coded by few mimiviruses. Mimicin contains three smaller predicted AMP sequences within it and has a broad in vitro antimicrobial activity against different bacteria, a yeast and two non-enveloped phages. In contrast, it has no activity against a marseillevirus, a mimivirus or human cell lines. When tested against bacterial endosymbionts co-cultured with Acanthamoeba terricola, Mimicin and its SIM-31 portion were able to control the attenuated Protochlamydia amoebophila but not the highly virulent Parachlamydia acanthamoebae. Based on deposited transcriptomic data, Mimicin is coded by an early gene more active during the beginning of the infection process. No structure could be predicted using Alphafold, while additional structural analysis indicate that Mimicin could be a highly disordered protein. In conclusion, we describe evidence of a biologically relevant antimicrobial activity derived from a giant virus. Mimicin highlights the relevance of AMPs from giant viruses for microbial ecology and opens the way for investigating their biotechnological and clinical potential.

microbiology↗

bioRxiv2025

Developmental Progression and Single-Cell Heterogeneity of Orientia tsutsugamushi during Intracellular Infection

Orientia tsutsugamushi, the causative agent of human scrub typhus disease, is an obligate intracellular bacterium transmitted by mites. It replicates exclusively within eukaryotic cells, inhabiting a range of host cell types in mammals and mites. Previously, we demonstrated that its extracellular, non-replicative form is developmentally distinct from the intracellular replicative form. In this study, we investigated spatial and temporal heterogeneity within the intracellular bacterial population. By quantifying bacterial DNA replication, protein synthesis, cell division, and subcellular localization, we defined four distinct intracellular growth stages: pre-active, lag, log, and maturation. Using microscopy-based single-cell analysis of selected transcripts and proteins, we further revealed transcriptional and translational heterogeneity within microcolonies. These findings highlight the complex, asynchronous nature of intracellular O. tsutsugamushi growth and underscore the importance of single-cell analysis in understanding its pathobiology. ImportanceScrub typhus is a serious, sometimes life-threatening disease caused by the bacterium Orientia tsutsugamushi, which is spread by the bites of tiny mites and is common in many rural parts of Asia. This bacterium can only survive and grow inside living cells, where hundreds of individual bacteria may infect a single host cell. Until now, it was unclear whether all the bacteria inside a cell behaved the same way or if they existed in different stages of growth. In this study, we show that O. tsutsugamushi goes through several distinct stages while growing inside host cells and that not all bacteria are at the same stage at the same time. Even within a single infected cell, some bacteria are actively dividing, while others are preparing to leave the cell. Understanding these stages provides a new framework for exploring how the bacterium develops, spreads, and causes disease -- insights that could help guide future research and improve ways to control or treat scrub typhus.

microbiology↗

bioRxiv2024

Convergent evolution of viral-like Borg archaeal extrachromosomal elements and giant eukaryotic viruses

Borgs are huge extrachromosomal elements of anaerobic methane-oxidizing archaea. They exist in exceedingly complex microbiomes, lack cultivated hosts and have few protein functional annotations, precluding their classification as plasmids, viruses or other. Here, we used in silico structure prediction methods to investigate potential roles for [~]10,000 Borg proteins. Prioritizing analysis of multicopy genes that could signal importance for Borg lifestyles, we uncovered highly represented de-ubiquitination-like Zn-metalloproteases that may counter host targeting of Borg proteins for proteolysis. Also prevalent are clusters of multicopy genes for production of diverse glycoconjugates that could contribute to decoration of the host cell surface, or of putative capsid proteins that we predict multimerize into hexagonal arrays. Features including megabase-scale linear genomes with inverted terminal repeats, genomic repertoires for energy metabolism, central carbon compound transformations and translation, and pervasive direct repeat regions are shared with giant viruses of eukaryotes, although analyses suggest that these parallels arose via convergent evolution. If Borgs are giant archaeal viruses they would fill the gap in the tri(um)virate of giant viruses of all three domains of life. One Sentence SummaryProtein analyses, informed by in silico protein structure prediction, revealed that Borgs share numerous features with giant eukaryotic viruses, suggesting that Borgs have a viral-like lifestyle and evolutionary convergence of large extrachromosomal elements across the Domains of Life.

microbiology↗

bioRxiv2024

Towards a unifying phylogenomic framework for tailed phages

BackgroundClassifying viruses systematically has remained a key challenge of virology due to the absence of universal genes and vast genetic diversity of viruses. In particular, the most dominant and diverse group of viruses, the tailed double-stranded DNA viruses of prokaryotes belonging to the class Caudoviricetes, lack sufficient homology in the genetic machinery that unifies them to reconstruct inclusive, stable phylogenies of these genes. While previous approaches to organize tailed phage diversity have managed to distinguish various taxonomic levels, these methods are limited in scalability and reproducibility, and they do not include modes of evolution, like gene gains and losses. ResultsHere, we present a novel, comprehensive and reproducible framework for examining evolutionary relationships of tailed phages. In this framework, we compare phage genomes based on presences and absences of a fixed set of gene families which is used as binary trait data that is input into maximum likelihood models, which include heterogeneous rates of trait losses and gains. Our resulting phylogeny stably recovers known taxonomic families of tailed phages, with and without the inclusion of metagenomic phages. We also quantify the mosaicism of replication and structural genes among known families. Our results suggest that these exchanges likely underpin the emergence of new families. Additionally, we apply this framework to large phages (>100 kilobases) to map emergences of traits associated with genome expansion. ConclusionTaken together, this evolutionary framework for charting and organizing tailed phage diversity improves the systemization of phage taxonomy, which can unify phage studies and advance our understanding of their evolution.

