79. A Précis on Penicillin Binding Proteins

Presenting: More on Penicillin-Binding-Proteins than you realistically ever need to know! 

• What is the difference between Gram positive and negative bacteria's cell walls?
• What is a Penicillin Binding Protein (PBP)?
• Why are they called PBPs?
• What do PBPs do?
• What makes MRSA resistant to (most) beta-lactams?

These any many more questions answered!

Notion page here - you'll need it for this one, check it out! 

Transcript

Callum: 0:20

Nah,

Jame: 0:23

I

Callum: 0:23

you're fine. It's fine. Everything is fine.

Jame: 0:27

fell down a rabbit hole.

Callum: 0:29

Oh, one of those.

Jame: 0:32

And like the dwarves in Moria, I went too deep.

Callum: 0:36

You delved too deep and too greedily.

Jame: 0:40

Hmm. Too great that Yeah, in my thirst to acquire knowledge.

Callum: 0:44

Jame, all we have to decide is what to do with the time that's given to us.

Jame: 0:50

Well, let's take the next 20 to 25 minutes depending on how quickly I can say all this to get all this stuff out of my head. We're talking about PBPs today.

Callum: 0:59

That's penicillin binding proteins.

Jame: 1:01

right, we don't need to explain everything Callum.

Callum: 1:02

What happened? How did it come to this?

Jame: 1:05

I came across a paper.

Callum: 1:07

Well, look, before that.

Jame: 1:11

So I was, yeah how did this come about? So you know who I really blame, Callum, is Ollie Bannister. Ollie Bannister, our guest on our Prepping to Pass Part 2 episode

Callum: 1:24

Episode 40.

Jame: 1:25

We were chatting and he was like, you know, what I really want is for somebody to explain PBPs to me in like proper amounts of detail, like which bugs have which PVPs and which ones are useful and why, which be the latter times target, which ones. And then that's that idea has lived rent free in my head for the past year and a half. And eventually, just late one night, the damp burst and I just started Googling.

Callum: 1:51

I texted you the other day and I said, did you have any papers? Because I was at work and someone was asking about, there was some weird strep resistance pattern. And we were talking about like penicillin binding proteins and how it mediates resistance. And then someone asked me a couple of questions. I was like, I don't know the answer to these questions. And this annoys me. And usually what I do when I don't know the answer to a question, it's annoying me. Like I did today at work. is I text Jane when I says, have you got any, have you got any good papers about this? So I did that. So maybe, maybe I sort of, that was about two weeks ago. And shortly afterwards, you're like, I've had an idea for a podcast episode. And I was like, Oh, what have I done?

Jame: 2:32

Well, that's what triggered it this time because I've been you know, I thinking about doing research on it for ages for the past couple of years, really. And then that question. I think it was something about Entercockle resistance to aminopenicillins and that's done through PVP modification. And it's possible to overcome that by, if you're, if you've got a urine enterococcus by dosing aamoxicillin because it concentrates into the urine to the point where, you know, it's a, it's a dose response relationship and at that point the the resistance can be overcome. But yes, and, and I suppose it was also in my mind because I had recently been explaining beta lactam resistance to the general medicine SHOs that I, that I work with in my day job. And the way that I explained it was that, you know, gram negatives have beta lactamases and gram positives thinking mostly about Staph Aureus. Here I do PBP modification, but of course that's a gross oversimplification of it. But certainly the other big mechanism of Beal lactam resistance is modification of PPPs. And we've already done an episode on Beales, in Beales and inhibitors. And so this is sort of like the follow up to that.

Callum: 3:50

So that's why we've come to this. And I guess why we think it might be important to spend a little bit of time talking about it or listening to us talk about it is that. To understand and best use antibiotics to treat infections, it's worth understanding how resistance works. Because if you understand how something works, you can maybe better get around resistance or, or manage patients with resistance.

Jame: 4:16

Yeah. Although, to be honest, there's a lot of here that I didn't know before CCTing. Maybe that says more about me than it does about this knowledge and how useful it is. But there we go. But Callum, what is the cell wall?

Callum: 4:31

Well, cell wall how far back do you want to go? So let's go all the way back and we say we've got procars, new carats. So procars are single cellular organisms that don't have intracellular organelles, and they have a cell wall. So basically water moves on a concentration dependent curve and you get osmosis and water wants to move into the cell. And if it did that and there was no cell wall, the cell would just lies and bursts. But with the cell wall there, it holds the cell and stops it from bursting. Whereas eukaryotes rely on sequestering waters into vacuoles, which are small like pockets of sort of cytoplasm of the, the sort of cell membrane, which they then pummel the water and get all the other things out and then just shove it out the out of the cell. And that's called exocytosis, osmotic status. So that's a very basic overview, and so the cell wall we talk about this a lot in microbiology anyway maybe not directly, but we talk a lot about, and I'm sure everybody who's listening has heard about gram positive and gram negative.

