Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferential packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.

Polyamines and linear DNA mediate bacterial threat assessment of bacteriophage infection.

Monitoring the extracellular environment for danger signals is a critical aspect of cellular survival. However, the danger signals released by dying bacteria and the mechanisms bacteria use for threat assessment remain largely unexplored. Here, we show that lysis of Pseudomonas aeruginosa cells releases polyamines that are subsequently taken up by surviving cells via a mechanism that relies on Gac/Rsm signaling. While intracellular polyamines spike in surviving cells, the duration of this spike varies according to the infection status of the cell. In bacteriophage-infected cells, intracellular polyamines are maintained at high levels, which inhibits replication of the bacteriophage genome. Many bacteriophages package linear DNA genomes and linear DNA is sufficient to trigger intracellular polyamine accumulation, suggesting that linear DNA is sensed as a second danger signal. Collectively, these results demonstrate how polyamines released by dying cells together with linear DNA allow P. aeruginosa to make threat assessments of cellular injury.

Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa.

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages.

Tripartite interactions between filamentous Pf4 bacteriophage, Pseudomonas aeruginosa, and bacterivorous nematodes.

The opportunistic pathogen Pseudomonas aeruginosa PAO1 is infected by the filamentous bacteriophage Pf4. Pf4 virions promote biofilm formation, protect bacteria from antibiotics, and modulate animal immune responses in ways that promote infection. Furthermore, strains cured of their Pf4 infection (ΔPf4) are less virulent in animal models of infection. Consistently, we find that strain ΔPf4 is less virulent in a Caenorhabditis elegans nematode infection model. However, our data indicate that PQS quorum sensing is activated and production of the pigment pyocyanin, a potent virulence factor, is enhanced in strain ΔPf4. The reduced virulence of ΔPf4 despite high levels of pyocyanin production may be explained by our finding that C. elegans mutants unable to sense bacterial pigments through the aryl hydrocarbon receptor are more susceptible to ΔPf4 infection compared to wild-type C. elegans. Collectively, our data support a model where suppression of quorum-regulated virulence factors by Pf4 allows P. aeruginosa to evade detection by innate host immune responses.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates has differential impacts on quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Entropically driven aggregation of bacteria by host polymers promotes antibiotic tolerance in Pseudomonas aeruginosa.

Bacteria causing chronic infections are generally observed living in cell aggregates suspended in polymer-rich host secretions, and bacterial phenotypes induced by aggregated growth may be key factors in chronic infection pathogenesis. Bacterial aggregation is commonly thought of as a consequence of biofilm formation; however the mechanisms producing aggregation in vivo remain unclear. Here we show that polymers that are abundant at chronic infection sites cause bacteria to aggregate by the depletion aggregation mechanism, which does not require biofilm formation functions. Depletion aggregation is mediated by entropic forces between uncharged or like-charged polymers and particles (e.g., bacteria). Our experiments also indicate that depletion aggregation of bacteria induces marked antibiotic tolerance that was dependent on the SOS response, a stress response activated by genotoxic stress. These findings raise the possibility that targeting conditions that promote depletion aggregation or mechanisms of depletion-mediated tolerance could lead to new therapeutic approaches to combat chronic bacterial infections.

Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.

Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo.

Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

Pseudomonas phage inhibition of Candida albicans.

Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.

Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.

VIBES: a workflow for annotating and visualizing viral sequences integrated into bacterial genomes.

Bacteriophages are viruses that infect bacteria. Many bacteriophages integrate their genomes into the bacterial chromosome and become prophages. Prophages may substantially burden or benefit host bacteria fitness, acting in some cases as parasites and in others as mutualists. Some prophages have been demonstrated to increase host virulence. The increasing ease of bacterial genome sequencing provides an opportunity to deeply explore prophage prevalence and insertion sites. Here we present VIBES (Viral Integrations in Bacterial genomES), a workflow intended to automate prophage annotation in complete bacterial genome sequences. VIBES provides additional context to prophage annotations by annotating bacterial genes and viral proteins in user-provided bacterial and viral genomes. The VIBES pipeline is implemented as a Nextflow-driven workflow, providing a simple, unified interface for execution on local, cluster and cloud computing environments. For each step of the pipeline, a container including all necessary software dependencies is provided. VIBES produces results in simple tab-separated format and generates intuitive and interactive visualizations for data exploration. Despite VIBES's primary emphasis on prophage annotation, its generic alignment-based design allows it to be deployed as a general-purpose sequence similarity search manager. We demonstrate the utility of the VIBES prophage annotation workflow by searching for 178 Pf phage genomes across 1072 Pseudomonas spp. genomes.

Adjuvanted Vaccine Induces Functional Antibodies against Pseudomonas aeruginosa Filamentous Bacteriophages.

Pseudomonas aeruginosa (Pa), a WHO priority 1 pathogen, resulted in approximately 559,000 deaths globally in 2019. Pa has a multitude of host-immune evasion strategies that enhance Pa virulence. Most clinical isolates of Pa are infected by a phage called Pf that has the ability to misdirect the host-immune response and provide structural integrity to biofilms. Previous studies demonstrate that vaccination against the coat protein (CoaB) of Pf4 virions can assist in the clearance of Pa from the dorsal wound model in mice. Here, a consensus peptide was derived from CoaB and conjugated to cross-reacting material 197 (CRM197). This conjugate was adjuvanted with a novel synthetic Toll-like receptor agonist (TLR) 4 agonist, INI-2002, and used to vaccinate mice. Mice vaccinated with CoaB-CRM conjugate and INI-2002 developed high anti-CoaB peptide-specific IgG antibody titers. Direct binding of the peptide-specific antibodies to whole-phage virus particles was demonstrated by ELISA. Furthermore, a functional assay demonstrated that antibodies generated from vaccinated mice disrupted the replicative cycle of Pf phages. The use of an adjuvanted phage vaccine targeting Pa is an innovative vaccine strategy with the potential to become a new tool targeting multi-drug-resistant Pa infections in high-risk populations.

Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferentially packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of mortality in patients with cystic fibrosis (pwCF), necessitating treatment with antibiotics. Pf is a filamentous bacteriophage produced by Pa and acts as a structural element in Pa biofilms. Pf presence has been associated with antibiotic resistance and poor outcomes in pwCF, although the underlying mechanisms are unclear. We have investigated how Pf and sputum biopolymers impede antibiotic diffusion using pwCF sputum and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf in sputum reduces the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of morbidity and mortality in patients with cystic fibrosis, necessitating treatment with inhaled antibiotics. Pf phage is a filamentous bacteriophage produced by Pa that has been reported to act as a structural element in Pa biofilms. Pf presence has been associated with resistance to antibiotics and poor outcomes in cystic fibrosis, though the underlying mechanisms are unclear. Here, we have investigated how Pf phages and sputum biopolymers impede antibiotic diffusion using human sputum samples and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf phages and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf phages in sputum reduce the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf phages and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

PICI thieves: Molecular piracy and cooperation.

Phage-inducible chromosomal islands (PICIs) steal structural proteins from helper phages. In two related studies, Penadés and coworkers reveal that PICIs are not parasites but mutualists. Some PICIs mobilize defense systems that restrict niche competitors, while other PICIs encode their own capsids and steal helper phage tails without affecting their fitness.

VIBES: A Workflow for Annotating and Visualizing Viral Sequences Integrated into Bacterial Genomes.

Bacteriophages are viruses that infect bacteria. Many bacteriophages integrate their genomes into the bacterial chromosome and become prophages. Prophages may substantially burden or benefit host bacteria fitness, acting in some cases as parasites and in others as mutualists, and have been demonstrated to increase host virulence. The increasing ease of bacterial genome sequencing provides an opportunity to deeply explore prophage prevalence and insertion sites. Here we present VIBES, a workflow intended to automate prophage annotation in complete bacterial genome sequences. VIBES provides additional context to prophage annotations by annotating bacterial genes and viral proteins in user-provided bacterial and viral genomes. The VIBES pipeline is implemented as a Nextflow-driven workflow, providing a simple, unified interface for execution on local, cluster, and cloud computing environments. For each step of the pipeline, a container including all necessary software dependencies is provided. VIBES produces results in simple tab separated format and generates intuitive and interactive visualizations for data exploration. Despite VIBES' primary emphasis on prophage annotation, its generic alignment-based design allows it to be deployed as a general-purpose sequence similarity search manager. We demonstrate the utility of the VIBES prophage annotation workflow by searching for 178 Pf phage genomes across 1,072 Pseudomonas spp. genomes. VIBES software is available at https://github.com/TravisWheelerLab/VIBES.

Longitudinal map of transcriptome changes in the Lyme pathogen Borrelia burgdorferi during tick-borne transmission.

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, adapts to vastly different environments as it cycles between tick vector and vertebrate host. During a tick bloodmeal, Bb alters its gene expression to prepare for vertebrate infection; however, the full range of transcriptional changes that occur over several days inside of the tick are technically challenging to capture. We developed an experimental approach to enrich Bb cells to longitudinally define their global transcriptomic landscape inside nymphal Ixodes scapularis ticks during a transmitting bloodmeal. We identified 192 Bb genes that substantially change expression over the course of the bloodmeal from 1 to 4 days after host attachment. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 outer surface lipoproteins embedded in the unusual protein-rich coat of Bb. As these proteins may facilitate Bb interactions with the host, we utilized mass spectrometry to identify candidate tick proteins that physically associate with Bb. The Bb enrichment methodology along with the ex vivo Bb transcriptomes and candidate tick interacting proteins presented here provide a resource to facilitate investigations into key determinants of Bb priming and transmission during the tick stage of its unique transmission cycle.

Coupled induction of prophage and virulence factors during tick transmission of the Lyme disease spirochete.

The alternative sigma factor RpoS plays a central role in the critical host-adaptive response of the Lyme disease spirochete, Borrelia burgdorferi. We previously identified bbd18 as a negative regulator of RpoS but could not inactivate bbd18 in wild-type spirochetes. In the current study we employed an inducible bbd18 gene to demonstrate the essential nature of BBD18 for viability of wild-type spirochetes in vitro and at a unique point in vivo. Transcriptomic analyses of BBD18-depleted cells demonstrated global induction of RpoS-dependent genes prior to lysis, with the absolute requirement for BBD18, both in vitro and in vivo, circumvented by deletion of rpoS. The increased expression of plasmid prophage genes and the presence of phage particles in the supernatants of lysing cultures indicate that RpoS regulates phage lysis-lysogeny decisions. Through this work we identify a mechanistic link between endogenous prophages and the RpoS-dependent adaptive response of the Lyme disease spirochete.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates impacts quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous bacterial virulence phenotypes. Most work on Pf phages has focused on strain Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf strains infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, virulence factor production, and virulence. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates, but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.