genomics↗

bioRxiv2023

Automated classification of giant virus genomes using a random forest model built on trademark protein families

Viruses of the phylum Nucleocytoviricota, often referred to as "giant viruses," are prevalent in various environments around the globe and play significant roles in shaping eukaryotic diversity and activities in global ecosystems. Given the extensive phylogenetic diversity within this viral group and the highly complex composition of their genomes, taxonomic classification of giant viruses, particularly incomplete metagenome-assembled genomes (MAGs) can present a considerable challenge. Here we developed TIGTOG (Taxonomic Information of Giant viruses using Trademark Orthologous Groups), a machine learning-based approach to predict the taxonomic classification of novel giant virus MAGs based on profiles of protein family content. We applied a random forest algorithm to a training set of 1,531 quality-checked, phylogenetically diverse Nucleocytoviricota genomes using pre-selected sets of giant virus orthologous groups (GVOGs). The classification models were predictive of viral taxonomic assignments with a cross-validation accuracy of 99.6% to the order level and 97.3% to the family level. We found that no individual GVOGs or genome features significantly influenced the algorithms performance or the models predictions, indicating that classification predictions were based on a comprehensive genomic signature, which reduced the necessity of a fixed set of marker genes for taxonomic assigning purposes. Our classification models were validated with an independent test set of 823 giant virus genomes with varied genomic completeness and taxonomy and demonstrated an accuracy of 98.6% and 95.9% to the order and family level, respectively. Our results indicate that protein family profiles can be used to accurately classify large DNA viruses at different taxonomic levels and provide a fast and accurate method for the classification of giant viruses. This approach could easily be adapted to other viral groups.

bioinformatics↗

bioRxiv2023

Assessing the biogeography of marine giant viruses in four oceanic transects

Viruses of the phylum Nucleocytoviricota are ubiquitous in ocean waters and play important roles in shaping the dynamics of marine ecosystems. In this study, we leveraged the bioGEOTRACES metagenomic dataset collected across the Atlantic and Pacific Oceans to investigate the biogeography of these viruses in marine environments. We identified 330 viral genomes, including 212 in the order Imitervirales and 54 in the order Algavirales. We found that most viruses appeared to be prevalent in shallow waters (<150 meters), and that viruses of the Mesomimiviridae (Imitervirales) and Prasinoviridae (Algavirales) are by far the most abundant and diverse groups in our survey. Five mesomimiviruses and one prasinovirus are particularly widespread in oligotrophic waters; annotation of these genomes revealed common stress response systems, photosynthesis-associated genes, and oxidative stress modulation that may be key to their broad distribution in the pelagic ocean. We identified a latitudinal pattern in viral diversity in one cruise that traversed the North and South Atlantic Ocean, with viral diversity peaking at high latitudes of the northern hemisphere. Community analyses revealed three distinct Nucleocytoviricota communities across latitudes, categorized by latitudinal distance towards the equator. Our results contribute to the understanding of the biogeography of these viruses in marine systems.

microbiology↗

bioRxiv2021

Endogenous giant viruses shape intraspecies genomic variability in the model green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is an important eukaryotic alga that has been studied as a model organism for decades. Despite extensive history as a model system, phylogenetic and genetic characteristics of viruses infecting this alga have remained elusive. We analyzed high-throughput genome sequence data of C. reinhardtii field isolates, and in six we discovered sequences belonging to endogenous giant viruses that reach up to several hundred kilobases in length. In addition, we have also discovered the entire genome of a closely related giant virus that is endogenized within the genome of Chlamydomonas incerta, the closest sequenced phylogenetic relatives of C. reinhardtii. Endogenous giant viruses add hundreds of new gene families to the host strains, highlighting their contribution to the pangenome dynamics and inter-strain genomic variability of C. reinhardtii. Our findings suggest that the endogenization of giant viruses can have important implications for structuring the population dynamics and ecology of protists in the environment.

microbiology↗

bioRxiv2021

Pseudomonas aeruginosa infection reveals a Caspase-1-dependent neutrophil pyroptosis pathway that restrains damaging Histone release

