Understanding the Role of Peptidoglycan Metabolism in Bacterial Predation

We are studying the natural, friendly bacterium Bdellovibrio bacteriovorus - roughly translated as "leech-like bacteria-eater" - which is able to kill other less-friendly bacteria. The control of bacterial populations is important in many areas of human existence - namely tackling undesirable pathogenic species in healthcare (e.g. superbugs), crop pestilence, food safety, biofouling and water quality management. The potential exists to use i)knowledge gained from Bdellovibrio study, ii)protein products (enzybiotics) or iii)whole cells/cultures in a therapeutic manner. Unlike other predatory bacteria, Bdellovibrio kills its targets from within - entering them, breaking them down, reproducing inside and then bursting them to release daughter cells and begin the cycle anew.

Bdellovibrio arose from a non-predatory ancestor bacterium, and thus developed specialized tools that allow it to enter and kill other bacteria. These tools are protein enzymes - we aim to investigate the form and function of these so that we understand the killing process better, and perhaps even enhance the potential of Bdellovibrio as an antibacterial agent. Such proteins can be very useful to let us target the invasion of pathogen cells and break them in a controlled way.

Prior investigation by our laboratories revealed that one such enzyme (Bd3459) was targeted to the prey, and acted to change prey shape. We showed that Bd3459 achieved this by cutting particular regions of the prey wall (known as peptidoglycan), causing the wall to partially collapse and so alter the shape of the bacterium that it previously supported - much like sawing away at the support walls of a house! The shape change serves to provide the optimal space for Bdellovibrio invasion of prey, which also signals to fellow invaders that this particular "home" is occupied and that further entry would be wasteful. There are more "special" Bdellovibrio enzymes that "chip away" at the cell walls of bacterial prey- we would like to work on these to develop a fuller picture of what goes on when Bdellovibrio starts to kill its prey and to allow people to use these for biotechnology.

We will look at the enzymes themselves in atomic detail (known as x-ray crystallography), using fluorescent versions of the enzymes to track where they exert their effects (do they "chew up" the host or prevent unwanted destruction of self?), monitoring the precise nature of the function (known as enzyme assays), and also testing the enzyme:location:function relationship by constructing mutant strains of bacteria (lacking the enzymes) to confirm/dispel the hypotheses arising.

The investigation of peptidoglycan-targeting enzymes has a very practical application - an intact wall is essential to most medically-relevant bacteria, and forms the basis of action of several very successful antibiotics (e.g. penicillin, vancomycin). Results from our study may inform on this process, and also have implications for microbial physiology (form and function) in general.

Bdellovibrio bacteriovorus is the model organism for investigating predatory bacteria; it breaches the outer membrane of Gram-negative prey (examples include many pathogens) and reproduces within the periplasm, modifying but not destroying the prey cell wall until it is lysed at the end of Bdellovibrio reproduction. Bdellovibrio has many specialized proteins for this purpose, one such example of which is the specialized peptidoglycan endopeptidase, Bd3459. We showed recently that this enzyme is the elusive peptidase that converts the rod-shaped prey into the spherical niche upon invasion. Our studies revealed that Bd3459 is a member of the DacB/PBP4 DD-transpeptidase family, but had several modifications presumably related to its promiscuous use in hydrolysing prey walls. This work represented the first molecular-level investigation into the evolution of predation, and we aim to further explore the processes of Bdellovibrio prey-wall-modification/self-protection.
We will:-
1)Determine which features of Bd3459 (deviations/modifications from "housekeeping" DacBs especially) are most important for substrate recognition and cell-rounding function
2)Investigate whether the co-expressed Bd3460 ankyrin protein is responsible for self-protection or further prey manipulation (bioinformatics strongly suggests a protein:protein interaction role for Bd3460)
3)Test the predatory roles of specifically predatorily upregulated peptidoglycan LD-transpeptidases (localization, structural features, specific activity)
4)Determine how peptidoglycan deacetylases act on prey peptidoglycan and whether this regulates its degradation (pertaining to a phenotype described ~30 years ago to which no gene product has been ascribed) -including proteins Bd0468/Bd3279. We have an interesting double knockout of these that suggests they are indeed the sought-after deacetylases. These enzymes act to modify host wall hydrolytic sensitivity and putatively aid in differentiating host versus self.

Bdellovibrio-based projects have significant relevance to almost all of the strategic priorities of the BBSRC - the natural antibiotic action of this species pertinent to animal health, food security and ageing-related disease (long term bacterial infections such as diabetic ulcers). Bdellovibrio prey of particular interest include Pseudomonas, Acinetobacter, Burkholderia, Proteus, Salmonella and Klebsiella. The diverse, bacterially-degredative enzymes of Bdellovibrio may have uses and impacts outside of whole cell applications, and the advent of synthetic biology means that engineered therapeutic strains are now possible (e.g. the recent use of pyocin-producing E. coli).

Industry: Antibacterial usage of Bdellovibrio has potential benefits in healthcare, farming (crops and livestock) and bioremediation. The lifestyle of Bdellovibrio means that it is also a rich source of unique enzymes, with potential for technology development (the bacterium itself representing a natural nanoscience solution to bacterial control and manipulation).

Basic Scientists: A substantial direct impact would be on predatory bacteria researchers - Bdellovibrio is the model organism in this field and our wider aim is to annotate the features revealed by its genome sequence and transcriptome analysis.
Of equal significance are the insights we will obtain into its prey, as E. coli is undoubtedly "the" model bacterium and we will uncover details of its cell wall physiology and regulation. The relationship between predator and prey is also relevant as a "simplified" model of intracellular growth adaptations, and as such will impact related pathogenesis/symbiosis fields.

Evolutionary microbiologists will be very interested in the series of peptidoglycan modofying enzymes that are required to firstly enter diverse bacterial cells and keep their walls expanded yet stable and also to "prepare" them for later synchronous lysis. Evolution for predation has required a large complement of such peptidoglycan active enzymes.

Students: Elements of the proposal (and associated spin-off findings) will make excellent small-scale lab projects for students - both the Lovering and Sockett groups find that Bdellovibrio elicits an enthusiastic and often awed response from students and we look upon this work as an ideal vehicle for fostering wider student appreciation of microbiology and structural biology.