The Bacterial Invasion Port of Bdellovibrio

There is a great need to find new ways to destroy bacterial pathogens now that many are becoming resistant to antibiotics and antibacterials. It is important to prevent plant crops and farm animals against diseases, as well as to protect human health. Some predatory bacteria that are harmless to humans naturally kill bacterial pathogens by building a molecular "port" in them and entering through this port to kill them. Importantly, because this access port is build from several molecular parts, it is not easy for the pathogens to adapt to not fit one of the parts and so escape being invaded. Also the things that are pumped through the port are good at attacking bacterial membranes- there arent many antibiotics that do that. Because Bdellovibrio only attack bacteria they don't harm humans or their membranes.

We recently identified some of the parts of the natural invasion port of the predatory bacterium Bdellovibrio, and some control proteins that work with the port. We now want to find out how those parts work together to build and co-ordinate what the port does so we know how bacterial membranes are invaded. Then we will be able to use all these parts to build a toolkit to make pathogenic bacteria be invaded and killed.

In this project we are trying to find out what protein chemicals are pumped through the port into the bacteria and when, and what attaches the port to the bacteria while the predatory bacteria are pumping in the membrane damaging proteins.
We can use a simple coloured test to see if a protein is damaging the membrane of a bacterium. If the membrane is damaged then the coloured dye goes pink. In this way we can test proteins coded by the DNA of the bacteria and see which make the dye go pink when exposed to bacteria, to see if they are damaged by each protein.

We also think that some of the same proteins that pull the Bdellovibrio inside the bacteria can then do a transformation by bolting on different proteins and start to act as a pump to pump more degrading proteins into the bacteria to break them down. We will test this too.

From the project we will learn how to tackle pathogenic bacteria in farms or in public or in hospital buildings by attacking their outer membranes with Bdellovibrio-derived materials. These could go on to help as working antibiotics become scarcer.

We will build on a recent discovery by David Milner in our lab (PLoS Genetics 2014) that there is a hub of proteins at the invasive pole of Bdellovibrio bacteriovorus. This includes MglA and TPR Bd2492 which interact with a) Type IV pilus controlling pilus extrusion/assembly/ratcheting, but also possibly controlling T4P mediated secretion, b) a TamAB secretion system which may control autotransporter secretion. Together we view these systems as an invasion port for Bdellovibrio.

We will discover particularly OM damaging proteins of Bdellovibrio, by screening for changes in what is secreted or surface displayed in TPR/MglA defective mutants and by screening directly for OM damage.

We aim to discover new OM active agents and how they work together to make a port through which Bdellovibrio enter Gram negative bacteria.

The work combines genetics microbiology and microscopy of the Sockett lab with protein structure function expertise of the Lovering lab. We will also collaborate with Yi Wei Chang in Grant Jensen's lab at Cal Tech to use cryoelectrontomography to define predator prey contact points at the OMs. We will use a new 3DSIM microscope at Nottingham to also interrogate these contacts and have piloted methods to apply this to this work.

The project aims to produce an inventory of genes to encode an OM-active antibacterial system that could in future be used in synthetic applications as new antibacterials.

Impact Summary
This project arose because of the impact of research work done by David Milner, a BBSRC Quota Student who carried out the work that led to this project for his PhD thesis and published it with the PI named Tec, CoI and PI in PLos Genetics, a Ref 3* level journal. This shows that the project proposal is born out of impact. We will now hope to develop and transfer knowledge from this original project to define a subset of genes encoding an OM breaching system. Such systems are useful as they are different to conventional antibiotics and multifactorial so bacterial resistance to them is not commonplace.
Bdellovibrio-based projects have significant relevance to almost all of the strategic priorities of the BBSRC - the natural antibiotic action of this bacterium is relevant to animal health (Salmonella and E.coli infection control), food security (Pseudomonas spoilage of foods like mushrooms) 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 OM-active enzymes of Bdellovibrio may have applications in synthetically engineered Cells as Therapies"- part of a new government priority.

Industry: Antibacterial usage of Bdellovibrio OM breaching enzymes has potential benefits in healthcare, farming (crops and livestock) and bioremediation. Bdellovibrio genomes encode an unpexloited 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 major direct impact will be on predatory bacteria researchers - Bdellovibrio is the model organism in this field and our wider aim is to understand the co-ordination of features revealed by transcriptome analysis of these new MglA mutants that attach but cannot invade bacteria .. 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.
Students: Spin off experiments from the proposal will make excellent small-scale lab projects for students - both the Lovering and Sockett groups find that Bdellovibrio elicits an enthusiastic and often a "wow" response from students. Our research is very tangible and an ideal vehicle for fostering wider student appreciation of microbiology antibacterials, membrane properties pilus structure and structural biology/ genetics in general.