Biology subjects

Haddock, N. L.

Publications and source records attributed to Haddock, N. L..

7 recordsLinked to original sources

bioRxiv2025

Pseudomonas superinfection drives Pf phage transmission within airway infections in patients with cystic fibrosis

Pf bacteriophages, lysogenic viruses that infect Pseudomonas aeruginosa (Pa), are implicated in the pathogenesis of chronic Pa infections; phage-infected (Pf+) strains are known to predominate in people with cystic fibrosis (pwCF) who are older and have more severe disease. However, the transmission patterns of Pf underlying the progressive dominance of Pf+ strains are unclear. In particular, it is unknown whether phage transmission commonly occurs horizontally between bacteria within the airway via viral particles or if Pf+ bacteria are mostly acquired via new Pseudomonas infections. Here, we have studied Pa genomic sequences from 3 patient cohorts totaling 663 clinical isolates from 105 pwCF. We identify Pf+ isolates and analyze transmission patterns of Pf within patients between genetically similar groups of bacteria called "clone types". We find that Pf is predominantly passed down vertically within Pa lineages and rarely via horizontal transfer between clone types within the airway. Conversely, we find extensive evidence of Pa superinfection by a new, genetically distinct Pa that is Pf+. Finally, we find that clinical isolates show reduced activity of the type IV pilus and reduced susceptibility to Pf in vitro. These results cast new light on the transmission of virulence-associated phages in the clinical setting.

microbiology↗

bioRxiv2023

Rapid assessment of changes in phage bioactivity using dynamic light scattering

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. Here, we use Dynamic Light Scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings. Significance StatementPhages are promising for use in treating antibiotic-resistant infections, but their decay over time in refrigerated storage and higher temperatures has been a difficult barrier to overcome. This is in part because there are no suitable methods to monitor phage activity over time, especially in clinical settings. Here, we show that Dynamic Light Scattering (DLS) can be used to measure the physical state of phage preparations, which provides accurate and precise information on their lytic function - the key parameter underlying clinical efficacy. This study reveals a "structure-function" relationship for lytic phages and establishes DLS as a method to optimize the storage, handling, and clinical use of phages.

biophysics↗

bioRxiv2023

Bacteriophage populations mirror those of bacterial pathogens at sites of infection

Bacteriophages, viruses that parasitize bacteria, are known to be abundant at sites of bacterial colonization but the relationship between phages and bacteria at sites of infection is unclear. Bacteriophage are highly specific to their bacterial host species and so we hypothesize that phage populations would mirror those of bacterial pathogens within infected tissues. To test this, here we study publicly-available cell-free DNA generated using next generation sequencing of infected bodily fluids, including urine, joint fluid, peritoneal fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, and abscess fluid as well as uninfected control samples. These were analyzed using a computational pipeline for identifying bacteriophage sequences in cfDNA. We find that bacteriophage sequences are present in both infected and uninfected bodily fluids and represent a variety of bacteriophage morphologies and bacterial hosts. Additionally, phages from E. coli, Streptococcus, and S. aureus are overrepresented both in terms of proportion and diversity in fluids infected with these same pathogens. These data indicate that phages reflect the relative abundance of their bacterial hosts at sites of infection. Bacteriophage sequences may help inform future investigative and diagnostic approaches that utilize cell-free DNA to study the microbiome within infected tissues. ImportanceBacteriophages are an active area of investigation in microbiome research but most studies have focused on phage populations at sites of bacterial colonization. Little is known about bacteriophage ecology at sites of active infection. To address this gap in knowledge, we utilized a publicly available dataset to study bacteriophage populations in cell free DNA collected from sites of infection. We find that phages reflect the relative abundance of their bacterial hosts at sites of infection. These studies may lead to future investigative and diagnostic approaches that incorporate phages as well as bacterial cell free DNA.

microbiology↗

bioRxiv2022

A filamentous phage triggers antiviral responses in cystic fibrosis basal airway epithelial cells

