I will warn you all now- this is a challenge to myself, and the journey is long.
I’ve decided to present to you, my Ph. D Oration/Completion Seminar… But aimed at a more lay audience, as opposed to my colleagues.
I presented this back in August 2019, when life was a little simpler (hahah, no it was still a bit hectic). It took about 35-40 minutes to go through it all, so I’m going to divide it into sections, to reflect the actual structure of my talk.
‘Life in the recycling bin’ was the lay title I gave to my Ph. D project. The actual Thesis title is a lot longer:
‘Understanding the mechanisms utilised by Coxiella burnetii for intracellular replication within the host phagolysosome’
I had to pay extra to get all that printed on to the front cover and spine. 🙄
So, what the heck does that mean?
To start with, let me give you a run down of my bug of choice- Coxiella burnetii.
Obviously some creative licencing taken over there with that cartoon. 😅
Not many people have heard of this little Bacterial species. Over the years I’ve only encountered a select few who knew of their existence before I spoke to/accosted them. Most of these people had some sort of agricultural or veterinary background, because that’s where infection is most likely to occur- in animal settings.
But before I get into the disease, let’s take a step back and have a wee history lesson.
Coxiella actually has a nice Australian origin to it.
The first described case dates back to 1935, which, if you think about diseases, isn’t that long ago.
There was this mysterious outbreak at an abattoir, in Brisbane.
The workers were getting sick, but no one had any idea how they were catching this bug.
A lot of them were experiencing fever, but it just didn’t seem like your typical ‘flu’.
So this mystery illness was named ‘Query (Q) fever‘, by a scientist named Edward Derrick, because… well- why not? Beats ‘Mystery fever’, I guess. 😅🤣
Derrick couldn’t isolate the bug that was causing the illness, so he decided to reach out and seek help from a scientist, who is very famous in the medical field here in Australia, Frank MacFarlane Burnet.
Some of you may have heard of him- or maybe you’ve heard of the Burnet Institute, which is named after him.
Burnet, alongside colleague Mavis Freeman, were then based at the Walter and Elisa Hall Institute (WEHI). With the samples sent from Derrick, they managed to culture, or grow the organism through infection of guinea pigs.
They saw, under the microscope, some organisms that looked like Rickettsia (which are bacteria found in ticks), in the spleens of these infected guinea pigs.
They found this microbial organism in ticks that were found in Nine Mile, Indiana.
Now, an unfortunate, but somewhat fortuitous event then happened.
A visiting researcher was accidentally infected with this organism. I’ve read that this was actually the then Head of the National Institute of Health (NIH) in some publications, which, if it’s true, is kind of funny- but also not- but also funny, only because they only got mildly sick. But like, seriously- he had one job. 🤣
Anyway, this researcher showed the same symptoms as the abattoir workers in Brisbane, so a connection was born.
The two teams, one in Australia and the other in the U.S., worked in collaboration, and Cox and Davis sent samples of blood from the infected researcher to Burnet and Freeman. Burnet and Freeman were able to show that the researcher had built an immune response against this mystery organism, and the immune mechanisms present in the blood was able to protect against infection from the organism found in Q fever patients from Australia.
Now, immune systems, especially the ‘adaptive immune response’ that takes time to build up, is very specific. It’s built to target one bug, or ‘non-you’ thing, and that’s it.
So if the researcher’s blood could protect against whatever was causing Q fever, then it was very likely that the organisms were the same, or very closely related.
With this discovery, the organism was officially named.
Initially, because they all thought it was Rickettsia, they named it Rickettsia burnetii.
… But then, many years later, they studied the organism more closely, at the genetic level, and they realised that in fact, this organism wasn’t related to Rickettsia at all.
Sometimes, things that look similar aren’t actually related. I guess as a kid, you’d think a shark and whale are closely related, because they both have fins and swim in the ocean. But then you grow up, and you learn that one is a fish (a cartilaginous one, at that), and the other is a mammal.
The same sort of thing happened here. It turns out, the bacteria were more closely related to Legionella, which causes Legionnaire’s Disease. They weren’t close enough to be called Legionella, though- they were their own distinct group. So, with all of that in mind, they decided to rename the organism, to Coxiella burnetii, to honour some of the prominent researchers that led to its discovery.
So with the history lesson out of the way, let’s start by talking about the disease itself.
Q fever, as mentioned earlier, is what’s called a ‘zoonotic‘ disease.
That’s just a fancy word to say, ‘you can catch it from animals’.
Coxiella, for the most part, is found in livestock.
Specifically, those that are categorised as ‘ruminants‘.
These animals don’t typically show any outward symptoms… except during pregnancy.
Coxiella has this uncanny ability to cause mass abortions. Sometimes, that’s the only sign of infection in a herd.
Here’s where it gets a bit gross- the aborted material/birthing products have heaps of bacteria attached to them. This is how they spread. They can also be excreted through poop, wees, and sometimes milk.
Once outside, the bacteria slowly dry up, or desiccate, and while most things don’t do so well in low moisture conditions, this is where Coxiella thrive. Coxiella can change its shape and structure to turn into a ‘spore-like form’, which is super resistant to dying. They can withstand drying out, but also UV exposure, which is handy if you’re having to sit outside for prolonged periods of time.