Jame: 5:44

Yeah. And we've got a picture in there. In the prep notes, yeah. Denoting the differences in structure between the gram-positive and Gram-negative cell wall.

Callum: 5:53

That's quite a complicated discussion in itself, but on a very basic level, both of them have a cell wall, the gram positive bacteria have this big thick cell wall, which is primarily, although not entirely made up of something called peptidoglycan. And because it's so big, and it doesn't have an outer cell membrane, when you stain it, it takes up the stain readily and then the stain sticks into it.

Jame: 6:19

And it's about 90% peptic glycan, the cell wall of the gram positive, whereas in the gram-negative, it's about 10% peptic glycans. That's the difference between the two and the gram-negative one's much more complex.

Callum: 6:32

yeah, Gram Negatives have got an outer cell membrane, they've got this thin cell wall, so it's harder for stains to stick into it, and they wash off more easily. So when you're doing your staining, your sort of darker stain comes off on the Gram Negatives, that's, so basically Gram Positive and Gram Negative in a, in a very, very brief description.

Jame: 6:52

That's interesting to know and there's a little bit more information in the prep notes about the gram negative cell wall structure that was that's like a side rabbit hole to the rabbit hole that I was already falling down Callum.

Callum: 7:03

This is a rabbit

Jame: 7:04

but now I want to talk a little bit about peptidoglycan. So like I said, it's about 90 percent of the gram positive cell wall. The gram negative is, it's about 10%. And there are two components to it. Nam and nag. And NAM is N acetylmuramic acid and NAG is N acetylglucosamine. Do you remember any of this? No, good, because I am pretty sure I never memorized this. And as the greater microbiologist of the two of us, Callum, I'm always comparing myself to you, as you well know. So these are a couple of, you know, PTO do glycans components and the alternate. So, to a the chamber will go nam nag, nam nag, nam nag, nam nag, nam nag, et cetera, et cetera. And the interesting thing about Nam is it's got a paint, a peptide chain. And NAG doesn't. And this pentapeptide chain is the thing that gets crosslinked between individual peptidoglycan chains. So they're laid down alternately by a process called transglycosylation. And how they are crosslinked will provide the bacterium with its shape and structure. So if it's circular, it'll be a cocci, if it's a rod, it'll be a bacilli, etc, etc. So it's the manner of the crosslinking that that happens.

Callum: 8:22

Slow down a second. So, cause I can just get my head around all this. And I'm sure listeners are as well because it's even harder when you're just listening. So, There's two different components, so we always talk about peptidoglycan, and there's two components of it, NAM and NAG, and you have this chain of peptidoglycan, and in that you've got a NAG and then a NAM, a NAG and then a NAM, and the NAM has got a thing called a pentapeptide chain, so that's five peptide chain, pentapeptide, and that's sort of five amino acids that are hanging off it. in a little linked

Jame: 9:00

and we'll, we'll talk about which ones they are in a sec, because that's important too.

Callum: 9:03

And and they sort of dangle down and then when you build your pepsi glycan up, you have these like long lines, imagine them going vertically, long lines of your namnags that are all parallel to each other and then you cross link them, the chains, and that's what forms a sort of rigid structure. So that's when we say cross linking. Okay, know I've just said the same thing again but I think it's worth repeating because it's a bit complicated.

Jame: 9:30

fair enough. And then there's the second stage, after you've laid down the Peptiglycan chains, is the cross linking, which is done by something called transpeptidation. And this is what PBPs do. So the pentapeptide chain is five amino acids in the following order. Alanine, glutamine, lysine, alanine, and alanine. Okay? But The glutamine and the last two alanines are dextroform. So if you remember your basics of chemistry, molecules tend to come in either a left handed or a right handed side, just like the basic outline of your hands is the same, but you've got a left side and a right hand side. So this is a, a feature of three dimensional structures. And so we call these D for dextro or right and L for levo or left hand form. so quite a lot of molecules will be racemic mixtures, which is a mixture of the two things. So, for example, Omeprazole. is a racemic mixture of D and L form, whereas Esomeprazole is only the L form, I think, because the D form is the bit that has slightly worse side effects. And so they when they were making Esomeprazole, they just made sure that it was all the, the right, the right shape of a Meprazole. a me too drug, it's a me again drug. The same is true of amino acids. So in nature, almost all amino acids are L form with two exceptions. One is the glutamine in this chain. And the other is the alanines that are at the end of this chain. And this is the only time that they're found of nature. And it is a feature not just of prokaryote, but only of bacteria. So arcanes don't have this. Eukaryotes don't have this. Fungi don't have this. Viruses don't have this. This is the only time that they appear in nature. Isn't that cool?