Multiple neutrophil death programs contribute to host defense against infections. Although expressing all necessary components, neutrophils specifically fail to undergo pyroptosis, a lytic form of cell death triggered by the activation of the pro-inflammatory complex inflammasome. In the light of the arm race, we hypothesized that intrinsic neutrophil pyroptosis resistance might be bypassed in response to specific microbial species. We show that Pseudomonas aeruginosa (P. aeruginosa) stimulates Caspase-1-dependent pyroptosis in human and murine neutrophils. Mechanistically, activated NLRC4 inflammasome supports Caspase-1-driven Gasdermin-D (GSDMD) activation, IL-1{beta} cytokine release and neutrophil pyroptosis. Furthermore, GSDMD activates Peptidyl Arginine Deaminase-4 which drives an "incomplete NETosis" where neutrophil DNA fills the cell cytosol but fails crossing plasma membrane. Finally, we show that neutrophil Caspase-1 account for IL-1{beta} production and contributes to various P. aeruginosa strains spread in mice. Overall, we demonstrate that neutrophils are fully competent for Caspase-1-dependent pyroptosis, which drives an unsuspected "incomplete NETosis". SummaryNeutrophils play an essential roles against infections. Although multiple neutrophil death programs contribute to host defense against infections, they fail to undergo pyroptosis, a pro-inflammatory form of cell death. Upon Infections, pyroptosis can be induced in macrophages or epithelial cells upon activation of pro-inflammatory complexes, inflammasomes that trigger Caspase-1-driven Gasdermin dependent plasma membrane lysis. In the light of host-microbe interactions, we hypothesized that yet to find microbial species might hold the capacity to overcome neutrophil resistance to inflammasome-driven pyroptosis. Among several bacterial species, we describe that the bacterium Pseudomonas aeruginosa specifically engages the NLRC4 inflammasome, which promotes Caspase-1-dependent Gasdermin-D activation and subsequent neutrophil pyroptosis. Furthermore, inflammasome-driven pyroptosis leads to DNA decondensation and expansion into the host cell cytosol but not to the so called Neutrophil Extracellular Trap (NET) release as DNA fails breaching the plasma membrane. Finally, in vivo P. aeruginosa infections highlight that Caspase-1-driven neutrophil pyroptosis is functional and is detrimental upon P. aeruginosa infection. Altogether, our results unexpectedly underline neutrophil competence for Caspase-1-dependent pyroptosis, a process that contributes to host susceptibility to P. aeruginosa infection.

immunology↗

bioRxiv2021

A Phylogenomic Framework for Charting the Diversity and Evolution of Giant Viruses

Large DNA viruses of the phylum Nucleocytoviricota have recently emerged as important members of ecosystems around the globe that challenge traditional views of viral complexity. Numerous members of this phylum that cannot be classified within established families have recently been reported, and there is presently a strong need for a robust phylogenomic and taxonomic framework for these viruses. Here we report a comprehensive phylogenomic analysis of the Nucleocytoviricota, present a set of giant virus orthologous groups (GVOGs) together with a benchmarked reference phylogeny, and delineate a hierarchical taxonomy within this phylum. We show that the majority of Nucleocytoviricota diversity can be partitioned into 6 orders, 32 families, and 344 genera, substantially expanding the number of currently recognized taxonomic ranks for these viruses. We integrate our results within a megataxonomy that has been adopted for all viruses to establish a unifying framework for the study of Nucleocytoviricota diversity, evolution, and environmental distribution.

microbiology↗

bioRxiv2019

Dynamic Genome Evolution and Blueprint of Complex Virocell Metabolism in Globally-Distributed Giant Viruses

The discovery of giant viruses with large genomes has transformed our understanding of the limits of viral complexity in the biosphere, and subsequent research in model virus-host systems has advanced our knowledge of intricate mechanisms used by these viruses to take over host cells during infection. The extent of the metabolic diversity encoded by these viruses in the environment is less well-understood, however, and their potential impact on global biogeochemical cycles remains unclear. To address this, we generated 501 metagenome-assembled genomes (MAGs) of NCLDVs from diverse environments around the globe and analyzed their encoded functional diversity and potential for reprogramming host physiology. We found that 476 (95%) of the MAGs belonged to the Mimiviridae and Phycodnaviridae families, and of these we recovered 96% from aquatic environments, highlighting the diversity of these viral families in global freshwater and marine systems. MAGs encoded diverse genes predicted to be involved in nutrient uptake and processing, light harvesting, central nitrogen metabolism, and the manipulation of cell death, underscoring the complex interplay between these viruses and their hosts. Surprisingly, numerous genomes encoded genes involved in glycolysis, gluconeogenesis, and the TCA cycle, including one genome with a 70%-complete glycolytic pathway, suggesting that many of these viruses can even reprogram fundamental aspects of their hosts central carbon metabolism. Phylogenetic trees of NCLDV metabolic genes together with their cellular homologs revealed distinct clustering of viral sequences into divergent clades, indicating these metabolic genes are virus-specific and were acquired in the distant past. Our findings reveal that diverse NCLDV genomes encode complex, cell-like metabolic capabilities with evolutionary histories that are largely independent of cellular life, strongly implicating them as distinct drivers of biogeochemical cycles in their own right.

microbiology↗