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Life in the recycling bin (part 2)

Previously, on ‘A Bug’s Life – Ph. D Blog’, I set a challenge to myself to try and summarise my Ph. D Oration- and present it to you, my readers, in a not-so-science-jargon-filled way.

If you missed the start, you should definitely head there, because you can’t really get the whole story without the introduction.

Part 2 of this series will be on the first aim of my Ph. D project. As a reminder, here’s the slide of my Ph. D Oration…

Note the beautiful CCVs in green, with all my bacteria in red

So we’re focusing on Aim 1, currently written as:

‘Metabolic comparison of axenically and intracellularly cultivated WT C. burnetii

Which in this instance means,

‘What are the metabolic differences between Coxiella (my bug) that are grown inside the host cell (i.e. during as close to natural infection as possible), and those that are grown artificially in liquid broth in a lab (i.e. no host cell)?

There were also other questions we set out to answer, that’s also part of this aim, but we’ll get to them shortly.

First, let’s take a step back at the overarching aim of my entire project, which is to basically ask, ‘how do Coxiella survive, and thrive, inside such a harsh environment?

If you recall, Coxiella resides in the host ‘lysosome’, a compartment that can only be described as the recycling bin of our cells. It’s highly acidic, it’s very degradative (things get broken down), and most living things that end up there- die.

But the great benefit of living in such a harsh environment, is that the host (i.e. us) does all of the hard work in chewing and digesting all the necessary food items for a wee bacteria to grow big and strong. Any nutrient source that gets taken in by the host cell, gets taken to the lysosome, broken down into its basic components, and then shuttled around the cell to be made into something else.

These nutrients (or metabolites) can be…

  • Carbohydrates (or sugars)
  • Proteins (broken down into amino acids)
  • And other compounds such as fats (also known as lipids)
From memory, glucose (left) and glutamate (right), a sugar and an amino acid, respectively, written as a simple chemical structure

Every living thing needs nutrients/food to grow, and the same goes for Coxiella.

But what we don’t know, is what types of foods they like to eat (‘Uptake‘).

And how that food gets broken down (‘Utilisation‘ i.e. how is it used?)

All of the above are interesting questions, especially given that Coxiella typically resides within the recycling bin of the host cell. It’s kind of like asking, ‘what would Oscar the Grouch eat?’, given he lives inside a trash can. πŸ˜‚

Oscar the Grouch puppet | Smithsonian Institution

But there’s no point just looking at one thing, when you’re doing studies like these. So we also threw in some additional questions, including:

What sort of differences would we see, between a population of Coxiella that were grown inside cells…

…and another that were grown artificially, in liquid broth, with no host cells?

And,

Would we see a difference between Coxiella that are replicating really fast (peak performance),

and those that have used up all their food sources and have figuratively passed out from exhaustion?

Which corresponds to these phases in a Bacterial growth curve/life cycle:

Log phase‘ is when Bacteria are replicating really fast. They’re undergoing what’s called exponential growth, where one Bacterial cell divides into two, then four, then eight, then… well, you get the picture-

They’re basically going gang-busters and are at their peak performance, but they’ll also be using up lots and lots of food… hopefully. You gotta eat if you wanna work hard.

Stationary phase‘ is the exact opposite of that.

Stationary phase is when they’ve used everything up- not necessarily themselves, but they’ve used up all the surrounding resources. It’s kind of like what’s happening with our planet right now. All the food’s run out, the place is crowded, and they can’t really go elsewhere to find greener pastures. πŸ˜… Once they’re placed in a fresh, food-filled environment, though, they’ll be good as gold- but for now, they’re sort of passed out. There’ll be little to no growth happening at this phase, either way, so there won’t be as much food expenditures… hopefully.

So what the hell did I do to compare all these things?

Well, I grew Coxiella, both inside cells, and outside of it (in liquid broth)…

And then I harvested them (that’s the technical term) at day 3 (log phase) and at day 6 (stationary phase) post-infection (for those that are inside cells) and post-inoculation (for those that are in liquid broth). ‘Inoculating is adding Bacteria to your culture medium, whether that be liquid broth or agar jelly.