Callum: 11:33

That's very cool.

Jame: 11:34

And so they're very unique and they have to be, manufactured there. And that means this pentapeptide chain, very unusual and the remainder, the other two amino acids are L formed, so the first alanine and the lysine, they're levo formed. And the cross linkage hands between these two, and in particular, in the gram negative cell wall, a peptide bond will be formed between the L lysine residue of one pentapeptide chain, and the D alanine of another one chain. Thank you. So the last D alanine. And then for the gram positive cell wall, an interpeptide bridge of five glycine residues will link the L lysine and the D alanine of adjacent chains. So, gram positive and gram negatives cross link in different ways. And so the example that I've got here is from Staph aureus, and the gram negative example is in E. coli. So you know the the two common ones. But the end result is the same, the peptidoglycan chains are cross linked.

Callum: 12:36

So, so in the gram negatives, that pentapeptide chain, they just, both of them sort of flop up and link together. Whereas in the gram positives, instead of them directly linking, you've then got five glycines linking between the pentapeptides.

Jame: 12:50

Yes, that's right.

Callum: 12:52

Right.

Jame: 12:53

not entirely sure why there's a difference between gram positive and gram negative, but there we go. And so that process is called transpeptidation, and it's done by PBPs. And so that's how you get to PBPs, but why, why were they called that in the in the first place, Callum?

Callum: 13:13

Because they are proteins that bind penicillin. I'm being a bit facetious. I know the answer is more complicated than that. But basically, so penicillin is discovered and then there's a lot of research about, what exactly is penicillin. And the penicillin binding proteins are found. in the sort of periplasmic space. So that's the bit between the cell membrane and the cell wall. And that's where they're doing the work. So that's where they're doing the cross linking. So it's not surprising that are found there. And when you radiolabel penicillin to find out what it does, you find that it binds to these proteins. So before they really understood what they did, you get the radiolabeled penicillin and then you find, Oh, look, it's binding to these proteins. Let's call them penicillin binding proteins. Because it really, it would probably make more sense to call it, you know, that's not their purpose. Their purpose isn't to bind penicillins. Their purpose is to cross link the peptidoglycans. So, if you'd, if you'd named them, if you were going back and naming them again, you might say, actually, let's name these, you know peptidoglycan cross linking protein. That would make more sense.

Jame: 14:30

Well, you would call them a transpeptidase or a subset of a sulcerin transferases,

Callum: 14:35

Yeah, yeah, there we go.

Jame: 14:36

went back and did after they found out what they were doing.

Callum: 14:39

Exactly. Yeah. So if we go back to when it was discovered when we do western blot, so that's a way of looking at proteins The types that were first identified in 1975 using WesternBot listed in descending molecular weight, so that's how the numbers came about, were penicillin binding proteins or PBP1A1B23456. That makes sense, doesn't it? Name them in numbers. And later on, there were more PVPs identified, so 7, 8, and then DACD, AMPC, or AMP C, we've talked about that before, let's come back to that AMP H, and PVP 1C. And so, so there are, there's a huge range of penicillin binding proteins, and there's a way to classify them, but different bacteria have different penicillin binding proteins, and that can be altered leading to resistance.

