Biology subjects

Dennehy, J. J.

Publications and source records attributed to Dennehy, J. J..

11 recordsLinked to original sources

bioRxiv2026

Selective Packaging of Rotavirus Double-Layered Particles into Host Microvesicles

Rotavirus is a non-enveloped RNA virus traditionally thought to exit host cells through lytic or conventional vesicular pathways, but recent evidence shows that it also exploits host microvesicles for non-lytic transmission. To characterize this transmission pathway, we combined high-resolution microscopy (TEM, confocal microscopy, immunogold labeling) with biochemical and infectivity assays. Our work revealed that rotavirus infection robustly increased microvesicle production, and individual vesicles frequently contained multiple viral particles. Quantitative analysis revealed striking differential partitioning of particle types. Mature triple-layered particles predominated within infected cells, whereas extracellular microvesicles were strongly enriched for immature double-layered particles. Although free double-layered particles lack the outer capsid required for classical receptor-mediated entry, microvesicle-associated particles initiated productive infections, whereas disruption of the vesicular membrane abolished infectivity. These findings suggest an evolutionarily advantageous dual transmission strategy. By using host-derived membranes to transmit otherwise non-infectious intermediates, rotavirus can bypass outer-capsid-dependent entry, potentially reducing the energetic cost of producing fully mature infectious particles while enabling collective transmission. Maintaining a free-virus pathway may nevertheless be important because transmission by individual mature virions imposes population bottlenecks that limit the propagation of defective or cheating genomes and preserve high-fitness genotypes. Thus, partitioning viral progeny between vesicle-associated double-layered particles and free triple-layered particles may balance the immediate benefits of collective spread with long-term genetic quality control. Whether double-layered particle enrichment arises from active sorting or spatial coupling between viral assembly and microvesicle biogenesis remains unresolved.

microbiology↗

bioRxiv2026

Genomic Characterization and Therapeutic Potential of the Lytic Bacteriophage Curly against Klebsiella pneumoniae in Human Innate Immune Cells and a Murine Pneumonia Model

Klebsiella pneumoniae is an important cause of severe respiratory and systemic infections, and the increasing prevalence of multidrug-resistant strains has created an urgent need for alternative antibacterial strategies. In this study, nine K. pneumoniae-infecting bacteriophages isolated from diverse environmental sources were characterized genomically and functionally. Genome analyses revealed substantial genomic and proteomic diversity among the isolates. Functional screening against the clinical K. pneumoniae isolate JJD85 identified Curly as the most active phage, producing the highest plaque-forming titer and rapid suppression of bacterial growth in liquid culture. Curly was predicted to have a virulent lifestyle and encoded structural, genome-packaging, and DNA replication-associated proteins. In primary human monocyte-derived macrophage cultures, Curly markedly reduced bacterial burden in both cell-associated and cell-free fractions, while treatment of primary human neutrophil cultures produced an approximately 10^6-fold reduction in total recoverable bacterial burden. Transmission electron microscopy demonstrated phage-like particles within bacterial profiles located in both extracellular and macrophage-associated intracellular compartments. In a C57BL/6J murine pneumonia model, intranasal Curly treatment reduced pulmonary bacterial burden in a dose-associated manner, with approximately 10-fold and 100-fold reductions at the low and high doses, respectively. Curly treatment also attenuated infection-associated lung inflammation and preserved pulmonary architecture. These findings identify Curly as a promising bacteriophage candidate against K. pneumoniae and support further evaluation of its host range, resistance profile, and therapeutic potential.

