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The oxygenation of unsaturated fatty acids by dioxygenases occurs in all kingdoms of life and produces physiologically important lipids called oxylipins. The biological roles of oxylipins have been extensively studied in animals, plants, algae and fungi, but remain largely unidentified in prokaryotes. The bacterium Pseudomonas aeruginosa displays a diol synthase activity that transforms several monounsaturated fatty acids into mono- and di-hydroxylated derivatives. Here we show that oxylipins derived from this activity inhibit flagellum-driven motility and upregulate type IV pilus-dependent twitching motility of P. aeruginosa. Consequently, these oxylipins promote bacterial organization in microcolonies, increasing the ability of P. aeruginosa to form biofilms in vitro and in vivo (in Drosophila flies). We also demonstrate that oxylipins produced by P. aeruginosa promote virulence in Drosophila flies and lettuce. Our study thus uncovers a role for prokaryotic oxylipins in the physiology and pathogenicity of bacteria. Oxygenated fatty acids known as oxylipins play important roles in mammals, plants and fungi. Here, the authors show that oxylipins, produced by the pathogenic bacterium Pseudomonas aeruginosa , promote biofilm formation and virulence.

Pseudomonas aeruginosa is a leading cause of life-threatening infections in burn patients, yet the molecular cues driving its hypervirulence remain poorly understood. Here, we identify the Oxylipin Dependent Quorum Sensing (ODS) system as a key regulator of P. aeruginosa pathogenicity in the burn wound environment. Using a murine burn model, we show that thermal injury significantly increases free oleic acid levels in skin, which P. aeruginosa converts into oxylipin autoinducers (10-HOME and 7,10-DiHOME) via OdsA and OdsB. These molecules activate the ODS regulon, promoting bacterial invasion of burned tissue and dissemination to internal organs. ODS-deficient mutants exhibited markedly reduced skin colonization, impaired translocation across endothelial barriers, and attenuated mortality compared to wild-type strains, confirming the role of ODS in hypervirulence. Importantly, immunization with recombinant OdsA or treatment with a small-molecule OdsA inhibitor significantly improved survival and reduced bacterial dissemination in burned mice. High-throughput screening identified AB012 as a potent OdsA inhibitor, which competitively binds the enzyme’s catalytic site and suppresses oxylipin synthesis, ODS gene expression, and biofilm formation without affecting bacterial growth. In vivo, AB012 reduced bacterial burden and systemic spread following burn injury. Collectively, these findings reveal that P. aeruginosa exploits host-derived oleic acid to activate ODS and enhance virulence, and they highlight OdsA as a promising target for therapeutic intervention to prevent sepsis in burn patients.

Pseudomonas aeruginosa is a major cause of burn-related infections. It is also the most likely bacterial infection to advance to sepsis and result in burn-linked death. Pf is a filamentous bacteriophage integrated in the chromosome of most clinical isolates of Pseudomonas aeruginosa . Under stress conditions, mutations occurring in the Pf genome result in the emergence of superinfective variants of Pf (SI-Pf) that are capable of circumventing phage immunity; therefore, SI-Pf can even infect Pf-lysogenized P. aeruginosa . Here, we identified specific mutations located between the repressor and the excisionase genes of Pf4 phage in the P. aeruginosa PAO1 strain that resulted in the emergence of SI-Pf. Based on these findings, we genetically engineered an SI-Pf (eSI-Pf) and tested it as a phage therapy tool for the treatment of life-threatening burn wound infections caused by PAO1. In validation experiments, eSI-Pf was able to infect PAO1 grown in a lawn as well as biofilms formed in vitro on polystyrene. eSI-Pf also infected PAO1 present in burned skin wounds on mice but was not capable of maintaining a sustained reduction in bacterial burden beyond 24 h. Despite not lowering bacterial burden in burned skin tissue, eSI-Pf treatment completely abolished the capability of P. aeruginosa to disseminate from the burn site to internal organs. Over the course of 10 days, this resulted in bacterial clearance and survival of all treated mice. We subsequently determined that eSI-Pf induced a small-colony variant of P. aeruginosa that was unable to disseminate systemically. This attenuated phenotype was due to profound changes in virulence determinant production and altered physiology. Our results suggest that eSI-Pf has potential as a phage therapy against highly recalcitrant antimicrobial-resistant P. aeruginosa infections of burn wounds. IMPORTANCE Pseudomonas aeruginosa is a major cause of burn-related infections. It is also the most likely bacterial infection to advance to sepsis and result in burn-linked death. Frequently, P. aeruginosa strains isolated from burn patients display a multidrug-resistant phenotype necessitating the development of new therapeutic strategies and prophylactic treatments. In this context, phage therapy using lytic phages has demonstrated exciting potential in the control P. aeruginosa infection. However, lytic phages can present a set of drawbacks during phage therapy, including the induction of bacterial resistance and limited bacteria-phage interactions in vivo . Here, we propose an alternative approach to interfere with P. aeruginosa pathogenesis in a burn infection model, i.e., by using an engineered superinfective filamentous phage. Our study demonstrates that treatment with the engineered Pf phage can prevent sepsis and death in a burn mouse model.

