Regulation of carbon flux through the glyoxylate shunt in the opportunistic pathogen, Pseudomonas aeruginosa.

The last two decades have seen an increasing realisation - at both national and international level - that we urgently need to identify new strategies for controlling and managing bacterial infections. Due to widespread antibiotic use and abuse, the "golden age" of antibiotics is over; resistance to most classes of antibiotics is on the rise, and at the same time, fewer new antibiotics are emerging out of the R&D pipeline. In particular, antimicrobial agents that target the so-called "Gram-negative" bacteria are desperately needed. These bacteria are hard to fight because they have TWO membrane-like layers separating their interior from the environment (this double layer makes drug penetration difficult) and they also often express several "multi-drug efflux pumps" which, as their name suggests, export any antibiotics that do happen to get into the cell before they have a chance to have any effect.

One particularly dreaded Gram-negative "superbug" is the opportunistic pathogen, Pseudomonas aeruginosa (hereafter, "PA"). This bacterium causes devastating infections that can kill in a matter of days. Worryingly, antibiotic resistance is also rampant in PA populations. One reason why PA causes so much tissue damage during infections is because it has a mechanism (called "Type 3 Secretion", or T3S) that allows it to secrete toxic protein molecules directly into host cells, thereby killing them. As little as a single molecule of injected toxin is all that is required to kill the host cell, making this the most potent virulence factor in the PA arsenal. T3S activity can be stimulated by simple physical contact between the bacterium and the host cell. However, recent work has also shown that T3S is also turned on when the bacterium senses that it is running out of oxygen (as is also the case at the site of many infections). Unexpectedly, the "signal" telling the cell to activate T3S in the absence of oxygen turned out to be a metabolic one, generated by a biochemical pathway called "the glyoxylate shunt".

So, what is the glyoxylate shunt? In bacteria such as PA, most types of food molecule can be used to either generate energy or to generate biomass. However, a problem arises with certain foodstuffs - especially simple molecules like acetate or molecules which are broken down to yield acetate (e.g., fatty acids). Normally, the central metabolic hub of the cell (the "TCA cycle") takes the 2 carbon atoms in each acetate molecule and fully oxidizes these to yield 2 molecules of carbon dioxide. The dividend is that energy is produced. However, it also means that all the carbon that goes in to the TCA cycle is lost as CO2 - no carbon can become "fixed" for incorporation into biomass. To circumvent this, bacteria have evolved a special "shunt" to bypass the CO2 evolving steps of the cycle, thereby "saving" carbon atoms and allowing these to be re-routed to generate biomass. Without the glyoxylate shunt, PA therefore fails to grow on many foodstuffs, and mutants defective in the glyoxylate shunt are unable to cause disease in infection models. The reasons for this are still not clear, although diminished T3S and metabolic insufficiency are both probable contributors. Consequently, the enzymes of the glyoxylate shunt are widely accepted as potential targets for the development of antimicrobial compounds.

The problem is that although the glyoxylate shunt has been well-characterized in certain model organisms, the TCA cycle/glyoxylate shunt branchpoint in PA has a different architecture and is clearly regulated in a very different manner. Indeed, nothing is known about how flux is regulated through the glyoxylate shunt in PA, in spite of its obvious role in controlling fitness and virulence. The aim of the proposed work is redress this issue by generating a working flux model, allowing us to explore the best ways(s) of disrupting metabolism through the glyoxylate shunt, and to examine the impact of this on T3S.

The opportunistic human pathogen, Pseudomonas aeruginosa (PA), is a major public health threat. PA causes disease by elaborating a welter of highly-active tissue degrading enzymes, toxins and secondary metabolites. One of the most potent virulence determinants associated with PA is the Type III Secretion System (T3SS), which introduces toxins directly into host cells. Recent work by the lab of the PI has revealed that the T3SS becomes expressed and active during conditions of oxygen limitation, and that this stimulation is dependent upon a metabolic signal generated as a consequence of isocitrate lyase (ICL) activity.

ICL is the first enzyme in the glyoxylate shunt, which was recently shown to be conditionally-essential for PA infection in a mouse pneumonia infection model. The mechanisms controlling flux through the glyoxylate shunt have been well-established in E. coli. However, the architecture of the TCA cycle / glyoxylate shunt branchpoint in PA (and many other pathogens) is different compared with E. coli. The main aims of the current work are to investigate the enzymology of the branchpoint in PA in considerable detail, with a view to understanding how flux through the glyoxylate shunt is regulated in this organism. This is important because the control of flux cannot be extrapolated from a knowledge of the E. coli enzymology. This information will be used to construct a predictive flux model, which in turn, will be used to investigate how different flux scenarios impact on T3S.

New anti-pseudomonal strageties are urgently needed and the glyoxylate shunt is a validated target for the development of these. However, without a better understanding of how flux through the shunt is regulated in PA, we will not be in a position to optimize downstream drug discovery efforts. The current proposal aims to lay the groundwork for such efforts and should also yield a good deal of novel biology along the way.

We plan to maximize the impact of this work by carrying out the following;

(1) Generating a high-quality, professionaly-finished informative animation that captures the outcome(s) of the work in an easy-to-digest form. In my experience, these kinds of animations can really enhance the uptake of a piece of work. To be presented as a frontispiece on the website and for use in lectures.

(2) Generating a high-quality gallery of photographic "stills" for use in dissemination and cover art, thereby raising the profile of the work.

(3) Generating a bank of "visual bytes" to accompany the work. These visual shorts (including animations and time-lapse) will be used as website primers and as material to accompany any press releases made.

We are requesting funds specifically to cover the costs involved in the above efforts, which all carry a price-tag. In addition, I will continue in my ongoing regular Public Engagement efforts and interactions with Interest Groups (see Pathways statement).

Additional funds (not related to Impact per se, but under the "travel" heading) are requested to cover the usual academic dissemination avenues such as conference attendance. Publication costs (Open Access) to be covered from Central University funds.

Other Impact-related activities will include participation in the annual Cambridge Science Festival (an event that my team routinely assist with and occassionally organise (at a Dept level)), interaction with industry to kick-start exploitation of the project outcomes, and career appraisals for the PDRA. Clearly, exciting "spin-off" investigations deriving out of this work could form the basis for some excellent PhD or MPhil projects, so funding/appointments will be sought for those on an if-and-when basis. Shorter-term spin-off work could form the basis for a summer studentship or even an undergraduate project, and again, these possibilities will be pursued on an ad hoc basis as they arise.