microbiology↗

bioRxiv2025

A Chemostat-Based Model for Growing Bacterial Biofilms

Biofilms are groups of microbes that live together in dense communities, often attached to a surface. They play an outsized role in all aspects of microbial life, from chronic infections to biofouling to dental decay. In recent decades, appreciation for the diversity of roles that biofilms play in the environment has grown. Yet, most bacterial studies still rely upon approaches developed in the 19th century and center on planktonic populations alone. Here we present a chemostat-based experimental platform to investigate not only biofilms themselves, but how they interact with their surrounding environments. Our results show that biofilms grow to larger sizes in chemostats as opposed to flasks. In addition, we show that biofilms may be a consistent source of migrants into planktonic populations. We also show that secondary biofilms rapidly develop, although these may be more susceptible to environmental conditions. Taken together, our data suggest that chemostats may be a flexible and insightful platform for the study of biofilms in vitro. IMPORTANCEBiofilms are the predominant way that bacteria live in natural environments and are characterized by three emergent properties: ubiquity, resilience, and impact. They can be found across all environments, both natural and human-made, and across all of recorded time, dating back at least 3.5 billion years. Biofilms also represent a major economic impact of over $5 trillion annually. Yet, most of what is known about bacteria is the result of studies using planktonically growing liquid cultures under laboratory conditions. Here, we propose a comprehensive experimental platform that allows for study of biofilms on the molecular, organismal, and community levels using chemostats.

microbiology↗

bioRxiv2025

Translation Efficiency Impacts Phage Lysis Timing and Its Precision in Single Cells

The timing of host cell lysis is a fundamental life history parameter in bacteriophages as it presents an evolutionary trade-off between maximizing intracellular phage replication and optimizing transmission to new host cells, ultimately determining viral fitness in dynamic bacterial populations. In the bacteriophage {lambda}, lysis time is dependent on the expression of a timekeeper protein, holin, which accumulates in the Escherichia coli inner membrane. Cell lysis is triggered when the membrane concentration of holin crosses a critical threshold level. In the present study, we investigated the effects of the rate of holin translation on lysis timing and its precision. We show that modulating holin translation efficiency through genetic modifications and antibiotic treatment alters bacteriophage lysis timing and its precision, providing insight into how phages optimize the evolutionary trade-off between replication and transmission. Reducing ribosomal binding affinity decreased holin expression and delayed lysis, while optimizing the Shine-Dalgarno sequence enhanced translation and accelerated lysis. Surprisingly, very low tetracycline concentrations may have improved translation efficiency and hastened lysis, whereas higher doses predictably delayed it. In all cases, longer lysis times corresponded with decreased timing variability. A model that incorporates stochastic gene expression with additional stochasticity in the initiation of holin expression was sufficient to explain the data. Thus, we demonstrated that modifying the rate of holin translation can be used as an evolutionary strategy to achieve optimum lysis timing. ImportanceOur prior work demonstrated that phage lysis timing is calibrated to optimize fitness based on environmental conditions affecting host availability and phage replication. We identified an optimal threshold--the critical holin concentration--that minimizes lysis time variability. Genetic manipulation of the holin gene altered the lysis threshold and generated phages with non-optimal early and late lysis times. The current study reveals that adjusting holin translation rates offers another mechanism for modifying lysis timing. Phages therefore have multiple means to adjust the timing of host lysis in order to maximize fitness under different selective pressures.

microbiology↗

bioRxiv2022

Risk of Infection Due to Airborne Virus in Classroom Environments Lacking Mechanical Ventilation

The COVID-19 pandemic highlighted the role of indoor environments on disease transmission. Enclosed spaces where pathogen-laden aerosols accumulate was strongly linked to increased transmission events. Here we employ a surrogate non-pathogenic virus, the bacteriophage phi6, to interrogate aerosol transmission in classroom environments that do not have any natural or mechanical ventilation in order to determine how effectively aerosols facilitate new infections. We find that virus-laden aerosols establish new infections over all distances tested within minutes and that the time of exposure did not change transmission rate. We further find that humidity, but not temperature nor a UV-based disinfection device, significantly impacted transmission rates. Our data suggest that, even without mechanical ventilation, relative humidity remains a highly effective mitigation strategy while UV air treatment did not. Practical ImplicationsTransmission of pathogens through airborne particles is a major source of disease transmission. People now spend much of their time indoors, thus understanding indoor airborne transmission is vital to managing outbreaks. Most classrooms in the U.S. do not have any mechanical ventilation systems and so here, we test airborne transmission of a virus in such classrooms. Infection transmission rates are not greatly impacted by distance, time or even some UV treatment, but are curbed by the amount of moisture in the air.