Streptococcus pneumoniae ( Spn ) is the leading cause of invasive disease. Importantly, only a subset of the 100 capsule types carried by Spn cause the majority of serious infections, suggesting that the biochemical properties of capsular polysaccharide are directly tied to virulence. The polysaccharide capsule that surrounds Streptococcus pneumoniae ( Spn ) is one of its most important virulence determinants, serving to protect against phagocytosis. To date, 100 biochemical and antigenically distinct capsule types, i.e., serotypes, of Spn have been identified. Yet how capsule influences pneumococcal translocation across vascular endothelial cells (VEC), a key step in the progression of invasive disease, was unknown. Here, we show that despite capsule being inhibitory of Spn uptake by VEC, capsule enhances the escape rate of internalized pneumococci and thereby promotes translocation. Upon investigation, we determined that capsule protected Spn against intracellular killing by VEC and H 2 O 2 -mediated killing in vitro . Using a nitroblue tetrazolium reduction assay and nuclear magnetic resonance (NMR) analyses, purified capsule was confirmed as having antioxidant properties which varied according to serotype. Using an 11-member panel of isogenic capsule-switch mutants, we determined that serotype affected levels of Spn resistance to H 2 O 2 -mediated killing in vitro , with killing resistance correlated positively with survival duration within VEC, rate of transcytosis to the basolateral surface, and human attack rates. Experiments with mice supported our in vitro findings, with Spn producing oxidative-stress-resistant type 4 capsule being more organ-invasive than that producing oxidative-stress-sensitive type 2 capsule during bacteremia. Capsule-mediated protection against intracellular killing was also observed for Streptococcus pyogenes and Staphylococcus aureus . We conclude that capsular polysaccharide plays an important role within VEC, serving as an intracellular antioxidant, and that serotype-dependent differences in antioxidant capabilities impact the efficiency of VEC translocation and a serotype’s potential for invasive disease. IMPORTANCE Streptococcus pneumoniae ( Spn ) is the leading cause of invasive disease. Importantly, only a subset of the 100 capsule types carried by Spn cause the majority of serious infections, suggesting that the biochemical properties of capsular polysaccharide are directly tied to virulence. Here, we describe a new function for Spn ’s capsule—conferring resistance to oxidative stress. Moreover, we demonstrate that capsule promotes intracellular survival of pneumococci within vascular endothelial cells and thereby enhances bacterial translocation across the vasculature and into organs. Using isogenic capsule-switch mutants, we show that different capsule types, i.e., serotypes, vary in their resistance to oxidative stress-mediated killing and that resistance is positively correlated with intracellular survival in an in vitro model, organ invasion during bacteremia in vivo , and epidemiologically established pneumococcal attack rates in humans. Our findings define a new role of capsule and provide an explanation for why certain serotypes of Spn more frequently cause invasive pneumococcal disease.

