Hemolytic Phospholipase C Inhibition Protects Lung Function during <i>Pseudomonas aeruginosa</i> Infection

Wargo, Matthew J.; Gross, Maegan J.; Rajamani, Sathish; Allard, Jenna L.; Lundblad, Lennart K. A.; Allen, Gilman B.; Vasil, Michael L.; Leclair, Laurie W; Hogan, Deborah A.

doi:10.1164/rccm.201103-0374oc

Cited by 75 publications

(85 citation statements)

References 69 publications

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“…Previous work has shown that PlcH degrades phospholipids in host membranes and in lung surfactant both in vivo and in vitro (8,41). Furthermore, we recently reported that the recovery of WT P. aeruginosa was 40-fold greater than that of ⌬plcHR mutant cells after oropharyngeal infections of the mouse lung with 10 6 CFU of P. aeruginosa (42). To understand the role of PlcH, if any, in colonization of and growth in the lung, we examined P. aeruginosa WT and ⌬plcHR mutant cells in an airway epithelial cell colonization assay.…”

Section: Resultsmentioning

confidence: 86%

Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

et al. 2013

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bPseudomonas aeruginosa hemolytic phospholipase C (PlcH) degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A ⌬plcHR mutant, known to be defective in virulence in animal models, was less able to colonize epithelial cell monolayers and was defective in biofilm formation on plastic when grown in lung surfactant. Microarray analyses found that strains defective in PlcH production had lower levels of Anr-regulated transcripts than the wild type. PC degradation stimulated the Anr regulon in an Anr-dependent manner under conditions where Anr activity was submaximal because of the presence of oxygen. Two PC catabolites, choline and glycine betaine (GB), were sufficient to stimulate Anr activity, and their catabolism was required for Anr activation. The addition of choline or GB to glucose-containing medium did not alter Anr protein levels, growth rates, or respiratory activity, and Anr activation could not be attributed to the osmoprotectant functions of GB. The ⌬anr mutant was defective in virulence in a mouse pneumonia model. Several lines of evidence indicate that Anr is important for the colonization of biotic and abiotic surfaces in both P. aeruginosa PAO1 and PA14 and that increases in Anr activity resulted in enhanced biofilm formation. Our data suggest that PlcH activity promotes Anr activity in oxic environments and that Anr activity contributes to virulence, even in the acute infection phase, where low oxygen tensions are not expected. This finding highlights the relationships among in vivo bacterial metabolism, the activity of the oxygen-sensitive regulator Anr, and virulence.

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Section: Resultsmentioning

confidence: 86%

Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

et al. 2013

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“…To test whether GbdR was necessary for the transcriptional changes of previously unverified transcripts, we grew cells as described for the microarray preparation, except that wild-type (WT) and ⌬gbdR mutant cells were exposed to pyruvate or 1 mM choline in MOPS pyruvate medium for 3 h. RNA was prepared as described for the microarray experiments. cDNA was generated using Superscript III (Invitrogen) and the 5=-NSNSNSNSNS-3= primer as described previously (19). Quantitative PCR (qPCR) was performed using the Luminaris color HiGreen fluorescein qPCR master mix (Thermo Scientific) with the primers described in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the GbdR Regulon in Pseudomonas aeruginosa

Hampel

LaBauve

Meadows

et al. 2013

Journal of Bacteriology

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Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundance of transcription regulators in the genome. We have been investigating P. aeruginosa's response to the host, particularly changes regulated by the hostderived quaternary amines choline and glycine betaine (GB). We previously identified GbdR as an AraC family transcription factor that directly regulates choline acquisition from host phospholipids (via binding to plcH and pchP promoters), is required for catabolism of the choline metabolite GB, and is an activator that induces transcription in response to GB or dimethylglycine. Our goal was to characterize the GbdR regulon in P. aeruginosa by using genetics and chemical biology in combination with transcriptomics and in vitro DNA-binding assays. Here we show that GbdR activation regulates transcription of 26 genes from 12 promoters, 11 of which have measureable binding to GbdR in vitro. The GbdR regulon includes the genes encoding GB, dimethylglycine, sarcosine, glycine, and serine catabolic enzymes and the BetX and CbcXWV quaternary amine transport proteins. We characterized the GbdR consensus binding site and used it to identify that the recently characterized acetylcholine esterase gene, choE (PA4921), is also regulated by GbdR. The regulon member not directly controlled by GbdR is the secreted lipase gene lipA, which was also the only regulon member repressed under GbdR-activating conditions. Determination of the GbdR regulon provides deeper understanding of how GbdR links bacterial metabolism and virulence. Additionally, identification of two uncharacterized regulon members suggests roles for these proteins in response to choline metabolites.

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“…Bacteria commonly produce secreted phospholipases C and/or D, which liberate phosphorylcholine or choline from choline phosphate-containing phospholipids and sphingolipids, respectively (32,33). These phospholipases are often considered virulence factors, when studied in opportunistic pathogens (reviewed in reference 33), that can alter bacterial survival, cause cell damage, induce inflammatory cytokine production, and suppress antibacterial responses of innate immune cells (34)(35)(36)(37). However, they could also be categorized as choline/nutrient acquisition systems beneficial for niche survival (38), with the side effects of host cell damage and potential hemolysis.…”

Section: Distribution and Biological Roles Of Choline And Gbmentioning

confidence: 99%

Homeostasis and Catabolism of Choline and Glycine Betaine: Lessons from Pseudomonas aeruginosa

Wargo

2013

Appl Environ Microbiol

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Most sequenced bacteria possess mechanisms to import choline and glycine betaine (GB) into the cytoplasm. The primary role of choline in bacteria appears to be as the precursor to GB, and GB is thought to primarily act as a potent osmoprotectant. Choline and GB may play accessory roles in shaping microbial communities, based on their limited availability and ability to enhance survival under stress conditions. Choline and GB enrichment near eukaryotes suggests a role in the chemical relationships between these two kingdoms, and some of these interactions have been experimentally demonstrated. While many bacteria can convert choline to GB for osmoprotection, a variety of soil-and water-dwelling bacteria have catabolic pathways for the multistep conversion of choline, via GB, to glycine and can thereby use choline and GB as sole sources of carbon and nitrogen. In these choline catabolizers, the GB intermediate represents a metabolic decision point to determine whether GB is catabolized or stored as an osmo-and stress protectant. This minireview focuses on this decision point in Pseudomonas aeruginosa, which aerobically catabolizes choline and can use GB as an osmoprotectant and a nutrient source. P. aeruginosa is an experimentally tractable and ecologically relevant model to study the regulatory pathways controlling choline and GB homeostasis in choline-catabolizing bacteria. The study of P. aeruginosa associations with eukaryotes and other bacteria also makes this a powerful model to study the impact of choline and GB, and their associated regulatory and catabolic pathways, on host-microbe and microbe-microbe relationships.

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Hemolytic Phospholipase C Inhibition Protects Lung Function during Pseudomonas aeruginosa Infection

Abstract: We have directly linked production of a single virulence factor in P. aeruginosa with effects on lung function, and demonstrated that the inhibitor miltefosine protects lung function from PlcHR-dependent surfactant dysfunction.

Cited by 75 publications

References 69 publications

Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

Characterization of the GbdR Regulon in Pseudomonas aeruginosa

Homeostasis and Catabolism of Choline and Glycine Betaine: Lessons from Pseudomonas aeruginosa

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