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Life in the recycling bin (part 3)

Welcome back to my long post series that aims to summarise my Ph. D Oration!

We’ve so far had part 1, the Introduction, and part 2, which addressed the first Aim of my Ph. D project.

This post will tackle the second Aim, so here’s that slide from my Oration…

…and you can see Aim 2:

‘Characterisation of glucose transport in C. burnetii

Also known as, ‘How do Coxiella eat sugar?’

So the broad questions I was asking in this section of my Ph. D was,

‘What’s required for Coxiella to eat sugar?’

‘What happens when they can’t take up sugar as easily?’

‘Is sugar essential for Coxiella to survive?’

Also, if you would like more information on genetic material and proteins, please read my post on the Central Dogma of molecular Biology.

So I demonstrated in my previous post (part 2) that Coxiella are able to use glucose, which is a type of sugar.

Taking in nutrients from the outside world, as a single-celled organism, requires proteins called ‘transporters‘. They act like a mouth for the Bacteria, and essentially forms a pore, or a hole, that the nutrients can be transported through.

Each transporter is specific to a type of nutrient, or may be very specific in that it only transports one substrate, or molecule. Some might transport multiple types of sugars (glucose, fructose, maltose, etc), while others might only transport glucose. It’s called ‘substrate specificity‘, if you wanna get technical about it.

So what type/s of glucose transporters exist in Coxiella? Well- no one’s looked at it, experimentally, until… me.

Many have speculated on them by looking at the genetic material (remember how I said that’s a great starter way to guesstimate what proteins an organism might have, in part 2?), but no one had actually confirmed their functions.

So, what’s on the genome (all of the genetic material combined), then?

One prime candidate for sugar transport in Coxiella was a protein called CBU0265.

CBU stands for C. burnetii, I think. 😅😂 The number is just an identifier.

Many scientists thought CBU0265 was a sugar transporter, because it looked an awful lot like a sugar transporter found in Escherichia coli, or E. coli, which is another Bacterial species found in our gut. Many of you may have heard of them when we talk about faecal contamination of water, because obviously the contents of your gut come out as poop, and high E. coli numbers in water = lots of poop in the water.

We also use E. coli a lot in the lab as a general work horse, because they’re very easy to grow (relatively speaking) and to genetically manipulate, but I digress.

So looking at the amino acid sequence (the amino acids that make up the transporter, which is made of protein), CBU0265 looked a lot like the E. coli sugar transporter, FucP.

It had the same key domain (called Fucose permease domain) that FucP had, which suggested to us that CBU0265 was also able to transport sugars.

kDa, or kilo Daltons is a unit of measurement to describe the ‘molecular weight’ of a protein. The larger the number, the bigger the protein. For an individual Coxiella protein, this is mid sized.

So how does one determine what function/s a protein might have?

Well, the best way to study any system’s function/s is to take away a single component, and see how the system changes to cope (or not).

And that’s exactly what we do when we study the function of proteins. We remove it from the system by either disrupting the gene (the section in the genetic material) that makes the protein, or we delete it (i.e. remove it completely). Then we observe what happens when:

  • It’s present as normal (wild type, or WT- genetically unaltered)
  • Missing (in our case, we used transposon mutants, which I’ll describe shortly)
  • or given back artificially (a complemented strain, which I’ll also describe shortly)

So what do the terms in bold mean?

A transposon mutant

Transposons are what’s described as a mobile genetic element. It’s a sequence of genetic material that can be inserted into one’s genome. Sometimes it doesn’t have any observable effect on the system, but other times, it might actually disrupt a gene, and prevent it from making the protein it represents. Think of it like a wedge that gets inserted into fabric. The surrounding fabric gets separated and sit either side of this wedge. It’s still there, but the pattern’s now ruined. When it comes to making proteins, the cell’s machinery will try to make the protein by reading the genetic material, but will get cut off once it hits the transposon. It won’t be able to read the rest of the instruction manual, because there’s a random wedge in there (and this wedge cannot be made into protein). Sure, the cell might be able to make a portion of the disrupted protein, but it might be 10% of the original size, or maybe 50%- sometimes it might even make 99% of it. It just depends on where that wedge got placed.

