Glycerol metabolism impacts biofilm phenotypes and virulence in Pseudomonas aeruginosa via the Entner-Doudoroff pathway.

Pseudomonas aeruginosa is a ubiquitous bacterium and a notorious opportunistic pathogen that forms biofilm structures in response to many environmental cues. Biofilm formation includes attachment to surfaces and the production of the exopolysaccharide Pel, which is present in both the PAO1 and PA14 laboratory strains of P. aeruginosa. Biofilms help protect bacterial cells from host defenses and antibiotics and abet infection. The carbon source used by the cells also influences biofilm, but these effects have not been deeply studied. We show here that glycerol, which can be liberated from host surfactants during infection, encourages surface attachment and magnifies colony morphology differences. We find that glycerol kinase is important but not essential for glycerol utilization and relatively unimportant for biofilm behaviors. Among downstream enzymes predicted to take part in glycerol utilization, Edd stood out as being important for glycerol utilization and for enhanced biofilm phenotypes in the presence of glycerol. Thus, gluconeogenesis and catabolism of anabolically produced glucose appear to impact not only the utilization of glycerol but also glycerol-stimulated biofilm phenotypes. Finally, waxworm moth larvae and nematode infection models reveal that interruption of the Entner-Doudoroff pathway, but not abrogation of glycerol phosphorylation, unexpectedly increases P. aeruginosa lethality in both acute and chronic infections, even while stimulating a stronger immune response by Caenorhabditis elegans.IMPORTANCEPseudomonas aeruginosa, the ubiquitous environmental bacterium and human pathogen, forms multicellular communities known as biofilms in response to various stimuli. We find that glycerol, a common carbon source that bacteria can use for energy and biosynthesis, encourages biofilm behaviors such as surface attachment and colony wrinkling by P. aeruginosa. Glycerol can be derived from surfactants that are present in the human lungs, a common infection site. Glycerol-stimulated biofilm phenotypes do not depend on phosphorylation of glycerol but are surprisingly impacted by a glucose breakdown pathway, suggesting that it is glycerol utilization, and not its mere presence or cellular import, that stimulates biofilm phenotypes. Moreover, the same mutations that block glycerol-stimulated biofilm phenotypes also impact P. aeruginosa virulence in both acute and chronic animal models. Notably, a glucose-breakdown mutant (Δedd) counteracts biofilm phenotypes but shows enhanced virulence and stimulates a stronger immune response in Caenorhabditis elegans.

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.