Tiny Biological Infection Needles

Why is this work important?

Its important to understand infectious diseases. If we know them better, we can learn to stop them more effectively. 

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Bacteria are becoming a big issue for us as they are rapidly becoming antibiotic-resistant. This means the medicine which stopped bugs in the past aren't working any more. The bacteria are beating us in this biological arms race.

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We need to better understand how micro-organisms like bacteria can cause disease in order so that we can in turn develop better preventions and treatments.

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The bacteria I am working on is called Pseudomonas. Its a large issue in itself as it can be an aggressive antibiotic-resistant infection in hospitals and it can pose a threat to agricultural crops. The knowledge we gain from this bacteria however can then potentially be applied to other nasty bugs containing the same infection system if they are similar enough - multiple birds with one PhD-sized stone.

Laboratory Techniques

Below is a list of the type of work I carry out in the lab as part of this project.

Molecular Biology

This is the manipulation and analysis of genetic material. For me this is in the form of DNA, the genetic code for all living things. 

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I'm often extracting and purifying DNA from bacteria, sequencing the DNA to reveal the code within or making changes to the DNA for future experiments to understand its function.

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I am working with DNA associated with the type III secretion system of Pseudomonas syringae, a bacterial disease of plants.

Protein Biochemistry

In living things, DNA encodes for proteins (biological structures made of things known as amino-acid polypeptide chains).

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At the base of the type III secretion system in Pseudomonas syringae is an ATPase protein. This acts like a little motor which drives the whole system and facilitates infection. ATPase means its a protein which breaks down ATP. ATP is energy in one of its rawest form for living things.

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I can isolate and purify this ATPase protein and any variants I make. From here, I test their biochemical function by carrying out reactions. 

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One example is an ATPase assay which measures how well, if at all, a protein is able to break down ATP. This is carried out in a 96-well plate and is measured using a plate reader which detects a change in light absorbance. This corresponds with the amount of ATP the protein has broken down.

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Plant Infection Assays

The type III secretion system and its ATPase are involved in bacterial infection. The best way to fully understand how they work is to see them in action!

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Pseudomonas syringae is a plant pathogen. The kind I work on infects tomato plants but it will also infect a plant that we prefer to use in science. This plant is called Arabidopsis thaliana and is a fast growing weed which has traditionally been used extensively in research due to its ease to work with.

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In a very strictly controlled environment (a sealed growth room), I can infect these plants with Pseudomonas syringae to measure the infection and observe the disease symptoms. 

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By creating variations in the type III secretion system and its ATPase, I can potentially see changes in infection when comparing to the original "wildtype". This in turn will help me understand the system better as I will learn what bits are important for what function.

Structural Biology

The ATPase associated with the type III secretion system, we think, could be controlled or impacted with a small signalling molecule that could be binding to it. 

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This molecule is known as cyclic-di-GMP or CdG for short.

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I can visualise the protein structure and see whether or not its binding CdG. This protein and signalling molecule is far too see under a standard microscope. Instead we have to use techniques which really get up close and personal.

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Two such techniques are known as x-ray crystallography and cryo-EM which can visualise what protein structures look like.

Biophysical Analyses