microbiology↗

bioRxiv2021

A single-cell approach reveals variation in cellular phage-producing capacities

Virus burst size, a component of viral fitness, is the average number of viral particles released from a single infected cell. In this study, we estimated bacteriophage lambda ({lambda}) burst size mean and distribution at different lysis times. To estimate phage {lambda} burst sizes at single-cell level, we employed a lysis-deficient E. coli lysogen, which allowed chemical lysis at desired times after the induction of lytic cycle. Induced cultures of E. coli lysogen were diluted and aliquoted into the wells of 96-well plates. A high dilution rate results in mostly empty wells and minimizes the probability of having multiple cells in wells that do receive cells. Burst size was estimated by titering single-cell lysates obtained after chemical lysis at desired times. Our data shows that the viral burst size initially increases exponentially with the lysis time, and then saturates at longer lysis times. We also demonstrate that cell-to-cell variation or "noise" in lysis timing does not significantly contribute to the burst size noise. The burst size noise remains constant with increasing mean burst size. The most likely explanation for the experimentally observed constant burst size noise is that cell-to-cell differences in burst size originate from differences in cellular capacity to produce phages. The mean burst size measured at different lysis times is positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicates that there are other factors in addition to cell size that determine this cellular capacity. ARTICLE IMPORTANCEPhages produce offspring by hijacking a cells replicative machinery. Previously, it was noted that the variation in the number of phages produced by single infected cells far exceeded cell size variation. It has been hypothesized that this variation is a consequence of variation in the timing of host cell lysis. Here we show that cell-to-cell variation in lysis timing does not significantly contribute to the burst size variation. We suggest that the constant burst size variation across different host lysis times results from cell-to-cell differences in capacity to produce phages. We find that the mean burst size measured at different lysis times is positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicates that there are other factors in addition to cell size that determine this cellular capacity.

microbiology↗

bioRxiv2021

Humidity Reduces Rapid and Distant Airborne Travel of Viable Viral Particles in Classroom Settings

The transmission of airborne pathogens via aerosols is considered to be the main route through which a number of known and emerging respiratory diseases infect their hosts. It is therefore essential to quantify airborne transmission in closed spaces and determine the recommendations that should be implemented to minimize exposure to pathogens in built environments. We have developed a method to detect viable virus particles from aerosols by using an aerosolized bacteriophage Phi6 in combination with its host Pseudomonas phaseolicola, which when seeded on agar plates acts as a virus detector that can be placed at a range of distances away from an aerosol-generating source. Based on this method we present two striking results. (1) We consistently detected viable phage particles at distances of up to 18 feet away from the source within 15-minutes of exposure in a classroom equipped with a state of the art HVAC system. (2) Increasing the relative humidity beyond 40% significantly reduces dispersal. Our method can be used to quantify the exposure to pathogens at various distances from the source for different amounts of time, data which can be used to set safety standards for room capacity and to ascertain the efficacy of interventions that aim to reduce pathogen levels in closed spaces of specified sizes and intended uses. SummaryWe present a method to experimentally determine the exposure to airborne pathogens in closed spaces.

microbiology↗

bioRxiv2021

Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns

Reassortment of the Rotavirus A (RVA) 11-segment dsRNA genome may generate new genome constellations that allow RVA to expand its host range or evade immune responses. Reassortment may also produce phylogenetic incongruities and weakly linked evolutionary histories across the 11 segments, obscuring reassortant-specific epistasis and changes in substitution rates. To determine the co-segregation patterns of RVA segments, we generated time-scaled phylogenetic trees for each of the 11 segments of 789 complete RVA genomes isolated from mammalian hosts and compared the segments geodesic distances. We found that segments 4 (VP4) and 9 (VP7) occupied significantly different treespaces from each other and from the rest of the genome. By contrast, segments 10 and 11 (NSP4 and NSP5/6) occupied nearly indistinguishable treespaces, suggesting strong co-segregation. Host-species barriers appeared to vary by segment, with segment 9 (VP7) presenting the least conservation by host species. Bayesian skyride plots were generated for each segment to compare relative genetic diversity among segments over time. All segments showed a dramatic decrease in diversity around 2007 coinciding with the introduction of RVA vaccines. To assess selection pressures, codon adaptation indices and relative codon deoptimization indices were calculated with respect to common host genomes. Codon usage varied by segment with segment 11 (NSP5) exhibiting significantly higher adaptation to host genomes. Furthermore, RVA codon usage patterns appeared optimized for expression in humans and birds relative to the other hosts examined, suggesting that translational efficiency is not a barrier in RVA zoonosis.

