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Life in the recycling bin (part 4)

Previously, on ‘A Bug’s Life – Ph. D Blog’, I challenged myself to summarise the contents of my Ph. D Oration into something a little more… palatable. For a general audience.

We’ve now (finally) reached the last Aim of my Ph. D project!

This, by far, was the hardest, most tedious, most frustrating section of my Ph. D…

And it’s actually what I started with.

So here’s that slide of Aims…

And there’s that third (and last) Aim:

‘Functional characterisation of a novel protein essential for intracellular replication of C. burnetii

Also known as, ‘what the f*** does this protein do?’

I’ve written about this section previously, as well, if you’re interested in reading it.

I mentioned in part 3 that my Ph. D supervisor made a whole bunch of transposon mutants during their time in the U. S.

If you’re lost as to what a transposon mutant is, I highly recommend reading part 3, because there’s an explanatory section in there.

Essentially it’s a way to make mutations in the genetic material of Bacteria, so that this mutant strain doesn’t make a specific protein. As explained in my previous post, this is one of the ways we scientists study a protein. If a component is missing, how does that impact the overall system? Based on the observed effects, can we then make an educated guess (hypothesis) about what this component is doing (i.e. what is its function)?

Anyway, many of these transposon mutants were initially screened for any defects in intracellular replication- a fancy phrase which in this instance means, ‘can Coxiella still infect and grow inside host cells as normal?’

During this screening process, my Supervisor came across this weird strain that was behaving very strangely.

Here’s a throwback to what an infected cell looks like, under normal circumstances:

As a reminder, the host cell nucleus, the pocket within the human cell containing our genetic material, is in blue. The green marks where the host recycling bin, or the lysosome, is. The red is Coxiella. The little splotches of blue elsewhere (outside of the nucleus) are actually also pieces of DNA- now why might it be present inside the pocket where Coxiella are? It’s because they also have DNA, too! Did you know that the basic building blocks of our genetic material are the same as what Bacteria have?

You can see the very spacious and super populated Coxiella-containing vacuole (CCV). For more information on that, please take a look at the Introduction, where I cover a lot of the Biology. Coxiella has the ability to fuse multiple recycling bins/lysosomes and similar compartments to make one giant balloon of a home, which is pretty cool if you think about it.

I think you have to realise that the host cell (our cells), usually have this kind of stuff very tightly regulated. Things shouldn’t be allowed to go haywire, because there could be dire consequences if they do.

So to have this single-celled organism just come in, and completely take over everything…

That’s incredible.

I think.

Just a reminder of that whole process, explained in part 1

Anyway, enough praise of our Bacterial overlords-

This is what my Supervisor saw when they looked at this particular mutant strain.

Can you see the Coxiella in red? Some of them might look a little yellow (red + green = yellow here)

They’re still wrapped up in green- which means they’ve made it to the host cell lysosome. They’re in their home ground environment, so in theory, they should have started replicating and growing into large numbers…

… but they haven’t.

Now that’s weird.

Clearly, whatever is missing in this mutant strain of Coxiella, was essential to initiate replication. Because when it’s gone… it’s not replicating.

But strangely enough, this inability to replicate was only limited to during infection. In other words, the Bacteria could replicate fine in liquid broth, in the absence of a host cell. Put them with a host cell, though… nothing.

I tracked growth of Coxiella every 24 hours for seven days straight in liquid broth. You’ve got your unaltered parent/wild type strain in red, the mutant (2072::Tn) in blue, and the complement in green. If the terms are confusing, I highly recommend reading part 3. You can see there’s no differences in growth between all three strains, which means the mutant has no problems growing in liquid broth.

The mutation, in this instance, was within a gene called cbu2072. It makes the protein, CBU2072.

What is CBU2072?

Well, it’s not an effector protein (see part 1), which are Bacterial proteins that get pumped into the host cell to manipulate it into doing the Bacteria’s bidding.

These coloured dots here in green, red and blue

We know this because my Supervisor looked into it. I’ve explained a BlaM translocation assay before, but essentially, you can artificially observe whether a Coxiella protein can be translocated, or pumped into, a host cell.

