Microbiology & Cell Biology
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Item Polyamines and linear DNA mediate bacterial threat assessment of bacteriophage infection(Proceedings of the National Academy of Sciences, 2023-02) de Mattos, Camilla D.; Faith, Dominick R.; Nemudryi, Artem; Schmidt, Amelia K.; Bublitz, DeAnna C.; Hammond, Lauren R.; Kinnersley, Margie; Schwartzkopf, Caleb M.; Robinson, Autumn J.; Joyce, Alex; Michaels, Lia A.; Brzozowski, Robert S.; Coluccio, Alison; Xing, Denghui David; Uchiyama, Jumpei; Jennings, Laura K.; Eswara, Prahathees; Wiedenheft, Blake; Secor, Patrick R.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.Item The Depletion Mechanism Actuates Bacterial Aggregation by Exopolysaccharides and Determines Species Distribution & Composition in Bacterial Aggregates(Frontiers Media SA, 2022-06) Secor, Patrick R.; Michaels, Lia A.; Bublitz, DeAnna C.; Jennings, Laura K.; Singh, Pradeep K.Bacteria in natural environments and infections are often found in cell aggregates suspended in polymer-rich solutions, and aggregation can promote bacterial survival and stress resistance. One aggregation mechanism, called depletion aggregation, is driven by physical forces between bacteria and high concentrations of polymers in the environment rather than bacterial activity per se. As such, bacteria aggregated by the depletion mechanism will disperse when polymer concentrations fall unless other adhesion mechanisms supervene. Here we investigated whether the depletion mechanism can actuate the aggregating effects of Pseudomonas aeruginosa exopolysaccharides for suspended (i.e. not surface attached) bacteria, and how depletion affects bacterial inter-species interactions. We found that cells overexpressing the exopolysaccharides Pel and Psl remained aggregated after short periods of depletion aggregation whereas wild-type and mucoid P. aeruginosa did not. In co-culture, depletion aggregation had contrasting effects on P. aeruginosa’s interactions with coccus- and rod-shaped bacteria. Depletion caused S. aureus (cocci) and P. aeruginosa (rods) to segregate from each other and S. aureus to resist secreted P. aeruginosa antimicrobial factors resulting in species co-existence. In contrast, depletion aggregation caused P. aeruginosa and Burkholderia sp. (both rods) to intermix, enhancing type VI secretion inhibition of Burkholderia by P. aeruginosa, leading to P. aeruginosa dominance. These results show that in addition to being a primary cause of aggregation in polymer-rich suspensions, physical forces inherent to the depletion mechanism can promote aggregation by some self-produced exopolysaccharides and determine species distribution and composition of bacterial communities.Item A Filamentous Bacteriophage Protein Inhibits Type IV Pili To Prevent Superinfection of Pseudomonas aeruginosa(American Society for Microbiology, 2022-02) Schmidt, Amelia K.; Fitzpatrick, Alexa D.; Schwartzkopf, Caleb M.; Faith, Dominick R.; Jennings, Laura K.; Coluccio, Alison; Hunt, Devin J.; Michaels, Lia A.; Hargil, Aviv; Chen, Qingquan; Bollyky, Paul L.; Dorward, David W.; Wachter, Jenny; Rosa, Patricia A.; Maxwell, Karen L.; Secor, Patrick R.Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in a variety of settings. Many P. aeruginosa isolates are infected by filamentous Pf bacteriophage integrated into the bacterial chromosome as a prophage. Pf virions can be produced without lysing P. aeruginosa. However, cell lysis can occur during superinfection, which occurs when Pf virions successfully infect a host lysogenized by a Pf prophage. Temperate phages typically encode superinfection exclusion mechanisms to prevent host lysis by virions of the same or similar species. In this study, we sought to elucidate the superinfection exclusion mechanism of Pf phage. Initially, we observed that P. aeruginosa that survive Pf superinfection are transiently resistant to Pf-induced plaquing and are deficient in twitching motility, which is mediated by type IV pili (T4P). Pf utilize T4P as a cell surface receptor, suggesting that T4P are suppressed in bacteria that survive superinfection. We tested the hypothesis that a Pf-encoded protein suppresses T4P to mediate superinfection exclusion by expressing Pf proteins in P. aeruginosa and measuring plaquing and twitching motility. We found that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), promoted resistance to Pf infection and suppressed twitching motility by binding the T4P protein PilC. Because T4P play key roles in biofilm formation and virulence, the ability of Pf phage to modulate T4P via PfsE has implications in the ability of P. aeruginosa to persist at sites of infection.Item Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Immunity, and Chronic