Now this is where it gets weird.

Hopefully, everyone can refer back to their high school chemistry days, and remember the element, Carbon.

Normally, carbon is found as 12C, or carbon-12. It has 6 protons and 6 neutrons, and 6 + 6 = 12. Hence carbon-12.

But- sometimes, you can get things called isotopes, where the carbon atom has some extra neutrons. I dunno why. They just like to roll around a bit heavier than usual. Who are we to judge?

So, you can get carbon-13, carbon-14, etc.

Now, why is that relevant to my Ph. D Oration?

Well- we scientists like to use naturally occurring things in the environment, to our advantage. If we can re-jig something for our own purposes, we will do it. Why invent something entirely new when you can repurpose something else?

Enter, 13C-stable isotope labelling

As mentioned above, these isotopes are a bit heavier than your usual carbon atoms.

Imagine, for a moment… say… glucose. C6H12O6.

Don’t get bogged down in the high school PTSD too much- just remember that glucose has some carbon atoms in it.

If you replaced all of the carbon atoms in glucose, from 12C to 13C, then the glucose molecule, as a whole, will become just a smidge heavier. With all those extra neutrons.

Again, let’s not throw judgement at the glucose- we made it get heavier. It’s not its fault.

Now, you can do this with other compounds, including amino acids. Don’t ask me for the specifics on how you can do this (this isn’t a chemistry blog)- just know that you can. You can buy all kinds of metabolites that have all had their 12C replaced with 13C.

But we don’t have scales that weigh each individual glucose molecule and tell us whether it’s heavier than usual…

Or do we?

Enter, a mass spectrometer. Specifically, a gas or liquid chromatography coupled mass spectrometer (GC/MS or LC/MS).

A mass spec doesn’t necessarily weigh a compound like your average set of kitchen scales, but they can identify individual compounds that pass through a tiny, tiny column.

Let’s focus on GC/MS (because that’s what I used here- I’ve used LC/MS elsewhere).

First, the gas chromatography part makes all the compounds in your sample volatile– or turns it into a gas.

Then, once they’ve turned into a gas, they’ll be forced to pass through that narrow column in a mass spectrometer.

Based on the time that it passes through the column (certain compounds pass through at certain/specific times), as well as the ion spectra (see below) that it scatters, you can figure out exactly what compound was in your original sample.

Sound complicated? That’s because it is!!

Even I, someone who has used the GC/MS, numerous times, can’t explain to you exactly how that machine works.

And sometimes, I think that’s okay. I use WiFi all the time but I’ve got no f***ing idea how it works. Same goes for this. I put sample in this slot- technician/collaborator sets the machine up, the machine does magic, and it spits out data that I then process with some help. πŸ˜‚ MAGIC.

Either way, I can see a whole bunch of straight lines on a screen.

mass spectrum
Exhibit A. A sample ion spectra.

And the exact pattern of these lines correspond to a particular molecule. Coupled with the retention time, or the time it passed through the column, you can figure out what compound it is.

In all honesty, the general gist I’d like you to get out of it is that GC and LC/MS help us identify compounds that are present in a sample. Sometimes that can be proteins, and other times, like in my case, it’s amino acids and simple carbohydrates/sugars that are very, very small in size. In forensics shows, it’s tiny chemicals that might be on someone’s clothes. A mass spec has many different uses.

Okay, so let’s try to put this together. You’ve got your 13C-stable isotope labelled molecules. They might still be glucose, but they’re just a little bit heavier than normal glucose that has 12C instead.

Then you’ve got your GC/MS. This technique is pretty specific, and you can even detect slight changes in mass for even the same molecule (e.g. glucose), if, say, its carbon atoms were replaced with slightly heavier ones.

This means that if I add this 13C-labelled glucose to the environment, Coxiella might eat it up (if it’s capable of doing so), and we can see how it gets converted into other molecules, all the way along Coxiella‘s metabolic pathway.