She chose another, already validated effector protein to use as comparison (CBU1780, which, funnily enough, was my Honours project). It’s marked as BlaM-1780. To put it simply, if you see protein getting pumped into the host cell, the bars on the bar graph get longer. If you get barely any or no protein getting pumped into the host cell- you’ll get barely a bar on the bar graph.

And this is what my Supervisor saw:

BlaM-1780 (again, CBU1780 is a known effector protein) is quite easily detected as having been pumped into the host cell.

But BlaM-2072… not so much (if at all). The bar is so small that we can’t actually tell if it’s getting translocated at all.

So at least, using this technique, it doesn’t look like CBU2072 is an effector protein.

What else did we know about this protein?

As mentioned in my previous post, one of the best ways to guesstimate what a protein might do is to look at its gene, or amino acid sequence, and see if it’s similar to any other known genes or proteins (respectively).

Unfortunately for us, CBU2072 wasn’t really similar to anything. 😅

Let’s break this figure down- starting with what the colours mean.

My protein, CBU2072, is actually in purple.

The dark blue bit denotes a small portion at the front (start) end of my protein, which resembles a sequence of amino acids called a ‘signal peptide domain‘.

What’s that, you ask? Well, it’s usually like a little barcode that tells the cell where the protein is supposed to go. Like the address for a letter, if you will. It can be read by other proteins that function like the postal service, and it then gets sent off to the relevant section of a cell (destination).

The green bit is just a bit similar (like, 33% similar, based on amino acids) to a metabolic enzyme found in E. coli, called SthA (in light blue). That led us to think that CBU2072 might be involved in Coxiella metabolism (see the small link between my previous two Aims? Maybe? 😂).

The other similarities were in the structure of CBU2072. You can get computer programs to ‘predict’ (i.e. make an educated guess) on the shape of CBU2072, based on known shapes of other proteins that might share some similar amino acid sequences.

Based on this prediction, CBU2072 had similarities to proteins that are usually found on the outer surface of a Bacterial cell. This gave us a lead on where the protein might be, once it got to its final destination.

So, as mentioned previously, the best way to study a mutant is to artificially give the protein back to it, and see if it restores the mutant’s defect (in this case, intracellular growth) to wild type, or unaltered strain levels. This is what’s called a complemented strain.

So we made some complemented strains that made proteins that looked a bit like this…

Obviously some artistic licencing taken here. Proteins would normally twist and contort into very complex structures, but I’ve straightened it out for easier viewing (and illustrating).

The little, light blue box is what’s called a protein ‘tag‘. Like a little flag that gets attached to your protein of interest (in this case, CBU2072). Funnily enough, this tag in particular is actually called a ‘FLAG-tag‘, and there are three FLAG-tags, one after the other. Hence, the ‘3xFLAG’. Tags are handy, because you can use it to mark where your protein is. It makes the protein easier to detect, without having to do more costly (and time consuming) work. As you can see, I had a version of CBU2072 that had a tag at the beginning of the protein (the green protein), and at the end (the orange protein).

In addition to those, I also made a few mutated/altered versions of CBU2072.

First, the purple protein. This version of CBU2072 is missing that dark blue portion in the earlier schematic- the signal peptide sequence. In theory, if a protein is missing the signal peptide sequence, the Bacterial cell might not know where to send the protein, so it’s likely that it won’t function as it should- because it’ll be in the wrong place. Like a letter with no address. Where is the letter meant to go? Opening it won’t do you any good- it’ll just stay at the depot… forever unread… that’s actually kind of sad, now. Damn this metaphor!

Then there’s the pink and yellow proteins.

These proteins are a bit funky, in that it’s not ‘missing’ a portion- I’ve just altered it slightly.

Remember that green region of CBU2072, that had some similarities to a metabolic enzyme found in E. coli? Well, when you look at the two proteins (CBU2072 and SthA) at the amino acid level, some of the areas are a bit similar, and you can see that marked as asterisks (*) on the below diagram.

