Browsing by Author "Wozniak, Daniel J."

Now showing 1 - 7 of 7
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    A bacterial pigment provides cross-species protection from H2O2- and neutrophil-mediated killing
    Liu, Yiwei; McQuillen, Eleanor A.; Rana, Pranav S. J. B.; Gloag, Erin S.; Parsek, Matthew R.; Wozniak, Daniel J. (Proceedings of the National Academy of Sciences, 2024-01-03)
    Bacterial infections are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus cause chronic co-infections, which are more problematic than mono-species infections. Understanding the mechanisms of their interactions is crucial for treating co-infections. Staphyloxanthin (STX), a yellow pigment synthesized by the S. aureus crt operon, promotes S. aureus resistance to oxidative stress and neutrophil-mediated killing. We found that STX production by S. aureus, either as surface-grown macrocolonies or planktonic cultures, was elevated when exposed to the P. aeruginosa exoproduct, 2-heptyl- 4- hydroxyquinoline N-oxide (HQNO). This was observed with both mucoid and non-mucoid P. aeruginosa strains. The induction phenotype was found in a majority of P. aeruginosa and S. aureus clinical isolates examined. When subjected to hydrogen peroxide or human neutrophils, P. aeruginosa survival was significantly higher when mixed with wild-type (WT) S. aureus, compared to P. aeruginosa alone or with an S. aureus crt mutant deficient in STX production. In a murine wound model, co-infection with WT S. aureus, but not the STX-deficient mutant, enhanced P. aeruginosa burden and disease compared to mono-infection. In conclusion, we identified a role for P. aeruginosa HQNO mediating polymicrobial interactions with S. aureus by inducing STX production, which consequently promotes resistance to the innate immune effectors H2O2 and neutrophils. These results further our understanding of how different bacterial species cooperatively cause co-infections.
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    A Combination of Zinc and Arginine Disrupt the Mechanical Integrity of Dental Biofilms
    Gloag, Erin S.; Khosravi, Yalda; Masters, James G.; Wozniak, Daniel J.; Amorin Daep, Carlo; Stoodley, Paul (American Society for Microbiology, 2022-12-06)
    Mechanical cleaning remains the standard of care for maintaining oral hygiene. However, mechanical cleaning is often augmented with active therapeutics that further promote oral health. A dentifrice, consisting of the "Dual Zinc plus Arginine" (DZA) technology, was found to be effective at controlling bacteria using in vitro laboratory studies, translating to clinical efficacy to deliver plaque and gingivitis reduction benefits. Here, we used biophysical analyses and confocal laser scanning microscopy to understand how a DZA dentifrice impacted the mechanical properties of dental plaque biofilms and determine if changes to biofilm rheology enhanced the removal of dental plaque. Using both uniaxial mechanical indentation and an adapted rotating-disc rheometry assay, it was found that DZA treatment compromised biofilm mechanical integrity, resulting in the biofilm being more susceptible to removal by shear forces compared to treatment with either arginine or zinc alone. Confocal laser scanning microscopy revealed that DZA treatment reduced the amount of extracellular polymeric slime within the biofilm, likely accounting for the reduced mechanical properties. We propose a model where arginine facilitates the entry of zinc into the biofilm, resulting in additive effects of the two activities toward dental plaque biofilms. Together, our results support the use of a dentifrice containing Dual Zinc plus Arginine as part of daily oral hygiene regimens. IMPORTANCE Mechanical removal of dental plaque is augmented with therapeutic compounds to promote oral health. A dentifrice containing the ingredients zinc and arginine has shown efficacy at reducing dental plaque both in vitro and in vivo. However, how these active compounds interact together to facilitate dental plaque removal is unclear. Here, we used a combination of biophysical analyses and microscopy to demonstrate that combined treatment with zinc and arginine targets the matrix of dental plaque biofilms, which destabilized the mechanical integrity of these microbial communities, making them more susceptible to removal by shear forces.
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    Glycoside hydrolase processing of the Pel polysaccharide alters biofilm biomechanics and Pseudomonas aeruginosa virulence
    Razvi, Erum; Whitfield, Gregory B.; Reichhardt, Courtney; Dreifus, Julia E.; Willis, Alexandra R.; Gluscencova, Oxana B.; Gloag, Erin S.; Awad, Tarek S.; Rich, Jacquelyn D.; da Silva, Daniel Passos; Bond, Whitney; Le Mauff, Francois; Sheppard, Donald C.; Hatton, Benjamin D.; Stoodley, Paul; Reinke, Aaron W.; Boulianne, Gabrielle L.; Wozniak, Daniel J.; Harrison, Joe J.; Parsek, Matthew R.; Howell, P. Lynne (Nature Portfolio, 2023-02-02)
    Pel exopolysaccharide biosynthetic loci are phylogenetically widespread biofilm matrix determinants in bacteria. In Pseudomonas aeruginosa, Pel is crucial for cell-to-cell interactions and reducing susceptibility to antibiotic and mucolytic treatments. While genes encoding glycoside hydrolases have long been linked to biofilm exopolysaccharide biosynthesis, their physiological role in biofilm development is unclear. Here we demonstrate that the glycoside hydrolase activity of P. aeruginosa PelA decreases adherent biofilm biomass and is responsible for generating the low molecular weight secreted form of the Pel exopolysaccharide. We show that the generation of secreted Pel contributes to the biomechanical properties of the biofilm and decreases the virulence of P. aeruginosa in Caenorhabditis elegans and Drosophila melanogaster. Our results reveal that glycoside hydrolases found in exopolysaccharide biosynthetic systems can help shape the soft matter attributes of a biofilm and propose that secreted matrix components be referred to as matrix associated to better reflect their influence.
