A putative lipase affects Pseudomonas aeruginosa biofilm matrix production.

Pseudomonas aeruginosa is an opportunistic pathogen that is widely known for infecting patients with underlying conditions. This species often survives antibiotic therapy by forming biofilms, in which the cells produce a protective extracellular matrix. P. aeruginosa also produces virulence factors that enhance its ability to cause disease. One signaling pathway that influences virulence is the nitrogen-related phosphotransferase system (Nitro-PTS), which consists of an initial phosphotransferase, PtsP, a phosphocarrier, PtsO, and a terminal phosphate receptor, PtsN. The physiological role of the Nitro-PTS in P. aeruginosa is poorly understood. However, PtsN, when deprived of its upstream phosphotransfer proteins, has an antagonistic effect on biofilm formation. We thus conducted a transposon mutagenesis screen in an unphosphorylated-PtsN (i.e., ∆ptsP) background to identify downstream proteins with unacknowledged roles in PtsN-mediated biofilm suppression. We found an unstudied gene, PA14_04030, whose disruption restored biofilm production. This gene encodes a predicted phospholipase with signature alpha/beta hydrolase folds and a lipase signature motif with an active-site Ser residue. Hence, we renamed the gene bipL, for biofilm-impacting phospholipase. Deletion of bipL in a ∆ptsP background increased biofilm formation, supporting the idea that BipL is responsible for reducing biofilm formation in strains with unphosphorylated PtsN. Moreover, substituting the putative catalytic Ser for Ala phenocopied bipL deletion, indicating that this residue is important for the biofilm-suppressive activity of BipL in vivo. As our preliminary data suggest that BipL is a lipase, we performed lipidomics to detect changes in the lipid profile due to bipL deletion and found changes in some lipid species. IMPORTANCE Biofilm formation by bacteria occurs when cells secrete an extracellular matrix that holds them together and shields them from environmental insults. Biofilms of bacterial opportunistic human pathogens such as Pseudomonas aeruginosa pose a substantial challenge to clinical antimicrobial therapy. Hence, a more complete knowledge about the bacterial factors that influence and regulate production of the biofilm matrix is one key to formulate more effective therapeutic strategies. In this study, we screen for factors that are important for reducing biofilm matrix production in certain genetic backgrounds. We unexpectedly found a gene encoding a putative lipase enzyme and showed that its predicted catalytic site is important for its ability to reduce biofilm formation. Our findings suggest that lipase enzymes have previously uncharacterized functions in biofilm matrix regulation.

PtsN in Pseudomonas aeruginosa Is Phosphorylated by Redundant Upstream Proteins and Impacts Virulence-Related Genes.

The bacterial nitrogen-related phosphotransfer (PTSNtr; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and phosphorylation. The Nitro-PTS comprises an enzyme I (EI), PtsP; an intermediate phosphate carrier, PtsO; and a terminal acceptor, PtsN, which is thought to exert regulatory effects that depend on its phosphostate. For instance, biofilm formation by Pseudomonas aeruginosa can be impacted by the Nitro-PTS, as deletion of either ptsP or ptsO suppresses Pel exopolysaccharide production and additional deletion of ptsN elevates Pel production. However, the phosphorylation state of PtsN in the presence and absence of its upstream phosphotransferases has not been directly assessed, and other targets of PtsN have not been well defined in P. aeruginosa. We show that PtsN phosphorylation via PtsP requires the GAF domain of PtsP and that PtsN is phosphorylated on histidine 68, as in Pseudomonas putida. We also find that FruB, the fructose EI, can substitute for PtsP in PtsN phosphorylation but only in the absence of PtsO, implicating PtsO as a specificity factor. Unphosphorylatable PtsN had a minimal effect on biofilm formation, suggesting that it is necessary but not sufficient for the reduction of Pel in a ptsP deletion. Finally, we use transcriptomics to show that the phosphostate and the presence of PtsN do not appear to alter the transcription of biofilm-related genes but do influence genes involved in type III secretion, potassium transport, and pyoverdine biosynthesis. Thus, the Nitro-PTS influences several P. aeruginosa behaviors, including the production of its signature virulence factors. IMPORTANCE The PtsN protein impacts the physiology of a number of bacterial species, and its control over downstream targets can be altered by its phosphorylation state. Neither its upstream phosphotransferases nor its downstream targets are well understood in Pseudomonas aeruginosa. Here, we examine PtsN phosphorylation and find that the immediate upstream phosphotransferase acts as a gatekeeper, allowing phosphorylation by only one of two potential upstream proteins. We use transcriptomics to discover that PtsN regulates the expression of gene families that are implicated in virulence. One emerging pattern is a repression hierarchy by different forms of PtsN: its phosphorylated state is more repressive than its unphosphorylated state, but the expression of its targets is even higher in its complete absence.

An acute animal model of aortic stenosis: initial attempts at leaflet modification.

BACKGROUND AND AIM OF THE STUDY: Percutaneous valve insertion is an emerging treatment for aortic stenosis (AS). To date, no large animal model exists that replicates human calcific AS; moreover, the absence of any valve pathology in currently available animal models prevents their use in any realistic assessment of percutaneous aortic valve therapy. Hence, the aim of the present study was to create an acute large animal model in which human calcific AS could be simulated. METHODS: Ten domestic swine underwent open-heart surgery utilizing cardiopulmonary bypass (CPB) and cardioplegic arrest. The aortic valve annulus and leaflets were injected with cyanoacrylate, after which epicardial echocardiography was used to assess the creation of AS. At the time of animal sacrifice, the hearts were harvested for gross and histopathological examination. RESULTS: The leaflet and annular injections were performed successfully in all animals. Subsequently, seven animals were weaned from CPB and underwent post procedural echocardiographic evaluations, whereby the treated valves were harvested for gross and histological examination. CONCLUSION: Cyanoacrylate can be injected into the porcine aortic valve and annulus to create a model that resembles human calcific AS in the acute setting. Additional long-term follow up studies must be conducted, however, before this model can be utilized in the development of percutaneous valve therapy.