Streptococcus pneumoniae ( Spn ) is the leading cause of community-acquired pneumonia. PspA and PspC are among its most important virulence factors, and these surface proteins carry the proline-rich domain (PRD), whose role was unknown until now. Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC, also called CbpA) are major virulence factors of Streptococcus pneumoniae ( Spn ). These surface-exposed choline-binding proteins (CBPs) function independently to inhibit opsonization, neutralize antimicrobial factors, or serve as adhesins. PspA and PspC both carry a proline-rich domain (PRD) whose role, other than serving as a flexible connector between the N-terminal and C-terminal domains, was up to this point unknown. Herein, we demonstrate that PspA binds to lactate dehydrogenase (LDH) released from dying host cells during infection. Using recombinant versions of PspA and isogenic mutants lacking PspA or specific domains of PspA, this property was mapped to a conserved 22-amino-acid nonproline block (NPB) found within the PRD of most PspAs and PspCs. The NPB of PspA had specific affinity for LDH-A, which converts pyruvate to lactate. In a mouse model of pneumonia, preincubation of Spn carrying NPB-bearing PspA with LDH-A resulted in increased bacterial titers in the lungs. In contrast, incubation of Spn carrying a version of PspA lacking the NPB with LDH-A or incubation of wild-type Spn with enzymatically inactive LDH-A did not enhance virulence. Preincubation of NPB-bearing Spn with lactate alone enhanced virulence in a pneumonia model, indicating exogenous lactate production by Spn -bound LDH-A had an important role in pneumococcal pathogenesis. Our observations show that lung LDH, released during the infection, is an important binding target for Spn via PspA/PspC and that pneumococci utilize LDH-A derived lactate for their benefit in vivo . IMPORTANCE Streptococcus pneumoniae ( Spn ) is the leading cause of community-acquired pneumonia. PspA and PspC are among its most important virulence factors, and these surface proteins carry the proline-rich domain (PRD), whose role was unknown until now. Herein, we show that a conserved 22-amino-acid nonproline block (NPB) found within most versions of the PRD binds to host-derived lactate dehydrogenase A (LDH-A), a metabolic enzyme which converts pyruvate to lactate. PspA-mediated binding of LDH-A increased Spn titers in the lungs and this required LDH-A enzymatic activity. Enhanced virulence was also observed when Spn was preincubated with lactate, suggesting LDH-A-derived lactate is a vital food source. Our findings define a role for the NPB of the PRD and show that Spn co-opts host enzymes for its benefit. They advance our understanding of pneumococcal pathogenesis and have key implications on the susceptibility of individuals with preexisting airway damage that results in LDH-A release.

Streptococcus pneumoniae (Spn) colonizes the nasopharynx and can cause pneumonia. From the lungs it spreads to the bloodstream and causes organ damage. We characterized the in vivo Spn and mouse transcriptomes within the nasopharynx, lungs, blood, heart, and kidneys using three Spn strains. We identified Spn genes highly expressed at all anatomical sites and in an organspecific manner; highly expressed genes were shown to have vital roles with knockout mutants. The in vivo bacterial transcriptome during colonization/disease was distinct from previously reported in vitro transcriptomes. Distinct Spn and host gene-expression profiles were observed during colonization and disease states, revealing specific genes/operons whereby Spn adapts to and influences host sites in vivo. We identified and experimentally verified host-defense pathways induced by Spn during invasive disease, including proinflammatory responses and the interferon response. These results shed light on the pathogenesis of Spn and identify therapeutic targets.

Objectives/Goals: The goal of this study is to explore the lysogenized bacteriophages of clinical isolates of Pseudomonas aeruginosa (Pa) for their capacity to affect intraspecies competition. This study also aims to explore the capacity of using clinical isolates as a source of new candidates for phage therapy. Methods/Study Population: We compiled a collection of 207 Pa isolates and screened them for the presence of lysogenic phages using polymerase chain reaction (PCR). A subset of 96 phage containing clinical isolates was tested for intra-strain infectivity. Using a representative subset of isolates producing infective and non-infective phage, we evaluated competitive dynamics in liquid co-cultures, bacterial biofilms and in a murine chronic wound model. Results/Anticipated Results: Among all isolates, 74.3% contained detectable phages in their genome, and 63.7% contained detectable phage in their supernatants. Among those tested for intra-strain infectivity, 82.2% of strains demonstrated inhibition of growth on lawns of at least one other strain, while 94.8% of these strains were susceptible to at least one other supernatant. In in vitro co-culture competition experiments, strains producing infective phages outcompeted competitors more effectively than strains producing non-infective phage. Infective phage producing strains were more resistant to biofilm invasion than non-infective counterparts. In vivo competition experiments showed that PAO1 was significantly reduced when co-infecting with an infective phage-producing strain compared to a non-infective one. Discussion/Significance of Impact: Collectively, these data demonstrate that clinical isolates of Pa frequently encode active bacteriophages that can mediate intraspecies competition and confer a competitive advantage during infection. Future work will center on combining these phages as therapeutic cocktails targeting acute Pa infections.