Jame: 15:36

Let's talk about the classification first. So the, the acylserine transferases, of which the PPPs are, one kind. They're divided into class A, B, and C. Class A and B are high molecular weight PPPs. And class C is low molecular weight. And there's about In class A, there's about 430 different PVPs and in class B there's about 350 and you might think what's the difference if they're, they're both high molecular weight, these two, what's the difference between them? Class A ones can do the transpeptidase reaction, but also the transglycosylase reactions. They're responsible for the elongation of peptidoglycan. This is an enzymatic function done by a different part of the, of the enzyme, which PBPs don't interfere with. They only interfere with transpeptidase crosslinking. Whereas the class B's do transpeptidase only, they don't do any transglycosylation. And then the low molecular weight class C, of which there are about 250, do a bunch of different enzymes. So their enzymatic functions, their DD carboxypeptidase or DD endopeptidase activity, which And I'm just reading directly from the papers here, play a major role in the control of cell diameter and correct septum formation. So when the cell is dividing, it forms a septum, which will then eventually divide. So they sort of play about with that kind of thing. So PVPs are not just doing the cross linking willy nilly. They're doing it in specific areas. And in particular, for the for the class A's in terms of as a beta lactam target the transpeptidase reaction is the target for penicillin and other beta lactam the structure of the D ala D ala bit at the end of the pentapeptide chain. And after cleavage, the beta lactam ring continues to occupy this active site, serine residue of PBPs, and thereby inhibiting them. So they're, they're kind of suicide inhibitors, the same as the beta lactamases. Okay. The first generations like clavulanic acid. They'll, you know, each penicillin molecule will go in and will kill an individual PBP enzyme. And then in the The other thing to say about the high molecular weights of the class A and class B's is that that's PBP 1, 2, and 3 are high molecular weight. Whereas PBPs, you know, 4, 5, 6, DACD, AMP C, AMP H, they're all low molecular weight, class Cs. And there's another class of course, which we've not talked about before now, but it's the beta lactamases. So the beta lactamases are also acylserine transferases, they just happen to have no activity, PbP activity. So they don't do the transpeptidase function, instead their target is beta lactams. And so they, they react with beta lactams but not with the D ala D ala dipeptide at the end of the peptide chain. And they hydrolyze the beta lactam ring to release the active enzyme and an inactive compound. They are not suicide inhibitors. Got it?

Callum: 18:49

Yeah so, high molecular weight and low molecular weight. High molecular weight is class A and B. Low molecular weight is class C.

Jame: 19:00

Yep.

Callum: 19:00

In class A, the enzymes do transpeptidase, which is cross linking, and they also do trans Transglucosylase, which is elongation of peptidoglycan, whereas class B do transpeptidase only. They have different binding. But I guess, why, why are we, why are we mentioning the classes in the classification? Because that's a bit complicated. So, I guess, essentially if you're going back to the organisms, we know that Each bacterial cell, they must have at least one class A and one class B penicillin binding protein at any one time for viability.

Jame: 19:35

And in fact Strep Pneumo needs two Class Bs and one Class A in order to remain viable but the minimum is one of each so yeah.

Callum: 19:45

So we're now going to go and move on to talk a little bit about which organism use which penicillin binding proteins, but it's worth bearing in mind that there are some minimum requirements for the bacteria to have the penicillin binding proteins in order to survive. So they can't just go willy nilly changing them up and mixing it up so that they become resistant.

Jame: 20:02

Yeah.

Callum: 20:03

There, there is, there's some limitations on what, what they, they can do and still survive. Because I guess with lots of, of bacterial adaptation to antibiotic resistance, it sometimes comes with a fitness cost.

Jame: 20:17

A fitness cost, yeah. Yeah we'll talk about a couple of examples here I think. Let's do E. coli first. So in terms of its class A, and I have to say before I go on this table that we're about to look at, it's taken from the the paper that, that Kicked me in the backside and made me fall down the rabbit hole in the first place. It's a Sahari and moon 2014. It's a paper that I found in a very sort of niche journal called the international journal of scientific and engineering research from 2014. It's an open access journal. It's not very big. Obviously, Callum barely has an impact factor. I couldn't find the doi for this paper. So I've just put the reference in. For people to find. The, the paper sort of smacks very much of a first year PhD let's do a review on, on the, the thing that your PhD is going to be in. It's, it's kind of got all the hallmarks of that. And consequently, it's really well detailed and I think quite well researched. And I really enjoyed reading it. So this table is taken from there, but e coli has in Class A, it's got PPP one A, one B, and one C. And in class B it's got PPP two and three. So it's got sort of multiple redundancies. And so if you've got a a beta LATAM targeting, you know, one, it might be able to, to kinda switch to the other contrast that if you like, with NIO gonorrhea, which has PPP one and PPP two. And that's it. And so there's less redundancy, and so it does vary by species a little bit. For Staph aureus, their class A that they've got, they've only got one, and it's PBP2. And then for their class B, they've got PBP1 and PBP3. Because remember, PPPs one, two, and three can be either class A or class B. But interestingly, you guys have probably all heard about PPP two A. PPP two A is a class B enzyme, but it can take over the transpeptidase function for PPP two. So. PVP2A is the thing that MRSA acquires. It doesn't lose PVP2, it acquires PVP2A as well as the PPP. So PVP2 would still be doing the transglycosylation function, but maybe its transpeptidase function would be impaired by flucloxacillin, kefoxitin, you know, keflexin, kefazolin, whatever you want to try and use it at. Well The MRSA will get around that by using PPP2A for the transpeptidase function. And just to sort of hammer the point home, we don't have a drug which targets the transglycosylation function of PPPs. If we did, I think that would be a very powerful antibiotic indeed, but we don't have that right now.