The dried up bacteria then get carried off by the wind, and the next animal will inhale these bacterial cells. Creating a new infection cycle.
You don’t need many bugs to cause an infection. In fact, some shady studies (shady in that you’d never get ethics/permission to do this these days) done in the 1960s have shown that you can have as little as 10 (if not less) bacterial cells, to cause an infection. Can you imagine that? Bacterial cells are even smaller than our own cells (which you’ll see later)- and you only need less than 10 of them to be infected.
So, coupled with the hardiness and aerosol transmission, Coxiella are highly infectious, so they’re actually listed as a Biological Agent (Category B). 😱 They’ve been used in studies (predominantly in the Cold War era) to trial them for use as actual Biological Weapons.
Okay, that’ll do for the history lesson (number 2).
One is called acute Q fever, and the predominant symptoms are much like ‘flu’. Bit of fever, maybe pneumonia, maybe some hepatitis (just to make things a bit different)… Usually it takes around 2-3 weeks to get symptoms, but for the most part, most people don’t show any symptoms. Only around 40% of those infected actually show any outward symptoms at all, so many go undiagnosed. Maybe even yourself, if you’ve been exposed to livestock for prolonged periods of time- you might have already been infected.
The second category is called chronic Q fever, and that’s when things can get a bit nasty.
It only happens to around 5% of people that get infected.
The transition from acute to chronic Q fever is not really well studied, so no one really knows exactly what triggers the shift. All we know so far is that people with chronic Q fever tend to have underlying health conditions, usually relating to issues with their heart or their blood circulation. They used to say that the most common symptom of chronic Q fever was infected heart valves (endocarditis), but there have been reports of other vascular (as in, relating to your blood circulation) infections in other outbreaks. Chronic Q fever is fatal without treatment, but the treatment itself is quite gruelling.
For two, solid years, you have to take antibiotics – twice a day. Every. Single. Day.
Some people have side effects to the antibiotics, so it’s definitely not ideal.
The other big issue with Q fever is the chronic fatigue. Many people, regardless of whether it’s acute or chronic Q fever, experience chronic fatigue. You can imagine, then, a farmer, who catches Q fever, being unable to work because they’re constantly exhausted and unable to get out of bed…
It would suck.
Bringing it back to the biological weapon thing, you can imagine the effects of a population if the working age group is completely knocked out by chronic fatigue. Productivity would plummet.
So while it doesn’t kill a lot of people, it can be debilitating. Your quality of life is reduced.
Those sorts of diseases can be described as having low mortality, but high morbidity.
A great example of all of this is what’s probably the biggest outbreak of Q fever, ever.
The Netherlands experienced around 4,000 confirmed cases of Q fever between 2007 and 2010. That’s confirmed, as in, the people who actually bothered to get tested. You can imagine then, that the real number would be much higher.
It cost around 300 million euros to contain the outbreak, and around 50,000 goats and sheep had to be culled.
A wee bit costly, both economically and agriculturally.
And with climate change causing many areas in the world to become dry, windy, and harsh… transmission rates could go up.
Especially here, in Australia.
Although Q fever is found pretty much worldwide (except, so far, in New Zealand and French Polynesia).
So that’s the disease side of things. But what we were really interested in, was what happens once the Bacteria gets inside the host cell. In other words, we wanted to look at the intricate interplay between our cells (the host), and the Bacteria, during infection.
So here’s a crash course on Coxiella‘s intracellular (‘inside cells’) life cycle.
As mentioned, Coxiella get inhaled, into our lungs, where they encounter the first line of defence in our immune system. Alveolar macrophages.
Alveolar is just a fancy word to say ‘lungs’.
Now, Macrophages are like the ‘Pac-man’ of the immune system. They specialise in engulfing foreign objects and ‘nuking’ it within themselves. There are videos online of these types of cells chasing after bacteria on a slide.
So let’s go through what happens after getting engulfed, step by step.
Here’s a Macrophage I prepared earlier. They kinda look blobby, so it’s shown as a giant grey blob.
Obviously there’s a lot of artistic licencing going on here- a cell doesn’t look this empty!
Now, there’s a particular process in cells- in the diagram it’s referred to as the ‘Endocytic Pathway‘. It’s just a fancy word for, ‘how to get stuff into me/a cell’. It’s a perfectly normal process for a cell to get material that’s on the outside of the cell, inside it.
But what’s interesting is that the stuff that gets brought in, are progressively acidified, and eventually, broken down. The break down products can then be recycled for use around the cell. Maybe it’ll be used to build up proteins. Maybe it’ll get recycled to generate energy? Either way, with each of the steps, these little pockets, called ‘vacuoles‘, progressively become more and more hostile towards its contents.
