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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.

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.

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

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

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 bi

Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 (CoV2) are the leading causes of death due to infectious disease. Although Mtb and CoV2 both cause serious and sometimes fatal respiratory infections, the effect of Mtb infection and its associated immune response on secondary infection with CoV2 is unknown. To address this question we applied two mouse models of COVID19, using mice which were chronically infected with Mtb. In both model systems, Mtb-infected mice were resistant to the pathological consequences of secondary CoV2 infection, and CoV2 infection did not affect Mtb burdens. Single cell RNA sequencing of coinfected and monoinfected lungs demonstrated the resistance of Mtb-infected mice is associated with expansion of T and B cell subsets upon viral challenge. Collectively, these data demonstrate that Mtb infection conditions the lung environment in a manner that is not conducive to CoV2 survival.

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.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.