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My roller coaster Ph. D project

I still remember sitting in my Ph. D supervisor’s office, way back in March 2016, being shown a figure depicting this peculiar Coxiella burnetii mutant.

Usually in a bacterial growth curve, you see something like this.

Bacterial growth, detachment and cell size control on polyethylene ...
Thanks, Google

There’s an initial lag (I like to think the bacteria are just revving their engines) followed by a logarithmic phase where the bacteria grow exponentially. This is then followed by the stationary phase, where the bacteria have essentially run out of nutrients, space, etc. and can no longer continue to multiply (they’ve hit ‘peak’ growth). Eventually, provided you don’t supplement them with more food or space, they will drop off and slowly die.

Anyway, this Coxiella mutant, missing a protein called CBU2072, couldn’t replicate inside host cells at all. The curve basically flat-lined after lag phase and didn’t go up significantly. The beauty of it was, when you gave the protein back to the bacteria (i.e. the mutant could once again make CBU2072), growth was restored to normal (wild type) levels, thus illustrating that the lack of growth was solely due to the absence of this protein.

On the left is normal (those big circles are hundreds of bacteria replicating inside the host cell), and on the right is the mutant. It just sits quietly on its own.

A protein so important that the bacteria can’t grow inside cells without it- sounds pretty funky and cool to me! is pretty much what I thought in that moment. I also got told that this project will be Metabolomics heavy, meaning I would get to learn how to study bacterial metabolism. It was a foreign concept (and associated technique/s), so I thought I should try and diversify my skill set and agreed to take on this project.

Four years of craziness followed.

This project has been with me since the beginning, and it’s been full of many ups and downs. We found out that the front portion of the protein contained a signal that was required for its function, and that the lack of growth was only seen during intracellular replication (i.e. growth inside the host cell). We found that you could ‘rescue’ growth of the mutant by infecting the same cell with a normal, ‘wild type’ strain, meaning whatever the protein was doing, it could allow replication to resume in strains that still couldn’t make protein themselves. I remember my brain just felt like it was melting all the time.

Usually when we find a novel protein, we run database searches to see if the protein is similar to other, perhaps more characterised proteins. Maybe it contains amino acid residues that are similar to a particular enzyme? Maybe it has a motif (i.e. barcode) that tells the protein where to go? Is it structurally similar to another protein? All of these things can be calculated computationally- so, I went ahead and checked all the databases, but, the rather unfortunate (but also cool) thing about Coxiella proteins is, most of them are highly unique, and are unlike proteins found in other organisms. It was just a huge, needle in a haystack situation.

When you’re at the centre of a project, it can feel like you’re at the centre of this crazy maelstrom of stress. It can affect your mindset, and ideas for experiments don’t come out naturally anymore. Especially at the beginning, I was just trying really hard to learn how to science, so things often felt really hard. Every failed experiment chipped away at your self-confidence, and every constructive criticism felt like a personal attack. Fortunately in my case, the vast majority of criticism was not personal, but I know that this isn’t always the case for others in my position.

So, what do you do when you just feel really drained, stressed, and genuinely depressed? Well, aside from learning how to dissociate your self-worth from your project, you just gotta keep crawling your way through. Try new approaches, consult experts, talk to your Ph. D committee and ask for advice… all that stuff that people tell you to do, that you kind of hand wave and dismiss at the time- turns out they help, little by little (and sometimes in leaps and bounds).

With this project, I tried so many different things. My Ph. D Thesis has more of these (inconclusive) experiments outlined, but eventually, they started to form a pattern.

It’s hard to describe everything without having a face to face discussion, but we discovered a couple years ago that when you don’t have CBU2072, even when you restored intracellular growth in the mutant, they still couldn’t pump out specialised bacterial proteins called ‘effectors’ through a bacterial syringe-like apparatus.

Pumping out effector proteins via the Dot/Icm type IV secretion system (T4SS) is absolutely essential for Coxiella to replicate inside cells

So if absence of CBU2072 meant effector proteins couldn’t get out, that suggests that there’s something wrong with the secretion system (T4SS in the diagram) itself… But unfortunately, by the time we bulked out the data with more experiments to try and support this claim, my Ph. D candidature was drawing to a close!

And I guess that’s the point of this post. Sometimes, the simplest conclusion (written essentially with a couple sentences) may take 3-4 years to become fairly solid, and even then, it might not answer your original question. I still have no idea what this protein really does- if I could just ask the bacteria, I would!

‘Excuse me, kind bug? Sorry to be a bother, but, WTF DOES THIS PROTEIN DO?!’

*shakes fist at sky angrily*

*Coxiella laughs and runs away into the distance, leaving me all sad*

There are plenty of other experiments that could be done to keep trying to answer that simple question, but it’s time for me to pass the baton on to somebody else. Maybe someone might figure it out one day, but at least I got to lay down the foundations for them to do so.

If you like closure, or a finality to things, science might not be a good fit. 99.99% of the time, experiments only churn out more questions. The story never really ends. It might curve around and take you on random tangents, but it’ll never come to a close.

So here’s to my last paper, and CBU2072, now named EirA (Essential for intracellular replication A). You’ve been a giant pain in the arse, but I’m glad I got to tell your story.

If anyone would like to read this paper, the link is here. Unfortunately this paper is not Open Access and will require purchasing to read the full text.

Categories: Ph D posts

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A Ph. D graduate in Microbiology, residing in Victoria, Australia. Currently working in multiple locations but still in the STEM field. 👀 🦠 🧫 🧬

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