Now, if two proteins share similarities at the amino acid level, then they might also share similar functions, because some areas, or amino acids of a protein, are essential for the protein to do whatever it needs to do. These key amino acids (or residues) are very important for a protein to fold properly, or to bind another protein (or molecule) it needs to interact with.

So, if I were to change it… ever so slightly…

Then the protein might not be able to function anymore.

And that could tell us what sections (or residues) of a protein is essential for its function.

So we sort of picked some residues at random (needle in a hay stack situation). I’ve marked them in red. You can see where they are, relative to the entire CBU2072 protein, in the diagram above. The ‘40AAAA43‘ and ‘84AVNVA88‘ just means that I changed the 40th to 43rd amino acid of the protein to alanines (‘A’), or that I changed the 84th and 88th amino acid of the protein to alanines, respectively. Originally they were ‘ 40PEIS43‘ (phenylalanine, glutamic acid, isoleucine, serine) and ‘84GVNVG88‘ (glycine, valine, asparagine, valine, glycine). The details don’t really matter. Just know that I changed them into something they’re not supposed to be. Just very subtly.

So, what happens to the growth of these cbu2072 mutants (2072::Tn), when they produce these varied versions of CBU2072? Can they once again grow inside a host cell, like it’s unaltered counterpart (wild type/WT)?

See part 3 for help on interpreting these graphs!

The answer is- sort of.

The wild type (red) grows as usual, and the 2072::Tn mutant (blue) doesn’t.

The 2072::Tn mutants given full versions of CBU2072, regardless of where the 3xFLAG-tag is (at its front or its bum), can grow like wild type. That’s a good thing, because it means that what we see in 2072::Tn is solely due to the absence of functional CBU2072 protein. When you give it back, everything is back to normal.

What about all the various, altered versions of CBU2072?

Well- the shortened one missing the signal peptide (purple), is still not able to replicate.

That means the signal peptide/address is really important for CBU2072 to function properly. It’s essential.

The weird amino acid mutations?

The pink version did nothing. But the yellow protein… not able to replicate.

The ‘GXXXG’ motif (where the ‘X’ is any old amino acid), as it’s called, has been linked with proper folding of a protein so that it can function properly. Without this motif, a protein might not fold correctly, and so it’s not structurally sound (bit wonky, if you will). This might explain why this mutated version of CBU2072 was unable to let the 2072::Tn mutant grow again, because a wonky shape might mean it doesn’t slot in correctly at its destination, or interact with other proteins as it should, because they can’t bind properly together.

Now, much like with part 3, we repeated these experiments with a host cell that more closely resembles Coxiella‘s usual host.

The above graph is a bit different, because it’s just comparing growth at day 3, relative to day 0 (i.e. how much has the Bacteria replicated in three days?).

And we saw very similar kinetics in growth. Which is great. That means that what we’re seeing in the lab might likely happen during a natural infection out in the world.

Can we use our Galleria model to make sure that this happens at an animal/insect level, as well?

Why yes we can. The wild type (red) and complement (green) will kill Galleria over the course of eleven days- but the mutant (blue) and shortened version of CBU2072 (purple) won’t. This once again demonstrates that if Coxiella can’t effectively replicate/grow inside the host, then there’s no disease. The host will be fine. And on top of that, that signal peptide sequence is also essential for disease progression with this protein. If you don’t have it, then the protein is useless.

I also looked at whether you can ‘rescue’ the growth in the 2072::Tn mutant, by infecting the same host cell with wild type/unaltered Coxiella. In theory, if whatever CBU2072 was doing during infection was affecting something ‘external’ to the individual Bacterial cell… then the wild type strain should be able to help the 2072::Tn mutant grow. If the function of the protein was ‘internal’, then only the individual Bacteria making CBU2072 would be helped, and so the wild type would be useless for the mutant.

It might be a bit hard to tell-

The mutant is in red, the parent/WT is not.

But- ALL Coxiella, regardless of mutation, is in green. This basically means that the green is both WT and mutant, while the red is just mutant.

So if you get more red signal, that means the mutant is replicating.

And that’s exactly what we saw.