Transposon mutagenesis is the name of the technique whereby scientists can artificially place these wedges into the genome of an organism. The insertion events happen at random, so you just have to cross your fingers and hope that they occur in useful spots that make studying the organism more interesting. In really unfortunate cases, nothing happens (no insertions occur). In even more unfortunate circumstances, the insertions occur but they don’t hit any interesting targets, or they all cluster to one (or very few) locations. It happens- they’re called hotspots.

My Ph. D supervisor made hundreds of these transposon mutants, during her Post-Doctoral (Post-Ph. D) work over in the U.S. Now we have hundreds of different mutant strains that have transposons inserted into various different spots on the Coxiella genome, which means we have hundreds of strains with different mutations in specific genes. If you know which gene is disrupted, then we can use them in studies like the one I’ve described.

Thankfully, we did have a mutant strain for CBU0265- and I managed to re-isolate it from our freezer stocks!

Sometimes you can lose strains- they might degrade over time, especially in transit from overseas. That can be really sad, given the amount of effort that went into making them.

Luckily this wasn’t the case for my CBU0265 mutant, which I’ve called 0265::Tn in my figures.

The italicised 0265 denotes the gene, cbu0265 (genes are always italicised), and the ::Tn means that the gene preceding it was disrupted by a transposon.

In our case, we saw that the gene was disrupted around one quarter of the way into the gene, meaning roughly 25% of the protein would be made (if it did, hypothetically speaking). Given this protein needs to fold into a big barrel/circle for it to work properly, I was quite confident that it wasn’t going to be functional with just one quarter of a barrel.

Red arrow is where the transposon went in. The internal >>> shows you which way the gene gets read, and the numbers denote the number of base pairs (i.e. individual DNA molecules) we’re at, from the start of the Coxiella genome

A complemented strain

I mentioned that you should have a strain that has the missing genetic component artificially introduced back. A complemented strain, ideally, should behave like the unaltered strain of Bacteria (wild type), because the missing element has been returned. But, like most things in science, it doesn’t always return the way we want it to- and it comes down to how we restore the genetic element.

In our case, we use a different kind of mobile genetic element called a plasmid. Plasmids are circular pieces of DNA that are found in Bacteria (not all, but many). Scientists can use this natural element to our advantage, and use it to artificially introduce genetic material into a cell. I’ve tried to describe plasmids previously so do check them out here, and here.

Anyway, using a plasmid, we can re-introduce cbu0265 (see the italicisation?) back into the 0265::Tn mutant. The plasmid essentially mimics Coxiella‘s genetics, and the Bacterial cell’s machinery will recognise what’s on it and make the protein.

But it’s still an artificial system, so sometimes, it might not make a lot of the protein… or maybe it’ll make tonnes. You never know until you check protein expression, i.e. how much protein has been made- using a technique called ‘western blotting‘. I’ve gone into extensive detail about this technique before, so do please check it out here.

Anyway, I got my 0265::Tn mutant, re-introduced cbu0265 via a plasmid, and thankfully- the Bacteria recognised it and made the CBU0265, the protein. In ample amounts.

So (phew!), now the stage is set. I’ve got my wild type (genetically unaltered), 0265::Tn and 0265::Tn complemented strains (0265::Tn pFLAG-0265) of Coxiella ready to go.

I then got my trusty 13C-glucose label (see part 2), and added them to these strains individually, and observed how the glucose got taken up and used…

Let’s take a moment to try and figure out what we should see- in an ideal world.

In general, we should know what the wild type (WT) strain is going to do, because I’ve shown it in part 2.

Hopefully, the complemented strain does similar things, because it should (hopefully) behave like wild type.

The mutant could behave in different ways, depending on what CBU0265 is used for.

Scenario A: CBU0265 is absolutely essential for glucose uptake, and without this protein, Coxiella can’t take up any glucose

  • We should see no 13C-labelling in this strain, because glucose transport has been blocked by the absence of CBU0265
  • This would suggest that, not only is CBU0265 a glucose transporter, but it’s the only glucose transporter that Coxiella has

Scenario B: CBU0265 is partially responsible for glucose uptake, and without this protein, Coxiella can only partially take up glucose

  • We should see a general reduction in 13C-labelling in this strain, because glucose transport has reduced
  • This would suggest that there’s another glucose transporter that Coxiella has (aside from CBU0265), that can compensate for this loss
  • Cool new protein/transporter to study

Scenario C: CBU0265 is in no way responsible for glucose uptake, and without this protein, Coxiella goes about business as usual

  • We won’t see any reduction in 13C-labelling, and the 0265::Tn strain will behave exactly like WT, because there’s been no impact on glucose transport without CBU0265
  • This would suggest that there’s another glucose transporter that is able to take up glucose
  • Cool new protein/transporter to study
  • All the scientists’ predictions were wrong

So, what did we actually see, for reals?

