# Novel phospholipase involved in outer membrane homeostasis in Pseudomonas aeruginosa

> **NIH NIH F31** · HARVARD MEDICAL SCHOOL · 2024 · $45,574

## Abstract

Project Summary
The Gram-negative outer membrane (OM) is an effective permeability barrier against numerous chemical
assaults, including antibiotics. The intrinsic barrier capacity of the outer membrane in part due to its asymmetric
nature; the inner leaflet consists of phospholipids (PLs) while the outer leaflet is composed of lipopolysaccharide
(LPS). Although the systems responsible for the maintenance of OM asymmetry have been well-characterized
in enterobacteria like Escherichia coli, little is known about the mechanisms maintaining OM barrier function in
the human pathogen Pseudomonas aeruginosa. E. coli encodes three pathways that remove PLs from the outer
leaflet of the OM: the Mla system, PagP, and PldA. The Mla system removes PLs from the outer leaflet of the
OM and promotes their retrograde transport back to the IM. PagP removes an acyl group from outer leaflet PLs
and attaches them to LPS, converting the glycolipid from a hexa-acylated to a hepta-acylated form. Finally, PldA
is an OM phospholipase that cleaves outer leaflet PLs. Much less is known about how the highly antibiotic
resistant pathogen P. aeruginosa maintains the asymmetry of its OM. Like E. coli, it also encodes an Mla system
and PagP. However, it lacks PldA. A transposon-sequencing (Tn-Seq) screen for P. aeruginosa mutants
hypersensitive to an antibiotic that blocks LPS synthesis identified the OM phospholipase PlpD as a novel factor
involved in OM barrier function. To further investigate the potential role of PlpD in OM asymmetry maintenance
I will investigate whether PlpD is playing a role in P. aeruginosa analogous to that of PldA in E. coli by developing
assays to detect phospholipids in the outer leaflet of the OM in P. aeruginosa. I will also perform cross
complementation experiments to test whether E. coli PldA can substitute for PlpD function in P. aeruginosa and
vice-versa. To complement these focused experiments, I will take advantage of unbiased genetic approaches to
investigate the function in PlpD in OM barrier function. Suppressor mutants that restore normal drug sensitivity
to a plpD mutant will be isolated and characterized. Additionally, I will use Tn-Seq analysis to identify genes that
when inactivated result in a synthetic lethal phenotype when combined with a plpD mutation. In both genetic
assays, the identity of the genes with suppressive or synthetic lethal interactions with plpD will inform us about
the nature of the OM defect in cells inactivated for PlpD and tell us under what conditions PlpD function becomes
essential. The genetic results also have the potential to identify new factors in P. aeruginosa that play important
roles in OM barrier function. Overall, the knowledge generated from this proposal will shed light on the
mechanisms that underlie OM barrier function and promote drug resistance in a problematic gram-negative
pathogen.

## Key facts

- **NIH application ID:** 10825970
- **Project number:** 1F31AI181511-01
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Johnathan Lee Kepple
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $45,574
- **Award type:** 1
- **Project period:** 2024-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10825970

## Citation

> US National Institutes of Health, RePORTER application 10825970, Novel phospholipase involved in outer membrane homeostasis in Pseudomonas aeruginosa (1F31AI181511-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10825970. Licensed CC0.

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