So this means that whatever CBU2072 does during infection, it has the ability to affect other Bacteria present inside the same compartment. If there are Bacteria present that are unable to make CBU2072, it gets rescued by those that can.

Now, something else we were wondering was whether those lone little 2072::Tn Coxiella inside the host cell…

…were still alive. Because the stains don’t tell you whether the Bacteria are alive or not.

Now, there are some stains available that tell you whether Bacteria are alive or dead, but most of them are for Bacteria grown in liquid broth, and even though I tried to use it on these guys, the results were… varied… to put it mildly (it was real bad).

So we went for a different approach, which takes advantage of the fact that host cells are relatively quick to clear dead material, especially in relation to the six to seven day growth cycle of your average Coxiella.

So you start by infecting some human cells with 2072::Tn Coxiella.

And then you leave them for five days. If the Bacteria are truly dead, then the host cell would have cleared them all up (digested them?).

Now, this is where the paths split.

For one condition, at this point, we re-infect the same cells with more 2072::Tn Coxiella. This, in theory, should have no effect, because we know that these guys can’t replicate.

But for the other condition, we infect again, but this time with WT- which we know can allow any living 2072::Tn Coxiella to start growing again…

…and because Coxiella take ages to grow, we have to leave them for a further three days before we can check their growth…

…at which point we can stain them as I did before during the co-infection experiments (WT and 2072::Tn at the same time).

And this is what we saw:

The graph just shows you how much ‘red’ (i.e. mutant) there was. The brighter the red, the more mutant there was. But you can quite clearly see in the photos that you have proper CCVs again that are full of 2072::Tn mutants.

So all of that combined means that while the 2072::Tn mutant is unable to replicate, it’s not dead.

In fact, there’s something inherently strong about Coxiella, that allows it to withstand the host lysosome- even when it’s not growing.

If given the right environment (i.e. wild type), the mutant will begin growing again.

Now, let’s backtrack a little for a quick refresher on how Coxiella establishes infection inside the host cell.

A typical infection requires the translocation (pumping out of) effector proteins into the host cell- this establishes the Coxiella-containing vacuole (CCV). No translocation machinery = No effectors getting out = no CCV.

In the 2072::Tn mutant, we don’t see any CCV, or replication of Bacteria- but we also don’t see any translocation of effectors by the 2072::Tn mutant.

Now, the big question here is:

Is the lack of translocation due to the fact 2072::Tn is unable to replicate…

… or is it because CBU2072 is required for the Type IVB secretion system (T4SS) to translocate effectors into the host cell?

I.e. Is the absence of CBU2072 the reason why there’s no translocation in this mutant strain?

If it’s the latter, then it means that CBU2072 might help translocate effector proteins into the host cell (somehow). It’s still not pinpointing the exact function, but it definitely starts to narrow down the field.

So how do you tease that apart?

Well, we know that if you co-infect the same cell with WT, then the mutant will begin to replicate.

IF CBU2072 is involved in translocation of effector proteins, then once replication is restored in the mutant, translocation will still be blocked- because CBU2072 is still absent.

But, if CBU2072 has no involvement with translocation, and replication is restored in the mutant, then translocation should occur as usual- because even without CBU2072, there’s no link between it and translocation. Its absence should be a hindrance.

So, did we see translocation of effectors during co-infection?

No!

Even with replication restored by the wild type, there was no translocation occurring in the mutant that carried a tagged, known effector protein (MceA, which my former housemate worked on during their Ph. D). You can see translocation occurring in the WT strain with MceA (far left), but nothing in the other conditions. Sure, you might see some blips, but it’s not consistent or strong enough for it to be really conclusive. If it did occur, it should be as noticeable as what you see in WT.

And you can see this visually, as well.

MceA (green) is known to go to the host mitochondria (‘the powerhouse of the cell’), which is red in the above images. You can see clearly, green specs in the WT with MceA condition (top left), in the same spots as host mitochondria in red. For the co-infection, though… nothing. You might see some specs of green, but they’re much more faint, and are likely to be what’s referred to as ‘background noise’ (i.e. not real signal).

This give a good case that CBU2072 has something to do with the translocation of effector proteins by the T4SS- indirectly or otherwise.