Drum roll, please

🥁

If you have a look at the above figure (for instructions on how to interpret this, please read part 2), you can see WT in red, 0265::Tn in light blue, and the complemented strain in dark blue.

And I hope you can appreciate that what we saw was Scenario B.

We can still see some 13C-glucose in the mutant strain- but it’s no where near as much as WT.

That means that CBU0265 is partially responsible for glucose uptake, because without it, there’s a visible reduction in glucose usage.

But because it’s not completely gone, it means that Coxiella has another way to transport glucose into itself.

The complement (dark blue) is a classic demonstration of what happens when you artificially re-introduce a gene via a plasmid. It’s using way more glucose, most likely because it’s making way more CBU0265 than what’s normally being made. Plasmids don’t get regulated in quite the same way as what’s naturally on the genome, so you can get these funky results. Still, though, at least we can see that CBU0265 is definitely back. With a vengeance. 😂

Now, it’s all good and well to look at stable isotopes nutrient levels-

But how does this translate to infection?

Does this drop in glucose usage impact the Bacteria’s ability to take over a host cell?

Let’s see!

Remember that classic Bacterial growth curve graph, with all the phases?

Well, here’s a real one.

The colours are the same as the graphs, for each strain.

And the short answer is, the growth in the mutant isn’t impaired. It behaves just like WT. 😂

Even visually, there was no difference.

All the asterisks indicate CCVs, which I described in part 1

Even in a different cell type, that more closely resembles those alveolar macrophages that Coxiella infects during normal human infection, there were no changes.

We also looked at infection in an animal!

Well… an insect…

WELL- an insect larvae…

I’ve mentioned Galleria before in all my ‘moth posts’ (see some here, here, here, and here), but they’re a very cheap and easy way of studying infection kinetics at a ‘multicellular’ (‘many-celled’) level.

In terms of Coxiella infections using WT (unaltered strains), over the course of around eleven days, the larvae begin to produce melanin (an immune response), and slowly succumb to infection.

So what did we see with our strains above?

PBS, or phosphate buffered saline, is what’s called a ‘negative control‘, where no bugs are present. The vast majority of the larvae injected with saline survived (all but one), but the ones infected with Coxiella, regardless of cbu0265 absence, died. Basically, the lack of cbu0265 (and therefore the protein, CBU0265) had no impact on Coxiella‘s ability to infect hosts.

Now, was that unexpected?

Not really- we saw that glucose uptake and usage was still occurring, even without CBU0265, and also more importantly, we don’t know how important glucose is to Coxiella during infection. Our previous data in part 2 made it look like they might prefer amino acids, which are more common inside a host lysosome.

So what was next?

Well, we knew that there was another glucose transporter out there, so we repeated this entire process, starting with investigating the genetic material.

And lo and behold! We found another potential suspect, I mean- transporter.

This next protein was called CBU0347. It had similarities to XylE, which transports a sugar called xylose into E. coli (and other Bacteria).

We had a transposon mutant for cbu0347 in our stocks, so I re-isolated it…

This gene runs backwards! You get that sometimes- it doesn’t affect the cell’s machinery in any way so the protein gets made without any issues

…made a complemented strain (which, by the way, takes a significant amount of time, as described here)…

Here’s a western blot- the dark bands mean there’s protein. There’s more explanation on how to interpret this in all my western blot posts.

…and looked at 13C-glucose usage- exactly as before.

And we have Scenario B once again!

But that’s good- because we already knew that CBU0265 could take up glucose (i.e. there was another transporter involved).

So this just confirmed that Coxiella had at least two glucose transporters on hand.