Because when a nutrient source (e.g. glucose) gets taken up, it gets converted into other molecules to be used in other areas of your metabolism. Glucose gets turned into glucose 6-phosphate (G6P), this in turn gets turned into fructose 6-phosphate (F6P) with some additional steps in between. All the while, those heavier carbon molecules get incorporated into these new compounds. The core material gets passed down, so you’ll always see that heavier, 13C in whatever gets made from the 13C-glucose. That 13C also diminishes as it passes down the metabolic pathway, too, so you might start with 100% 13C, and as it gets converted into something else, it’ll go down to 90%, then 80%, then 50%, and so on. By the end of the road, it might be down to 10% 13C. Either way, it allows us to track and map how and where that glucose molecule got used up, by following the % decrease.

So here we are

In my experiments, I used 13C-glucose and 13C-glutamate. A sugar…

and an amino acid…

Both are found in the host lysosome, although sugars are in much lower amounts.

Now why might that be?

Let’s divert for a moment.

Have a think. You’re the host cell. You’ve got your sugars and your proteins. Sugars are easier to metabolise and turn into energy, and is vital to your health. Proteins take a bit more effort to use around the cell, and first need to be broken down into its basic components (amino acids).

So… with that in mind- would you leave your precious sugar in the recycling bin (lysosome), or use it up before you chuck it in the trash?

And where would you leave your proteins? The stuff that needs to be broken down and recycled before it’s used?

With this in mind, what nutrient source (sugar vs amino acids) might Coxiella prefer to eat, given where it usually lives?

πŸ‘€

So I’ve harvested the Bacteria, again at day 3 and day 6 (remember why I picked those dates?), and then I add my labelled substrates to the Bacteria, and wait 10 minutes for these compounds to get gobbled up by the Bacteria (i.e. taken into the Bacterial cell to be used). 10 minutes is plenty of time for Bacteria to take up and start using both glucose and glutamate.

Now, the best way to preserve biological material is to chill it. If you want to stop your food from going off- you chuck it in the fridge.

The same goes for Bacteria. They won’t die at cold temperatures, but they’ll slow down their metabolism and sit in stasis.

I want to preserve my labelled Bacterial samples, so that I can extract all the nutrient samples/metabolites and analyse them on the GC/MS.

But- unfortunately, if I just chuck them in the fridge, or freezer, the Bacteria will undergo extra metabolism to help them adapt to the colder climate, and that might skew the results of my experiment.

Whatever I see might not be how the Bacteria use up glucose and glutamate normally- it might be the way they behave when you suddenly chuck them in Antarctica and tell them to get on with it.

That might be all good and well, but I want to see what they’re doing when they’re behaving normally. When they infect us, and when we’re a healthy ~37 degrees Celsius.

So how do I address this temperature issue?

By ‘quenching‘ their metabolism.

Essentially, I chill them so fast that the Bacteria don’t know what’s hit them. They freeze (not literally, but figuratively), and I can take a snapshot of their metabolism at the time of sampling, when they were still happy and not knowing what was about to happen to them.

There’s nothing glamorous about this technique. All you do is get a beaker of 100% ethanol and some dry ice- not so cold that it snap freezes, but cold enough that if you stir your tube of 13C-labelled bacteria hard, and dip it in this ethanol mixture, the Bacterial broth quickly drops temperature to just above 0 degrees Celsius.

You don’t want to freeze the Bacteria at this point, because freezing risks the cells rupturing- releasing the contents of the cell, including all the nutrients/metabolites it contained. I’d detect less material, simply because I broke open my ‘sample bag’ (i.e. Bacterial cell) too soon.

So it’s a fine art of dropping the temperature quickly, but stopping before it hits 0/freezing point.

The actual technique was like beating an egg mixture to make an omelette. That’s certainly what it sounded like.

*clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink*

*pause, check thermometer that I’m using to stir Bacterial culture*

*clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink*

*pause, check thermometer*

*clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink clink*

*Swear loudly because you can see ice shards and you’ve got it too cold*

*Repeat for the other 11 samples*

It’s also really important to keep your samples cold from this point onwards, so that you can preserve them at their best quality. While I’m working with them, I usually sat them in an esky of ice. If I was centrifuging (spinning) my samples down, then I would set the temperature at around -2 degrees. Again, much like you would with food- except this ‘food’ would go off in a few minutes if you kept it warm for long!

So we’ve quenched the metabolism of the Bacteria at that particular point in time, 10 minutes post-labelling.

We then move onto extracting the metabolites. That involved the process of rupturing the Bacterial cells with repeat exposure to dry ice-ethanol (like what we used for quenching, at around -80 degrees), then liquid nitrogen (-200 degrees), ten times. The huge difference in temperature will cause the cell walls to rupture, releasing the contents of said cell.

To me, this process sounded like I was deep frying something. My friend who taught me all of these techniques would agree, and we would sit there in the lab with our tummies grumbling, because it was likely to be mid afternoon and we wouldn’t have had lunch yet. Unfortunately we couldn’t really eat what we were preparing!

Anyway, after releasing the contents of the cells (by this time the Bacteria are well and truly dead), it’s a case of centrifuging, or spinning out the debris (the husk of the dead Bacteria πŸ˜‚), adjusting the chemical solution inside the tube to isolate all your metabolites, and then preparing the sample for GC/MS analysis.

Now, I won’t go into detail about how to analyse stuff on the GC/MS, because it’s very boring, and not at all important to the story. It’s basically me staring at a computer screen, checking that the algorithm on the program has picked out the right compounds, while watching Netflix– I mean- working hard? What?

So, if you’ve never seen a metabolic pathway map- this will be extremely overwhelming to you.

Metabolic pathways are like… this train map.

There’s a lot of s*** going on, and you can’t really keep track of where anything is.

So I’ve tried to simplify this as best as possible, and condensed it down to what’s important.

Cue flashbacks to high school Biology.

Here’s a pathway map. It’s showing two major metabolic pathways. Glycolysis, and the TCA cycle. They’re named slightly differently in humans, but the TCA cycle is essentially the Krebs Cycle in humans.

Either way. Both produce energy for the cell. Think of this as the engine.

Anything that has a graph in the box are metabolites that I could detect from my samples, on the GC/MS. The bars in orange are Coxiella that were grown in broth, and the bars in yellow are Coxiella that were grown inside cells. There are two sets of these orange or yellow bars, one for day 3 (log phase), and another for day 6 (stationary phase).

The y axis (the vertical scale) shows the % labelling- in other words, what percent of all carbon atoms in that compound (whether it be L-Glutamate, Succinate, etc), had 13C, instead of 12C. It basically corresponds to, ‘where did that 13C-substrate go?’

The above graph is for 13C-glutamate labelling experiments. You can see L-Glutamate down here…

The simplest way to interpret this is to find your starter substrate (13C-glutamate)…

And then follow the direction of the arrows on the map, showing how this substrate gets converted into other compounds, using special metabolic enzymes. In this case, we can see that L-Glutamate gets converted into 2-Oxoglutarate, then it can either turn into citrate/isocitrate, or succinyl-CoA.

So how do we tell which way the substrate went?

Well, we need to look at the amount of 13C-label, which should be decreasing as the carbon substrate moves along the pathway. For instance, for the liquid broth Coxiella in orange, you can either go from L-Glutamate to citrate/isocitrate, which has around 30% label incorporation, or succinate, which has around 80%.

If you look at the next metabolite along the chain, after citrate/isocitrate, it looks like the Bacteria bypass the TCA cycle and convert the product into L-alanine (L-Ala), or… in the more likely scenario, the 13C-substrate gets converted from L-Glutamate to succinate, then fumarate, malate, then perhaps to citrate/isocitrate, at which point it enters the glycolytic pathway (glycolysis).

So why is this observation important?

