Structural basis for bacterial peptidoglycan hydrolase activation in cell division and intrinsic resistance

Bacteria are surrounded by a strong scaffolding-like molecule called the peptidoglycan layer.

The peptidoglycan layer provides protection to each bacterium and defines their distinctive shapes.

In E. coli, and many other bacteria, the peptidoglycan layer also serves as a point of attachment for a second protective layer (termed the outer membrane) which confers resistance to antibiotics. The additional protection conferred by the outer membrane can make many bacterial infections difficult to treat.

We are interested in a system of bacterial proteins that are responsible for breaking down the peptidoglycan layer when bacteria reproduce.

By understanding this system in E. coli, we hope to gain substantial new insights into how bacteria divide, and also, how they maintain their protective outer membrane barrier.

The key enzymes involved are a pair of peptidoglycan hydrolases (AmiA and AmiB) that are regulated by an 'activator' (EnvC) and an inner membrane protein complex (FtsEX). Under normal circumstances, the peptidoglycan hydrolases are carefully controlled to prevent them from chewing up the peptidoglycan layer. However, during division, a signal provided by FtsEX and EnvC is used to switch these proteins 'on' so that the peptidoglycan layer can be broken and newly-formed daughter cells can be separated.

In this project we will use a combination of structural biology, biochemistry and microbiological techniques to investigate precisely how the peptidoglycan hydrolases are turned 'on' and 'off' by FtsEX and EnvC.

Firstly, using X-ray crystallography, we intend to obtain 3-dimensional images that will show exactly how binding of the activator causes the peptidoglycan hydrolase to be switched 'on'.

Secondly, we will isolate all the proteins involved and bring them together in a test tube so that we can study their biochemistry.

Thirdly, we will test our current understanding of the peptidoglycan hydrolase activation mechanism by making modified versions of the proteins in live cells and observing the effect on peptidoglycan hydsrolase activation.

Finally, we will explore the possibility of blocking the peptidoglycan hydrolase activation. Successful inhibition at any point along the hydrolase activation pathway would validate these proteins as potential targets for future drug development.

At the end of the project we would expect to establish a near-complete molecular understanding of how petidoglycan hydrolases are activated during division.

Activation of periplasmic peptidoglycan hydrolases defines a crucial phase in bacterial cell division where the peptidoglycan layer is partially broken to allow the separation of daughter cells.

In E. coli, two of the three peptidoglycan hydrolases linked to cell division (AmiA and AmiB) are each regulated by a periplasmic activator (EnvC) bound to a Type VII ABC transporter (FtsEX).

We have recently determined a crystal structure of EnvC bound to the periplasmic domains of FtsX and uncovered a novel autoinhibition mechanism at the level of EnvC. This work has led to a testable molecular mechanism for peptidoglycan hydrolase activation where the ATPase activity of FtsEX drives a transmembrane conformational change that relieves EnvC autoinhibition to promote binding and activation of the hydrolase.

In this project, we will use a combination of structural biology, biochemistry and microbiology to address the molecular mechanism of peptidoglycan hydrolase activation.

Our first objective is to determine a high-resolution structure of AmiA (or AmiB) bound to the activating domain of EnvC using X-ray crystallography. This should unambiguously define the mechanism by which the hydrolase is activated by its interaction with EnvC.

Our second objective is to reconstitute the complete FtsEX-EnvC-AmiA/B system in vitro so that we can probe the relationship between ATPase activity and peptidoglycan hydrolase activation.

Our third objective is to test the proposed mechanism for peptidoglycan hydrolase activation using site-directed mutagenesis of EnvC. In vivo complementation, bacterial 2-hybrid assays, and viability assays will dissect the effect of each mutation to confirm, refute or modify our hypothesis.

Finally, we will use knowledge of protein:protein interactions within the FtsEX-EnvC complex to design protein fragments that interfere with amidase activation when expressed into the bacterial periplasm.