evolutionary biology↗

bioRxiv2020

An optimal lysis time maximizes bacteriophage fitness in quasi-continuous culture

Optimality models have a checkered history in evolutionary biology. While optimality models have been successful in providing valuable insight into the evolution of a wide variety of biological traits, a common objection is that optimality models are overly simplistic and ignore organismal genetics. We revisit evolutionary optimization in the context of a major bacteriophage life history trait, lysis time. Lysis time refers to the period spanning phage infection of a host cell and its lysis, whereupon phage progeny are released. Lysis time, therefore, directly determines phage fecundity assuming progeny assembly rate is maximized. Noting that previous tests of lysis time optimality rely on batch culture, we implemented a quasi-continuous culture system to observe productivity of a panel of isogenic phage {lambda} mutants differing in lysis time. We report that {lambda} phage productivity in our experiments is maximized around an optimal lysis time of 63 min, which is the lysis time of the {lambda} "wildtype" strain. We discuss this finding in light of recent results that lysis time variation is also minimized in the {lambda} "wildtype" strain.

evolutionary biology↗

bioRxiv2020

Optimum Threshold Minimizes Noise in Timing of Intracellular Events

How the noisy expression of regulatory proteins affects timing of intracellular events is an intriguing fundamental problem that influences diverse cellular processes. Here we use the bacteriophage {lambda} to study event timing in individual cells where cell lysis is the result of expression and accumulation of a single protein (holin) in the Escherchia coli cell membrane up to a critical threshold level. Site-directed mutagenesis of the holin gene was used to generate phage variants that vary in their timing of lysis from 30 to 190 min. Observation of the lysis times of single cells reveals an intriguing finding - the noise in lysis timing first decreases with increasing lysis time to reach a minimum, and then sharply increases at longer longer lysis times. A mathematical model with stochastic expression of holin together with dilution from cell growth was sufficient to explain the non-monotonic noise profile, and identify holin accumulation thresholds that generate precision in lysis timing.

biophysics↗

bioRxiv2019

Intragenic Recombination Influences Rotavirus Diversity and Evolution

Because of their replication mode and segmented dsRNA genome, homologous recombination is assumed to be rare in the rotaviruses. We analyzed 23,627 complete rotavirus genome sequences available in the NCBI Virus Variation database, and found 109 instances of homologous recombination, at least 11 of which prevailed across multiple sequenced isolates. In one case, recombination may have generated a novel rotavirus VP1 lineage. We also found strong evidence for intergenotypic recombination in which more than one sequence strongly supported the same event, particularly between different genotypes of segment 9, which encodes the serotype protein, VP7. The recombined regions of many putative recombinants showed amino acid substitutions differentiating them from their major and minor parents. This finding suggests that these recombination events were not overly deleterious, since presumably these recombinants proliferated long enough to acquire adaptive mutations in their recombined regions. Protein structural predictions indicated that, despite the sometimes substantial amino acid replacements resulting from recombination, the overall protein structures remained relatively unaffected. Notably, recombination junctions appear to occur non-randomly with hot spots corresponding to secondary RNA structures, a pattern seen consistently across segments. In total, we found strong evidence for recombination in nine of eleven rotavirus A segments. Only segment 7 (NSP3) and segment 11 (NSP5) did not show strong evidence of recombination. Collectively, the results of our computational analyses suggest that, contrary to the prevailing sentiment, recombination may be a significant driver of rotavirus evolution and may influence circulating strain diversity.

evolutionary biology↗