Well, if the direction of the labelling indicated that Coxiella just used L-Glutamate to go around and around the TCA cycle (so, no graphs in the section labelled, ‘Glycolysis’), then it would mean that Coxiella just relies on L-Glutamate to make energy using just that one section of the metabolic pathway.

But if it can actually shunt L-Glutamate/an amino acid into glycolysis, then it means that Coxiella has the ability to convert amino acids into sugars and sugar phosphates. We can do that, but we didn’t know Coxiella could, too. Especially even those that were harvested from human cells.

This could be especially useful for a Bacterial species that lives in the host recycling bin- notoriously low on sugars. If it can make its own stock, then that’s less reliance on the host. Coxiella is an independent Bug that don’t need no host-

Actually, they absolutely need their host. They can’t replicate outside of a cell unless we put them in very special broth.

But I digress-

Sugars and sugar phosphates can be stocked up to make energy later, or be converted into other molecules that help the Bacteria replicate (like its genetic material). There’s no shortage of uses for sugar.

On top of that, by not sticking to rounds and rounds of the TCA cycle, it might help Coxiella keep its cool. The downside of the TCA cycle is that it builds up a lot of toxic material for a cell. This toxic material (reactive oxygen species, or ROS), is actually found in the host lysosome, and it’s generally not good news for living things, including our own cells. By reducing the amount of ROS that gets built up (by not using the TCA cycle so much), it might help the Bacteria survive for longer.

And you can see that, quite clearly, with the graphs above. Compared to the orange bars (liquid broth Bacteria), the Bacteria grown inside cells (in yellow) seem to be avoiding doing a loop around the TCA cycle. The liquid broth environment has no where near the same level of hostilities that a host lysosome has, so it kind of makes sense that the Bacteria don’t care that toxic substances might build up inside itself. It doesn’t have to worry about its outside environment trying to nuke it all the time.

A little bit of poison building up doesn’t matter, if you’re not already drowning in poison. πŸ˜‚ You’d want to keep the internal stresses to a minimum if the world around you was on fire.

Another interesting thing to note from that is how the log phase (day 3) Bacteria are more likely to have more label incorporation than the stationary phase (day 6) Bacteria…

…unless they’re grown inside cells.

Note the graph below is a little bit different to the above. Log phase (day 3) is now in yellow, and stationary phase (day 6) is in orange. ‘AX’ are liquid broth Bacteria, and ‘IC’ are Bacteria grown in host cells.

Now, why might the broth ones do less at day 6, compared to the ones grown inside cells?

One is grown in liquid broth.

The nutrients are finite. Once it’s spent, it’s spent. We don’t replenish it for them. The volume of the liquid doesn’t increase, so there’s only a finite amount of space for them to grow, too. Population density matters.

But what about the ones inside a host cell?

The host cell will keep on replenishing its nutrient stores on its own. A constant buffet for the Bacteria.

Once the Bacteria grow to a point that the cell can no longer cater to them, space wise, all they have to do is exit that cell and infect another one. Sure, they’ll eventually reach a point where all host cells are packed to the brim, but… it takes a bit more time for this to happen.

So this is why, even at day 6, the Coxiella that are grown in cells are able to still be more metabolically active. But only if they’re inside host cells that keep providing them with all the creature comforts.

Another funky thing we saw was labelling into lactate, which many might know as lactic acid.

We didn’t think Coxiella could make lactate, based on its genetic material telling us what types of enzymes it had in its arsenal. You can do that, you know? You can look at the genetics of an organism and predict what types of proteins (some of which are enzymes) it might be able to produce. It gives us scientists a guesstimate of what an organism might eat.

Anyway, Coxiella didn’t look like it could make lactate, but apparently it can make some… albeit only a little bit.

We still don’t really know why it makes lactate, and we’re not 100% sure how it makes it. Most experiments don’t really give you a conclusive answer- only a billion other questions to ask instead. Maybe it’s used for storing excess carbon (a snack to be eaten later)? Who knows. If anyone knows, please contact me!