The vacuoles have names, because- everything has to have a name in Biology. You’ve got your early endosome, which is like the initial pocket that forms- generally not super hostile, it’s just like a shopping bag with some fresh food in it. Then as the early endosome traffics along, more material get added to it, causing it to become a little more acidic and… peroxide filled (the late endosome). Think of it as me dumping acid and bleach into your shopping bag of fresh produce, just bit by bit. Maybe chuck in a few worms that are slowly eating the food up and breaking it down into worm poop. 😅😂 Eventually you keep dumping so much acid and bleach into the shopping bag that- it’s no longer the original food- it’s just… mushed up food in various states of decomposition. The only way you can reuse it is by neutralising all that acid and bleach and turning it into useful compost. 🤣
Practicalities aside (can you even neutralise bleach??), that’s what a host lysosome is like. By host, I mean, our cells, or the cell that could get infected. The lysosome is extremely hostile, and most living things that get placed in it tend to die. This means that for most other pathogens, they actively try to escape getting carted off down the endocytic pathway. They’ll sometimes cut a hole in the bag and get out that way, or they’ll just funnel all that acid and bleach outside the bag, either by stopping the cell from pouring it in, or by pumping it back out.
Now, Coxiella are super unique, in that they actively require this acidification/nuking process for it to start growing.
They want to be taken to the host lysosome- in fact, they won’t be able to grow unless this happens.
So instead of trying to escape the situation, they just sit still and wait for its little baggy to become increasingly hostile and… harsh. Everything else will either escape or tolerate it, but Coxiella actually requires it.
So here we are, again. The Bacteria is the yellow-centred orange circle inside a vacuole. You can also see the host lysosome (in yellow). The vacuole that the Coxiella are in will progressively become more and more like a lysosome, with all the acidity and such to go with.
And boom- that’s when Coxiella wake up from dormancy. The circular form of Coxiella is its ‘spore-like form’ that I mentioned earlier. It’s been hanging around outside, it’s had to adapt and go into survival mode, and it’s definitely not been replicating (that only happens inside a host cell, under normal circumstances). The act of being placed inside the lysosomal environment kicks off its metabolism, and the Bacteria transform from their spore-like, ‘small cell variant (SCV)‘ form to its active, ‘large cell variant (LCV)‘ form. They literally change shape, and rearrange its structure.
This large cell variant is more ‘rod-like’, in shape.
You can see a mix of spherical shapes (SCV, or maybe LCV as a horizontal cross-section) alongside these wibbly wobbly elongated ones (LCV). Taken using a Transmission Electron Microscope (TEM).
The metabolically active and kicking Coxiella then enters ‘hijack’ phase.
They have these cool syringe like structures…
It’s actually made up of lots of different proteins coming together to make one big structure.
Anyway, this needle like structure is called the ‘Dot/Icm or Type IVB secretion system (T4BSS)‘, and it’s absolutely essential for Coxiella to replicate inside the host cell…
…Because it pumps out about 150 Bacterial proteins, called ‘effector proteins’, into the host cell. These Bacterial proteins then go on to hijack the host cell and make it do things that help the Bacteria grow safely inside the cell.
That could be… by dampening the immune response, by stopping the cell from pressing the panic button and calling for other white blood cells to come rescue it, or blowing itself up in an act of self sacrifice (apoptosis, or programmed cell death). Stopping the host from dying is Parasite strategy 101, because then the parasitic entity (in this case, Bacteria) can keep on replicating and growing inside.
It could also make the host cell bring other vacuoles to the one it resides in, so that it can collect even more recycled material, made up of nutrients and building blocks, to make more Bacterial cells. This leads to the formation of one giant vacuole inside the host cell, which we call the ‘Coxiella-containing vacuole (CCV)‘.
And if you think the cartoon was a little simplistic 😅😂, here’s what it actually looks like.
All of these things are carefully orchestrated, because it’s quite energy dependent. Bacteria generate energy and use it up like we do, just at a significantly smaller scale. This means that they have to be very conscious about how and where they use their resources, so that they don’t waste anything unnecessarily.
So this CCV, as mentioned, is quite acidic (pH 5.2, where neutral water is pH 7), really good at fusing with other vacuoles/pockets inside cells, and contains lots of broken up material, because it’s pretty much like a host lysosome- abundant with the basic building blocks of a cell, like amino acids (makes proteins), sugars (makes carbohydrates), and lipids (or fat).
This leads onto the Aim/s of my Ph. D project.
Here it is as I had it in my Oration slide:
And now lets break it down into simpler terms.
- What metabolic differences are there between Coxiella that are grown inside the host cell, and those that are grown artificially in liquid broths in a lab (i.e. no host cell)? What kinds of nutrients, or food, do they eat? Do they have preferences? Does it differ between the two conditions?
- Does this difference change depending on whether the Bacteria are replicating/growing really fast, or when they’ve stopped running around and have passed out?
- How do Coxiella eat sugar? What’s required for them to do this? What happens when they can’t take up sugar as easily? Is sugar essential for Coxiella to survive?
- Trying to figure out what this weird, funky protein, called EirA/CBU2072 does for Coxiella, during infection (i.e. what is it used for?)
And there you have it- the Introduction. If you’ve made it this far- good job! The next part will be on Aim 1.
Categories: Ph D posts
A Ph. D graduate in Microbiology, residing in Victoria, Australia. Currently working in multiple locations but still in the STEM field. 👀 🦠 🧫 🧬