I also checked infection as before…

And it all looked pretty similar to what we saw with 0265::Tn, even with the other cell type…

And with our Galleria

But again- that was okay- because we know that Coxiella can compensate for the loss of CBU0347 by using CBU0265 instead, and vice versa.

Now, if you were me, what would be the next thing you’d want to investigate?

We’ve shown that Coxiella can use glucose, and that they take in glucose using two glucose transporters, CBU0265 and CBU0347. Mutants of each showed decreased glucose usage, but one must have compensated for the other because there was still glucose being used. Also, the loss of either didn’t seem to have any impact on infection, because the mutant behaved like WT.

Can we definitively say we’ve identified all glucose transporters?

How can we tell whether it’s just CBU0265 and CBU0347 in the mix, and not more?

Thinking back to the techniques I’ve introduced, hopefully people can appreciate that a double mutant, lacking both cbu0265 and cbu0347, would be super handy to have. Without both, if we still saw glucose usage, then we can be certain that there are other transporters at play.

And that’s precisely what one of the reviewers asked, when we submitted this to a scientific journal.

😂

The downside is, we didn’t have a double mutant. We would have to make one from scratch, and let me tell you- making a deletion mutant (i.e. deleting a gene) in Coxiella is no easy feat. I mentioned the struggles of making a complemented strain before, but even if you had the plasmid required to make the deletion already made-

FYI I have tried twice to make such a plasmid, for different genes- I gave up on the first one after five months, and the second one I abandoned after… 18 months

-even if everything runs smoothly, it takes a minimum of 100 days to make a Coxiella deletion mutant. That’s with no hiccoughs along the way.

And every scientist agrees that there are always hiccoughs along the way.

So we said no to a double mutant. I was coming up to less than 6-7 months before Thesis submission, so there was no feasible way to get this done.

But we can always speculate.

Even if you looked at the decreases for each mutant, added them together, and subtracted that value from the glucose usage seen in the WT strain, then there’s still a portion of glucose that gets taken up and used. That suggests there’s another glucose transporter at play.

But that’s assuming that CBU0265 + CBU0347 acts like 1+1=2, and not 1+1=20. I know that mathematically that’s not possible (cue a mathematician telling me it’s totally possible), but there are situations like that, and it’s described as being ‘synergistic‘. Two proteins act in synergy, and amplify each other’s functions so that’s it’s no longer additive- it’s more.

We will never know until we have a double mutant.

But even then, it’s also possible that there really is another transporter out there. Coxiella have these things called ‘ABC transporters‘ (ATP-binding cassette transporters), that are also known to transport sugars. Unfortunately they’re hard to identify specificity at the genetic level, so I can’t speculate on which proteins other might be responsible for glucose uptake.

Alrighty- so let’s try to summarise these first two parts, which are actually quite related.

Coxiella are able to take in both glutamate (an amino acid) and glucose (a sugar), and use it

And in order to take in glucose, it uses CBU0265…

And also CBU0347

Maybe there’s another transporter out there, too- but with our current information, it’s hard to determine whether that’s true, or what it might be

Having multiple glucose transporters suggests to us that glucose/sugar is actually more important as a nutrient source than we thought (based on just part 2).

What we think goes on is more like that scavenging lifestyle I alluded to. I think Coxiella are able to use whatever is in that recycling bin- which is actually quite clever, if you’re living in in that kind of a hostile environment.

Now, I’ve written about these two Aims in a previous post of mine, too (click here). I’m also very fortunate to have this published, because it adds a sense of closure to this whole section. Given that some of the data in part 2 was generated before me by someone else, all in all this whole section probably took about 5 years from start (planning and performing experiments) to finish (published). You wouldn’t know it by just looking at the end result, that’s for sure. 😅 It also got scooped (i.e. someone else published similar data first, taking novelty and value out of what we had), went to one scientific journal, got rejected, which meant we had to do more experiments to address the issues (i.e. reasons why it got rejected), submit to a different journal (because the first one didn’t want it back at all), and then go through revisions and such. It was an exhausting process, but no where near as exhausting as the last part… 👀

And that completes part 3. Thanks for reading, and stay tuned for part 4! 😊

Categories: Ph D posts

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ABugsLife

A Ph. D graduate in Microbiology, residing in Victoria, Australia. Now working part time at a secret location as a Communications and Data Officer. 👀 🦠 🧫 🧬

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