Transition from pneumococcal colonization to invasive disease is not well understood. Studies have shown that such a transition can occur as a result of influenza A virus (IAV) coinfection. We investigated the pneumococcal (serotype 19F, strain EF3030, and isogenic mutants) and airway epithelial transcriptomes with and without IAV (A/California/07 2009 pH1N1) infection. Pneumococcus and influenza coinfection leads to enhanced bacterial transcriptional programs related to growth, nutrient availability, and energy biosynthesis, suggesting conversion to an invasive phenotype. Influenza-induced secondary EF3030 infection influences human bronchial epithelial cell (HBEC) microtubules and extracellular matrix. Notably, sialic acid (NanR) utilization is a central regulon in EF3030 mono/coinfection with pH1N1 on HBEC. Downregulation of sialic acid utilization during influenza coinfection improved Spn pathogenicity ex vivo but did not alter disease in vivo, suggesting other metabolic cues are also important. This study uncovers critical metabolic features of the EF3030-pH1N1 interface to inform how Spn proliferates during IAV coinfection.

Biofilm development is a key component of the ability of Pseudomonas aeruginosa to evade host immune defenses and resist multiple drugs. Induction of the filamentous phage Pf, which usually is lysogenized in clinical and environmental isolates of P. aeruginosa , plays an important role in biofilm assembly, maturation, and dispersal. Despite the clinical relevance of Pf, the molecular biology of this phage is largely unknown. In this study, we found that rolling circle replication of Pf depends on UvrD, a DNA helicase normally involved in DNA repair. We also identified the initiator protein of Pf and found that it shares structural similarity with that of Vibrio cholerae phages CTXφ and VGJφ, which also use UvrD for replication. Our results reveal that, in addition to DNA repair, UvrD plays an essential role in rolling circle replication of filamentous phages among diverse bacteria genera, adding a new, previously unrecognized function of this accessory helicase. Pf is a lysogenic filamentous phage that promotes biofilm development in Pseudomonas aeruginosa . Pf replicates by a rolling circle replication system which depends on a phage-encoded initiator protein and host factors usually involved in chromosome replication. Rep, an accessory replicative DNA helicase, is crucial for replication of filamentous phages in Escherichia coli . In contrast, here we show that, instead of depending on Rep, Pf replication depends on UvrD, an accessory helicase implicated in DNA repair. In this study, we also identified the initiator protein of Pf and found that it shares similarities with that of Vibrio phages CTXφ and VGJφ, which also depend on UvrD for replication. A structural comparative analysis of the initiator proteins of most known filamentous phages described thus far suggested that UvrD, known as a nonreplicative helicase, is involved in rolling circle replication of filamentous phages in diverse bacteria genera. This report consolidates knowledge on the new role of UvrD in filamentous phage replication, a function previously thought to be exclusive of Rep helicase. IMPORTANCE Biofilm development is a key component of the ability of Pseudomonas aeruginosa to evade host immune defenses and resist multiple drugs. Induction of the filamentous phage Pf, which usually is lysogenized in clinical and environmental isolates of P. aeruginosa , plays an important role in biofilm assembly, maturation, and dispersal. Despite the clinical relevance of Pf, the molecular biology of this phage is largely unknown. In this study, we found that rolling circle replication of Pf depends on UvrD, a DNA helicase normally involved in DNA repair. We also identified the initiator protein of Pf and found that it shares structural similarity with that of Vibrio cholerae phages CTXφ and VGJφ, which also use UvrD for replication. Our results reveal that, in addition to DNA repair, UvrD plays an essential role in rolling circle replication of filamentous phages among diverse bacteria genera, adding a new, previously unrecognized function of this accessory helicase.

Oxygenated unsaturated fatty acids, known as oxylipins, are signaling molecules commonly used for cell-to-cell communication in eukaryotes. However, a role for oxylipins in mediating communication in prokaryotes has not previously been described. Bacteria mainly communicate via quorum sensing, which involves the production and detection of diverse small molecules termed autoinducers. Here we show that oleic acid-derived oxylipins produced by Pseudomonas aeruginosa function as autoinducers of a novel quorum sensing system. We found that this system controls the cell density-dependent expression of a gene subset independently of the quorum sensing systems thus far described in this bacterium. We identified a LysR-type transcriptional regulator as the primary receptor of the oxylipin signal. The discovery of this oxylipin-dependent quorum sensing system reveals that prokaryote-derived oxylipins also mediate cell-to-cell communication in bacteria. Eriel Martínez et al. report that the bacterial pathogen Pseudomonas aeruginosa can convert oleic acids into oxylipins for use in cell-cell communication. This quorum sensing system is regulated by the bacterial protein called oxylipin-dependent diol synthase regulator OdsR.