Basal airway epithelial cells are a multipotent stem cell population which gives rise to several airway cell types. Basal cells are known to be critical to airway epithelium homeostasis and repair, and altered basal cell phenotypes have been reported in cystic fibrosis and idiopathic pulmonary fibrosis. However, very little is known about how basal cells respond to stimuli in the cystic fibrosis airway environment. Cystic fibrosis patients experience chronic infection with Pseudomonas aeruginosa and contain both high quantities of lipopolysaccharide (LPS) as well as the filamentous bacteriophage Pf produced by biofilm-state P. aeruginosa in the airway. In this study, we sought to investigate the transcriptional responses of human basal cells from both healthy controls and patients with cystic fibrosis to LPS and Pf phage. Basal cells from wildtype and cystic fibrosis donors were cultured in vitro and exposed to LPS and/or Pf phage, followed by single-cell sequencing on the 10x platform. We report that basal cells show strong antiviral responses and neutrophil chemokine production in response to Pf phage. We validate these findings in additional donors by qRT-PCR and show that Pf phage is internalized by basal cells. We also show that Pf decreases basal cell migration and proliferation. We demonstrate that Pf phage, a bacteria-infecting virus which does not replicate in mammalian cells, is taken up by basal cells and activates immune responses. Further studies are needed to determine the impact of this antiviral response to bacterial clearance. Author SummaryWhen we experience a lung infection or injury, the stem cells of the airway--called basal epithelial cells--are crucial for healing the affected tissue. Basal cells proliferate and migrate to close gaps in the epithelium and replace injured and dead cells. We are also learning that basal cells can directly contribute to the immune response against lung pathogens, although little is understood about how basal cells sense viruses and bacteria, and what molecules they secrete in response. In our work, we sought to investigate the role that basal cells play in the course of an infection with the common human pathogen Pseudomonas aeruginosa. We stimulated basal cells with lipopolysaccharide (LPS), a potent immunogen produced by Pseudomonas, as well as the Pseudomonas-infecting virus Pf4. We have previously shown that Pf4, which is produced in high amounts by lung-infecting Pseudomonas, affects the immune response to this bacterium. In our work here, we show that Pf4 is recognized as a virus by basal cells and changes the transcriptional response to LPS, derailing the antibacterial immune response to Pseudomonas. These findings raise the question of whether other bacteria-infecting viruses alter immune responses, and how these viruses interact with other airway cells.

cell biology↗

bioRxiv2022

The circulating phageome reflects bacterial infections

Bacteriophage, viruses that infect bacteria, are abundant in the human body but the relationship between the phageome and bacterial population dynamics is unclear. Because bacteriophage are often highly specific to bacterial host strains and species, we asked whether bacteriophage present in cell-free DNA (cfDNA) reflect bacterial infections in sepsis. To address this, we generated a workflow for identifying and interpreting bacteriophage sequences in cfDNA and a bacteriophage characteristic dictionary. In two independent cohorts of infected patients and asymptomatic controls, we demonstrate that all individuals, septic and healthy, have a circulating phageome. Moreover, infection associates with overrepresentation of pathogen-specific phage, allowing for the study of bacterial pathogens. We further show that phage can identify pathovariant Escherichia coli infections and distinguish between closely-related pathogenic bacterial species such as Staphylococcus aureus and frequent contaminants such as coagulase-negative Staphylococcus. Phage DNA may have utility in studying bacteriophage ecology in infection.

microbiology↗

bioRxiv2020

The Impact of Pf Bacteriophages on the Fitness of Pseudomonas aeruginosa; A Mathematical Modeling Approach

Pseudomonas aeruginosa (Pa) is a major bacterial pathogen responsible for chronic lung infections in cystic fibrosis patients. Recent work by ourselves and others has implicated Pf bacteriophages, non-lytic filamentous viruses produced by Pa, in the chronicity and severity of Pa infections. Pf phages act as structural elements in Pa biofilms and sequester aerosolized antibiotics, thereby contributing to antibiotic tolerance. Consistent with a selective advantage in this setting, the prevalence of Pf+ bacteria increases over time in these patients. However, the production of Pf phages comes at a metabolic cost to bacteria, such that Pf+ strains grow more slowly than Pf- strains in vitro. Here, we use a mathematical model to investigate how these competing pressures might influence the relative abundance of Pf+ versus Pf- strains in different settings. Our model predicts that Pf+ strains of Pa can only outcompete Pf- strains if the benefits of phage production falls solely onto Pf+ strains and not onto the overall bacterial community in the lung. Further, phage production only leads to a net positive gain in fitness at antibiotic concentrations slightly above the minimum inhibitory concentration (i.e., concentrations for which the benefits of antibiotic sequestration outweigh the metabolic cost of phage production), but which are not lethal for Pf+ strains. As a result, our model predicts that frequent administration of intermediate doses of antibiotics with low decay rates favors Pf+ over Pf- strains. These models inform our understanding of the ecology of Pf phages and suggest potential treatment strategies for Pf+ Pa infections. ImportanceFilamentous phages are a frontier in bacterial pathogenesis, but the impact of these phages on bacterial fitness is unclear. In particular, Pf phages produced by Pa promote antibiotic tolerance but are metabolically expensive to produce, suggesting that competing pressures may influence the prevalence of Pf+ versus Pf- strains of Pa in different settings. Our results identify conditions likely to favor Pf+ strains and thus antibiotic tolerance. This study contributes to a better understanding of the unique ecology of filamentous phages and may facilitate improved treatment strategies for combating antibiotic tolerance.

microbiology↗

bioRxiv2020

Hyaluronan Synthesis Inhibition Impairs Antigen Presentation and Delays Transplantation Rejection

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

immunology↗