So, as mentioned above, you can predict what types of enzymes a Bacteria might be able to make, by studying its genetic material. But this, ‘predictions based on genetic material’ trick had some Coxiella scientists confused, about another aspect of its metabolism.

Glucose usage.

Coxiella looked like it could use glucose, but based on the genetic material, it looked like it was missing the first enzyme needed to convert glucose into glucose 6-phosphate (G6P). So for a while there, no one was really sure that Coxiella could even use glucose and undergo glycolysis. A lot of people just assumed they would convert amino acids into metabolites within glycolysis (so moving up the pathway), much like what we saw in the glutamate graphs.

Well- turns out- they can actually move down the pathway, too.

There’s 13C-glucose up the top, getting taken into the Bacterial cell by some sort of transporter (in purple, on the diagram)- kind of like a mouth that gobbles very specific nutrients.

Then, the glucose gets converted (quite readily) into G6P.

So yeah, now we have a mystery enzyme on our hands.

No idea what it might be, though. We can’t find anything in Coxiella that resembles one of these enzymes, so whatever it is- it might be very unique, or not similar to what other organisms use to convert glucose into G6P. Kinda cool, if you think about it. Why is it different, I wonder?

Anyway, when it comes to comparing the two growth conditions, it looks like the liquid broth (dark blue) uses glucose more readily than the Coxiella grown inside cells (lighter blue)…

…because there’s more label incorporation for the dark blue bars, when compared to the light blue, overall.

Why might this be?

Well, apparently the liquid broth contains about 5000x more glucose than what’s found in a host lysosome. Slightly concerning if you think about it. Just a room full of sugar when you’re usually used to a… single bag. πŸ˜…

So, you can imagine, then, that a population used to being exposed to a metaphorical tonne of sugar, might be more used to using it very readily. They don’t need a brief adjustment period to get used to the initial shock of a sugar hit. This is basically what we’re seeing.

Day 3 vs Day 6 was quite similar to what we saw for the 13C-glutamate, where the log phase bacteria were more metabolically active overall. Replicating like crazy until they run out of food and space in the broth.

The Bacteria grown inside cells were more likely to show variability, again, likely because they could just jump ship and infect new host cells, that would, in turn, keep providing more nutrients.

So, what did I find out, as I tried to address my first Aim?

Well… I found out that Coxiella, especially ones that are grown inside cells, like to convert their amino acids into sugars. This might help them while they reside in an environment that typically doesn’t have a lot of sugar around, but has a lot of amino acids, instead. Coxiella like their proteins, a lot.

But that doesn’t mean they can’t take up sugar- especially if they’re exposed to a lot of it. They’re quite happy to use both amino acids and sugar as a nutrient source, depending on what’s more readily available. A scavenging lifestyle, as I’d like to call it. πŸ˜‚

Coxiella seem to use their metabolic pathways quite well, in that they can prevent toxic materials from building up within itself…

…and they can do so, using enzymes and mechanisms that are unique to them.

Bacteria that are replicating and growing rapidly tend to use up more nutrients, and are more metabolically active…

…and all of the above were either first seen by me and my team, or by a handful of other people (who looked at liquid broth grown Bacteria), first. Nobody else in the world would have seen the data, so looking at it all on your laptop screen for the first time ever- it’s kinda cool. Like you figured out a secret.

FYI- There’s other data that’s in my Thesis, that I didn’t include in my Oration.

You can take a simple snapshot of all the nutrients present in your sample, without any 13C-label at all. It’s all the same process, just without any percentages- just a general, ‘I detected this compound’ type thing. It basically showed very similar profiles to what you saw with the labelling experiments, which, in a way, is good. Consistency is key when you’re doing scientific experiments, so it’s good that I saw somewhat similar metabolites in both the unlabelled and labelled experiments.

But that concludes the results of the first Aim! Congratulations for making it this far, and stay tuned for part 3, or Aim 2. πŸ˜ŠπŸ˜‰

Categories: Ph D posts

Tagged as:

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. πŸ‘€ 🦠 🧫 🧬

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