And for my Oration, that’s as far as I got to. I was still working on some additional data to provide more info on what was happening. But at the time, I had some ‘not very pretty but probably true’ data that showed CBU2072 was in the outer layer of the Coxiella cell.

Shown here in red.

What we thought might be happening was, without CBU2072, the T4SS can’t assemble properly, because something about the structural integrity of that outer layer was compromised.

Remember how I mentioned in part 1 that the T4SS is made up of lots of proteins? It’s a big, multi-protein complex.

And so when the 2072::Tn mutant infects a host cell, it still gets taken in and trafficked through the cell, and into a host cell lysosome (recycling bin). But because there’s no functional T4SS, no effectors can get out, and the CCV doesn’t get established- so there’s no Bacterial replication going on.

But when you infect the same cell with Coxiella that has CBU2072…

…and it has a functional T4SS that can translocate effector proteins…

The CCV is restored, and the mutant can replicate again.

Funnily enough, this is the same thing we see with mutants that lack certain, essential components of the T4SS machinery itself. If key components of the machinery aren’t there, then proper construction and assembly won’t happen- and that leads to Bacteria lying dormant. Not dead, but dormant.

And that’s as far as I got at the time of my Ph. D Oration. Since then, I got proper, prettied up figures that showed CBU2072 was found both internally inside Coxiella, but also in the outer (but not outermost) layer of the Bacteria (called the ‘inner membrane’)…

Western blots galore! ‘WCL’ stands for ‘whole cell lysate’, or ‘the entire Bacterial cell turned into mush’. ‘Cyto’, or ‘cytosol’, is essentially the liquid portion inside the Bacterial cell. ‘IM’, or ‘inner membrane’ is literally the first, or inner most membrane that surrounds a Bacterial cell, while ‘OM’ or ‘outer membrane’ is the second, and outer most membrane that surrounds a Coxiella cell.

CBU2072 is called ‘EirA’ here, but hopefully you can see that EirA (shown in the top most box for each strain) is found- obviously in the whole cell lysate, but also more specifically the cytosol and the inner membrane (indicated by dark bands). The cytosol/fluid portion of the Bacterial cell is where proteins are first manufactured, so it’s not too bothersome that we find it here, as well.

Unfortunately that shortened version of CBU2072 (called EirA24-165 in the above image) didn’t have any differences in where the protein could be found inside the Bacterial cell- so it means that the signal peptide isn’t needed to tell the protein what ‘area’ of the Bacterial cell it should go to- but it’s certainly still required for any finer details.

Maybe it’s needed to allow the protein to embed itself a certain way into the membrane? Maybe without it, its tail sticks out at the wrong angle? Who knows. Either way, we still know that it’s essential for CBU2072 to function- just not in terms of roughly where it should go.

We also found out that the absence of CBU2072 does create some changes to overall Coxiella metabolism (the figure is overwhelming so I won’t show it here), so there might yet be some sort of metabolic aspect to what it does.

And we also saw that- at least at this magnification, we couldn’t see any structural integrity issues in the absence of CBU2072.

The transmission electron micrographs that I showed a segment of in part 1. The 2072::Tn mutant (eirA::Tn) is in the middle column. By the way- these are actually super funky looking Bacteria. Other Bacteria don’t look quite so squiggly.

It would be great if we could look at the assembly (or lack thereof) of the T4SS at a higher resolution, but we didn’t have the time or resources available to us. Maybe one day, someone might look into it.

For now, it’ll remain a mystery.

So what did we end up finding out about CBU2072 (subsequently named EirA, or essential for intracellular replication A by us- which is kind of cool, being able to name something)?

Well- it’s essential for Coxiella to be able to establish an infection within the host cell- without CBU2072/EirA, there’s no infection. The Bacteria just sit, waiting for the right cue to begin replicating. They’re not dead, though.

It’s also essential for causing disease in an insect model, because without CBU2072, the moth larvae are fine. Maybe we might not see that at a higher order animal level (i.e. humans), but you never know- we might?

