Characterisation of autolytic programmed cell death in Pseudomonas aeruginosa biofilms

Bacteria mostly live in communities called biofilms that are stuck to surfaces and held together by a sticky slime that protects the bacteria from antibiotics and disinfectants. Biofilm slime often contains lots of DNA and other cellular debris that came from dead bacteria. In fact, some bacteria will suicide by exploding to produce the sticky slime that benefits the other members of the biofilm community. However, these suicide pathways also increase the sensitivity of bacteria to antibiotics that cause cells to turn on the suicide systems. It is important that we understand how bacteria control their programmed suicide systems so that we can develop better ways to inhibit biofilms and to develop effective antibiotic treatments that exploit bacterial suicide pathways. This project will explore programmed suicide systems of the important bacterial pathogen Pseudomonas aeruginosa that is a major cause of hospital-acquired infections and is one of our greatest threats due to high levels of antibiotic resistance.

This project will explore the mechanism and co-ordinate regulation of autolytic programmed cell death (PCD) pathways of Pseudomonas aeruginosa during biofilm development and expansion. We have shown previously that extracellular DNA (eDNA) is released by explosive cell lysis that requires the endolysin, Lys, encoded in the R-/F- pyocin gene cluster. We also showed that the release of eDNA during the early stages of biofilm formation involves 3 holins- Hol, AlpB and CidAB.

Our proposed model is that the AlpB-E and CidAB/LrgAB autolytic PCD pathways have co-opted the endolysin/spanin genes encoded in the R-/F- pyocin gene cluster to provide these necessary lysis functions. The holins Hol, AlpB and CidAB are each capable of translocating Lys to the periplasm to degrade peptidoglycan causing the cell to become a spheroplast whilst the spanins facilitate disruption of the outer-membrane. Each holin has a cognate anti-holin that inhibits holin auto-aggregation, to control timing of autolysis. In addition to regulation of the Phol promoter by PrtN, expression of the lysin/spanin genes is also regulated via AlpA and CidR.

The specific hypotheses we will examine in this project are:
- That PA0630 and PA0631 encode spanins required for autolytic PCD.
- That Hol, AlpB and CidAB are holins that translocate the endolysin Lys across the inner membrane
- That Hol, AlpB and CidAB are regulated at a post-translational level via cognate anti-holins.
- That PrtN, AlpA and CidR are transcriptional activators that can activate expression of the lys, PA0630, PA0631 lysis gene cassette to provide these necessary lysis functions to each PCD pathway.

These hypotheses will be explored in three connected work packages. The PI's well-established genetic tools and expertise in biofilms, molecular microbiology, biochemistry and advanced microscopy will allow for efficient and successful progress.