Crossing the periplasmic void, elucidating the mechanisms of phospholipid transport in Gram-negative bacteria

The ever-increasing resistance of microorganisms to antimicrobial therapies, in particular for Gram-negative bacteria, represents one of the greatest threats to global public health of the 21st century. In fact, in Europe alone, an avoidable 25,000 deaths and 2.5 million days in hospital are thought to be directly related to this rise in resistance, totalling a cost of £1.2 Billion. England's Chief Medical Officer, Professor Dame Sally Davies recently termed antimicrobial resistance "a catastrophic threat", and suggested that without the development of new antibiotics minor operations may become deadly. However, a recent report by the WHO highlighted the alarming lack of new antibiotics under development and found most new drugs in the pipeline to be modifications of existing classes of antibiotics.

The development of new antimicrobial agents requires an in depth understanding of how Gram-negative bacteria function and maintain homeostasis. Selecting which of the systems in the bacteria to target signifies a central concept of the drug development programme. One such attractive target is the bacterial cell envelope, with the mechanisms involved in its production and maintenance perhaps holding the key to generating novel antimicrobials. All Gram-negative bacteria possess two membranes (made of lipids) that enclose the cell, separated by a space known as the periplasm. The outer of the two membranes protects the bacterium from the environment and represents its first line of defence by forming a semi-permeable layer through which it controls the movement of molecules into and out of the cell. Furthermore, proteins within this membrane are essential for bacterial pathogenesis and drug resistance. As such they are viewed as the instruments of microbial warfare, mediating the processes responsible for infection and disease progression. Preventing the formation of this membrane through the identification of new compounds could lead to the development of the next generation of antimicrobials.

Recently a network of proteins found at both the outer and inner membranes, known as the Mla pathway, was identified as being responsible for the transport of lipids between the two membranes. Recent evidence suggests that the Mla system is important for maintaining both the structure and the function of the outer membrane and can replenish its lipid content by transferring it from the inner membrane. Consequently, the system has been identified as a key modulator of membrane function. It is therefore of critical importance to understand how this system works, as the design of new compounds that inhibit the activity of the Mla pathway could potentially disrupt outer membrane structure and thus inhibit many essential physiological, pathogenic and drug resistance functions of Gram-negative bacteria.

In this research project we plan to characterise the mechanistic processes the Mla pathway undertakes in order to transport lipids between the inner and outer membranes, we will identify how the different types of lipids are transferred and the details behind how they are removed from the inner membrane, thus providing valuable mechanistic insights that will aid in the discovery of molecular inhibitors and new classes of antimicrobial agents.

To elucidate the mechanistic details of Mla mediated phospholipid (PL) transport we will employ a number of innovative techniques:

1. Two surface based membrane systems (SMSs), either tethered or untethered, that allow reconstitution of Mla membrane protein complexes within a bilayer enabling PL transport processes to be monitored in real time, either via observing changes in mass or chemical group composition.
2. Neutron reflectometry (NR) to study the bilayer architecture and enable detection of fluctuations in PL density within each leaflet during Mla complex function.
3. SMALP technology to isolate Mla membrane complexes, allowing for functional and structural analysis under more native and user friendly conditions.
Using these systems we have developed the following work packages:

A) Map the direction of PL transport of the MlaA/OmpF complex. The use of our SMSs in combination with QCMD and FTIR will allow us to probe PL movement into or out of the membrane. Combining this with mutagenesis will allow identification of key residues involved in the PL transport process.

B) Elucidate MlaFEDB function. In order to establish how MlaFEDB functions and the role of ATP, intrinsic fluorescence, fluorescently tagged PLs and NR will be employed to characterise substrate binding, map ATPase modulation and a monitor PL population changes.

C) Probe global PL transport selectivity. Using our SMSs the effect of PL composition on transport rates will be probed allowing us to to identify whether the Mla pathway shows PL preference.

D) Identify how PL is exchanged between components. Co-incubation using a combination of lipid bound and lipid free species, or if necessary the inclusion of intermolecular disulphide bonds, cross-linking PLs and/or coupled constructs will be used to create complexes of MlaC-MlaD and MlaC-MlaA. Structural analysis of these complexes will allow identification of sites of interaction and mechanistic details of the PL transfer event.

Academic impact:
Academic researchers in a number of fields will be the principal beneficiaries of this research. The elucidation of the mechanisms by which the Mla pathway functions will enhance the UK knowledge economy and contribute to the global understanding of outer membrane biogenesis and therefore microbial pathogenesis, virulence and multidrug resistance. This will be of huge importance to immunologists and bacteriologists. Furthermore, the Mla pathway is a potential new target for the development of novel antimicrobials. With the emergence of multi-resistant bacteria and a lack of antimicrobials under development or likely to be available for clinical use in the near future , this is a key priority.
More generally, elucidating the mechanisms of outer membrane lipid biogenesis will be of interest to numerous researchers including those interested in biological membranes, lipid transport, signalling, trafficking, secretion, drug discovery and bacterial physiology.

Commercial impact:
Our research focuses on the mechanistic details of the Mla pathway and as such we do not anticipate our research to produce commercially exploitable results in the short term. However the results of this study will provide targets for the development of novel antimicrobials. An important beneficiary therefore will be the pharmaceutical industry which will be given the ability to rationally design inhibitors of Mla function and therefore new opportunities to attenuate bacteria in the pursuit of anti-infective agents.
The emergence of bacteria that are resistant to available antibiotics represents an enormous and growing global threat. New targets and strategies are therefore urgently needed. The prevalence of Mla homologues throughout Gram-negative bacteria provides a broad target for intervention, potentially allowing treatment of a wide range of species. For example, Gram-negative bacilli cause respiratory problems (Hemophilus influenzae, Pseudomonas aeruginosa), urinary problems (Escherichia coli, Proteus mirabilis), and gastrointestinal problems (Helicobacter pylori, Salmonella enteritidis) whilst Gram-negative cocci cause sexually transmitted disease such as Neisseria gonorrhoeae, and other diseases including meningitis, e.g. Neisseria meningitidis. Mla component homologues are also prevalent within mycobacteria hence elucidating mechanistic insights in to function could lead to new drugs for the treatment of tuberculosis.

Societal Impact:
Improved understanding of the Gram-negative outer membrane will have a fundamental impact on our society. This is the essential organelle that protects all Gram-negative bacteria and harbours the instruments of microbial warfare. Elucidating how it functions will lead to the development of new antimicrobials and treatments. This is urgently needed as antimicrobial resistance is increasing rapidly. A recent report by Professor Dame Sally Davies, the Government's Chief Medical Officer, has liken it to a 'ticking time bomb' and warned that routine operations could become deadly in 20 years if we lose the ability to fight infection. The generation of new antimicrobials is therefore desperately needed. Hospital acquired infections currently cost the NHS £1 billion a year and approximately 70% of all intensive care unit infections are the result of Gram-negative bacteria. The development of novel antimicrobials will therefore benefit every member of society, from those suffering from an infection, to the families of patients, carers and health professionals.