The front end portion that contains that signal peptide sequence is also essential for protein function, because we can see that the shortened protein doesn’t do anything for the mutant. The shorter protein can still get to similar places within Coxiella that the whole protein will go to (the inner most membrane of Coxiella, and also the liquid portion inside the cell), but it still can’t function properly. There’s also that weird GXXXG motif, too, that I showed was important for protein function.

CBU2072 might have some sort of metabolic function- although I do wonder… if we got a mutant that lacks a component of the T4SS, whether we would see similar changes in metabolism to the 2072::Tn mutant. Maybe what we saw in that mutant is just a by-product of not having a functioning T4SS? I’m sure the Bacterial cell would be a bit stressed if there’s a roadblock in its usual day to day activities. 😂 That might come out as changes in metabolism. New experiment alert!

We still need to confirm whether the T4SS is structurally intact. Maybe it’s fallen apart- maybe it’s missing a section? We need answers on why that mutant still can’t translocate effectors, even when allowed to start replicating.

Now, all these summaries don’t do justice to the countless hours spent on brain-storming, optimising, and repeating experiments before collating data to write up. The growth curves/infections of human cells took the first six or so months of my Ph. D, because I had to make all the strains needed. Sure, some were already made by others, but all those weird variations of CBU2072 were made by me. Then you have to repeat each experiment at least three times (minimum) to make sure it’s consistent. I think I ended up doing the first cell line five times, and the second one four times, but I know plenty of others who repeat six or seven times to be sure.

The Galleria infections were a rush job at the latter half of 2019, because by then I had the infections down pat and I could do them on my own. It still took a good few weeks to do because of the need to repeat everything.

The co-infections only show one time point, but in reality I actually surveyed growth across seven days, every 24 hours. So every day, I had to come in and take samples. And I did that three times.

The co-infection translocation assays stressed me out so much, because the wells that contain each condition are teeny tiny, and one slip up could cross-contaminate samples- ruining the experiment. Given it takes a week for Coxiella to grow, unless you had backup cultures, you could set yourself back by a week. That might not sound like much, but minor setbacks do add up.

The fractionation experiments- where I determined where CBU2072 was inside the Bacterial cell, took three years to get working well. I admit that a lot of the problems were due to my own stubbornness and lack of insight, but sometimes concepts or ideas just don’t fully ‘click’ when you want them to.

Now, this isn’t to say that there aren’t plenty of others who work even harder. I know people who regularly pull overnighters, or practically live in the lab. I’ve only had to pull one overnighter, but I still think that’s one too many.

Basically- I’m not whinging about how hard my Ph. D was, or something along those lines (it’s not a competition of how miserable or unfortunate you are- let alone hardworking). I just mean that- while one figure may look nice, or pretty, or even semi dodgy, I’m sure that for a lot of the time, it’s the result of hours, days, weeks, months, or even years worth of hard work.

And it’s never solo work. I know that I wouldn’t have been able to do the work without help from my Supervisors, my lab mates, my colleagues, and other friends. Even if a paper lists a certain number of authors- all of those people needed other people to help them with various different things. Sure, it might not be direct, but a suggestion when you hit an experimental road block, or sharing of lab consumables/equipment- even a kind drop off of coffee or snack (or a meal)…

The combined efforts of those that are around you is what makes science possible.

So, here’s a summary of my Ph. D, in picture form…

And a huge thank you to those that have read this far. I hope you’ve enjoyed learning about a very small aspect of what a scientist might be doing in their research.

And while I’ve moved on to other things now, Coxiella (and more broadly Bacteria) will always hold a special place in my heart. This project provided many challenges and obstacles to me (both physically and psychologically), but all in all I’d like to think it taught me to be a better scientist. I wouldn’t be the person I am today, if it weren’t for all the learning curves my Ph. D threw at me. I also wouldn’t know all the amazing people I know, if it weren’t for all the collaborative opportunities this project provided.

Here’s hoping my project will be useful to someone one day… even if it’s for the techniques used. You never know what kind of impact your work will have, but I hope it brings some sort of inspiration to someone.

Categories: Ph D posts

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ABugsLife

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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