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    Pseudomonas aeruginosa Initiates a Rapid and Specific Transcriptional Response during Surface Attachment
    Jones, Christopher J.; Grotewold, Nikolas; Wozniak, Daniel J.; Gloag, Erin S. (American Society for Microbiology, 2022-05)
    Chronic biofilm infections by Pseudomonas aeruginosa are a major contributor to the morbidity and mortality of patients. The formation of multicellular bacterial aggregates, called biofilms, is associated with increased resistance to antimicrobials and immune clearance and the persistence of infections. Biofilm formation is dependent on bacterial cell attachment to surfaces, and therefore, attachment plays a key role in chronic infections. We hypothesized that bacteria sense various surfaces and initiate a rapid, specific response to increase adhesion and establish biofilms. RNA sequencing (RNA-Seq) analysis identified transcriptional changes of adherent cells during initial attachment, identifying the bacterial response to an abiotic surface over a 1-h period. Subsequent screens investigating the most highly regulated genes in surface attachment identified 4 genes, pfpI, phnA, leuD, and moaE, all of which have roles in both metabolism and biofilm formation. In addition, the transcriptional responses to several different medically relevant abiotic surfaces were compared after initial attachment. Surprisingly, there was a specific transcriptional response to each surface, with very few genes being regulated in response to surfaces in general. We identified a set of 20 genes that were differentially expressed across all three surfaces, many of which have metabolic functions, including molybdopterin cofactor biosynthesis and nitrogen metabolism. This study has advanced the understanding of the kinetics and specificity of bacterial transcriptional responses to surfaces and suggests that metabolic cues are important signals during the transition from a planktonic to a biofilm lifestyle. IMPORTANCE Bacterial biofilms are a significant concern in many aspects of life, including chronic infections of airways, wounds, and indwelling medical devices; biofouling of industrial surfaces relevant for food production and marine surfaces; and nosocomial infections. The effects of understanding surface adhesion could impact many areas of life. This study utilized emerging technology in a novel approach to address a key step in bacterial biofilm development. These findings have elucidated both conserved and surface-specific responses to several disease-relevant abiotic surfaces. Future work will expand on this report to identify mechanisms of biofilm initiation with the aim of identifying bacterial factors that could be targeted to prevent biofilms.
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    Interbacterial Antagonism Mediated by a Released Polysaccharide
    Liu, Yiwei; Gloag, Erin S.; Hill, Preston J.; Parsek, Matthew R.; Wozniak, Daniel J. (American Society for Microbiology, 2022-05)
    Pseudomonas aeruginosa and Staphylococcus aureus are two common pathogens causing chronic infections in the lungs of people with cystic fibrosis (CF) and in wounds, suggesting that these two organisms coexist in vivo. However, P. aeruginosa utilizes various mechanisms to antagonize S. aureus when these organisms are grown together in vitro. Here, we suggest a novel role for Psl in antagonizing S. aureus growth. Psl is an exopolysaccharide that exists in both cell-associated and cell-free forms and is important for biofilm formation in P. aeruginosa. When grown in planktonic coculture with a P. aeruginosa psl mutant, S. aureus had increased survival compared to when it was grown with wild-type P. aeruginosa. We found that cell-free Psl was critical for the killing, as purified cell-free Psl was sufficient to kill S. aureus. Transmission electron microscopy of S. aureus treated with Psl revealed disrupted cell envelopes, suggesting that Psl causes S. aureus cell lysis. This was independent of known mechanisms used by P. aeruginosa to antagonize S. aureus. Cell-free Psl could also promote S. aureus killing during growth in in vivo-like conditions. We also found that Psl production in P. aeruginosa CF clinical isolates positively correlated with the ability to kill S. aureus. This could be a result of P. aeruginosa coevolution with S. aureus in CF lungs. In conclusion, this study defines a novel role for P. aeruginosa Psl in killing S. aureus, potentially impacting the coexistence of these two opportunistic pathogens in vivo. IMPORTANCE Pseudomonas aeruginosa and Staphylococcus aureus are two important opportunistic human pathogens commonly coisolated from clinical samples. However, P. aeruginosa can utilize various mechanisms to antagonize S. aureus in vitro. Here, we investigated the interactions between these two organisms and report a novel role for P. aeruginosa exopolysaccharide Psl in killing S. aureus. We found that cell-free Psl could kill S. aureus in vitro, possibly by inducing cell lysis. This was also observed in conditions reflective of in vivo scenarios. In accord with this, Psl production in P. aeruginosa clinical isolates positively correlated with their ability to kill S. aureus. Together, our data suggest a role for Psl in affecting the coexistence of P. aeruginosa and S. aureus in vivo.
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    Rampant prophage movement among transient competitors drives rapid adaptation during infection
    Marshall, Christopher W.; Gloag, Erin S.; Lim, Christina; Wozniak, Daniel J.; Cooper, Vaughn S. (AAAS, 2021-07-16)
    Interactions between bacteria, their close competitors, and viral parasites are common in infections, but understanding of these eco-evolutionary dynamics is limited. Most examples of adaptations caused by phage lysogeny are through the acquisition of new genes. However, integrated prophages can also insert into functional genes and impart a fitness benefit by disrupting their expression, a process called active lysogeny. Here, we show that active lysogeny can fuel rapid, parallel adaptations in establishing a chronic infection. These recombination events repeatedly disrupted genes encoding global regulators, leading to increased cyclic di-GMP levels and elevated biofilm production. The implications of prophage-mediated adaptation are broad, as even transient members of microbial communities can alter the course of evolution and generate persistent phenotypes associated with poor clinical outcomes.
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    Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms
    Rana, Pranav S. J. B.; Gloag, Erin S.; Wozniak, Daniel J. (MyJove Corporation, 2022-06-08)
    Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.