Infections(Frontiers Media SA, 2020-02) Secor, Patrick R.; Burgener, Elizabeth B.; Kinnersley, M.; Jennings, Laura K.; Roman-Cruz, Valery; Popescu, Medeea; Van Belleghem, Jonas D.; Haddock, Naomi; Copeland, Conner; Michaels, Lia A.; de Vries, Christiaan R.; Chen, Qingquan; Pourtois, Julie; Wheeler, Travis J.; Milla, Carlos E.; Bollyky, Paul L.Pf bacteriophage are temperate phages that infect the bacterium Pseudomonas aeruginosa, a major cause of chronic lung infections in cystic fibrosis (CF) and other settings. Pf and other temperate phages have evolved complex, mutualistic relationships with their bacterial hosts that impact both bacterial phenotypes and chronic infection. We and others have reported that Pf phages are a virulence factor that promote the pathogenesis of P. aeruginosa infections in animal models and are associated with worse skin and lung infections in humans. Here we review the biology of Pf phage and what is known about its contributions to pathogenesis and clinical disease. First, we review the structure, genetics, and epidemiology of Pf phage. Next, we address the diverse and surprising ways that Pf phages contribute to P. aeruginosa phenotypes including effects on biofilm formation, antibiotic resistance, and motility. Then, we cover data indicating that Pf phages suppress mammalian immunity at sites of bacterial infection. Finally, we discuss recent literature implicating Pf in chronic P. aeruginosa infections in CF and other settings. Together, these reports suggest that Pf bacteriophage have direct effects on P. aeruginosa infections and that temperate phages are an exciting frontier in microbiology, immunology, and human health.Item Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis(American Association for the Advancement of Science, 2019-04) Burgener, Elizabeth B.; Sweere, Johanna M.; Bach, Michelle S.; Secor, Patrick R.; Haddock, Naomi; Jennings, Laura K.; Marvig, Rasmus L.; Krogh Johansen, Helle; Rossi, Elio; Cao, Xiou; Tian, Lu; Nedelec, Laurence; Molin, Søren; Bollyky, Paul L.; Milla, Carlos E.Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% (n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% (n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF.Item Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive InfectionIn Vivo(American Society for Microbiology, 2017-01) Secor, Patrick R.; Michaels, Lia A.; Smigiel, Kate S.; Rohani, Maryam G.; Jennings, Laura K.; Hisert, Katherine B.; Arrigoni, Allison; Braun, Kathleen R.; Birkland, Timothy P.; Lai, Ying; Hallstrand, Teal S.; Bollyky, Paul L.; Singh, Pradeep K.; Parks, William C.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.Item Pseudomonas aeruginosa aggregates in cystic fibrosis sputum produce exopolysaccharides that likely impede current therapies(Elsevier BV, 2021-02) Jennings, Laura K.; Dreifus, Julia E.; Reichhardt, Courtney; Storek, Kelly M.; Secor, Patrick R.; Wozniak, Daniel J.; Hisert, Katherine B.; Parsek, Matthew R.In cystic fibrosis (CF) airways, Pseudomonas aeruginosa forms cellular aggregates called biofilms that are thought to contribute to chronic infection. To form aggregates, P. aeruginosa can use different mechanisms, each with its own pathogenic implications. However, how they form in vivo is controversial and unclear. One mechanism involves a bacterially produced extracellular matrix that holds the aggregates together. Pel and Psl exopolysaccharides are structural and protective components of this matrix. We develop an immunohistochemical method to visualize Pel and Psl in CF sputum. We demonstrate that both exopolysaccharides are expressed in the CF airways and that the morphology of aggregates is consistent with an exopolysaccharide-dependent aggregation mechanism. We reason that the cationic exopolysaccharide Pel may interact with some of the abundant anionic host polymers in sputum. We show that Pel binds extracellular DNA (eDNA) and that this interaction likely impacts current therapies by increasing antimicrobial tolerance and protecting eDNA from digestion.Item Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix(Proceedings of the National Academy of Sciences, 2015-08) Jennings, Laura K.; Storek, Kelly M.; Ledvina, Hannah E.; Coulon, Charlène; Marmont, Lindsey S.; Sadovskaya, Irina; Secor, Patrick R.; Tseng, Boo Shan; Scian, Michele; Filloux, Alain; Wozniak, Daniel J.; Howell, P. Lynne; Parsek, Matthew R.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.Item Entropically driven aggregation of bacteria by host polymers promotes antibiotic tolerance in Pseudomonas aeruginosa(Proceedings of the National Academy of Sciences, 2018-10) Secor, Patrick R.; Michaels, Lia A.; Ratjen, Anina; Jennings, Laura K.; Singh, Pradeep K.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.