Callum: 23:23

Hmm,

Jame: 23:25

The other example that I've got here is is PPP five for Enterococcus. Feum, which is not in the table. But it also uses PPP five in a similar way to staph using PPP two A Now, remind me of the numbers Calum. 90 percent of Fecalis is sensitive to Amox and 10 percent of faecium is sensitive to Amox?

Callum: 23:50

Yeah, I used to always think faecium was resistant, Fecalis is sensitive, I S at the end, that's how I remembered it, but it's not always that, and yeah, some, rarely faecium, Enterococcus Fecum can be sensitive to amoxicillin. You don't see it that often, but it does happen, so it's still worth testing.

Jame: 24:06

So those ones, the the Aamoxicillin Sens faeciums will not have PbP5. That's the issue there.

Callum: 24:14

So what are some other, so there's, there's lots of examples, and in the show notes Jame has put together, there are quite extensive notes for a series of bacteria about which penicillin biting proteins that they have, and where they can become resistant. So what other kind of things that you think might be interesting for listeners who have you know, come here for top penicillin binding protein knowledge and little facts. And I guess we're trying to sell why this is, is, is relevant and useful. Cause I think we do talk about it quite a lot when we're talking about antibiotics, but it's a bit niche maybe.

Jame: 24:47

Yeah. Well, I, yes, that, that's true. And I think that I think I just want to mention a couple of other things. And I think one would be strep pneumoniae, PBP modification. And the other would be pseudomonas.

Callum: 25:03

Okay. That sounds great. I like both of

Jame: 25:05

we can call it A. And then I So, Strep pneumoniae has What's it? It's got six PBPs normally. So it's got three Class As, one A, one B, and two A. Two Class Bs, two X and two B. And one Class C, which is PBP3. Which is not all that relevant. And when it is resistance, it it can happen as a point mutation, or it can acquire the resistant gene from other alpha hemolytic streps. So strep pneumonia is an alpha hemolytic strep, you know, similar to your mitis and your mutans and things like that. It just happens to like the upper respiratory tract and occasionally the lower respiratory tract. And it can get point mutations in either 1A, 2X, or 2B. And so if it was 2B, that might render it resistant to penicillin, but sensitive to other betal atoms. Which is why, if you've got a Pen R organism, you can get away with using Keflasporin sometimes, depending on what the MIC to penicillin is. If it's 2X, then it will get low level Keflasporin resistance, but it will still be sensitive to penicillins. And so you sometimes see that pattern. And when people do see that, people are always a bit kind of twitchy about, like, can I trust the penicillin if it's keflasporin resistant? And the, you know, I think that's because when people think about resistance, people think about beta lactamase resistance and gram negatives

Callum: 26:31

Which usually is that you start with a couple and yeah, yeah.

Jame: 26:37

Yeah, and their only example of PBP modification is MRSA. And if you've got, you know, MRSA because of PBP2A, all beta latterms are out with the exception of Keftaroline and Keftobiprol now. So people think of PBP modification as like an all or nothing thing. And, wiping out an entire class, I think, because of that. Or they will. In, think of it as a ladder because of the of, of Beal Lamas, but that's not necessarily the case. And the the other me modification is that you can get PPP one modification of strep pum and that will give you a pen MIC over one. So it will be considered resistant there, and it will increase third generation careful sporin, MIC to more than 0.5. And forgive me, Cal, I can't remember what the MIC is for. The SR breakpoint is for strep pneumo and keftriaxone, but it certainly is getting towards resistant if it's not there already.

Callum: 27:39

Yeah, 0. 5 milligrams per liter is the, is the cutoff if you're using Keftraxone for meningitis. And for other indications, it's the cutoff between sensitive and sensitive at increased exposure or increased dose. So what we might used to have called intermediate.

Jame: 27:55

Yeah. Yeah. So I guess if it was over 0. 5, you would not be wanting to use it for meningitis. That might make you. Go and use something else. Yeah. Now, I suppose the other one that I would want to we could talk about E. coli as well So they've got three classes, one A, one B, and one C, and they've got PBPs two and three. And they've got seven other class C's of, that we're not going to talk about. And they've got two methods of resistance. One is a PbP3 point mutation. And if they get that, then they will become resistant against all beta latams except carbapenems. So you sometimes see that kind of pattern and you would be forgiven for thinking actually that it was a beta latamase,

Callum: 28:38

So yeah, I think if I saw that normally, and it was resistance against all non carbapenems, I would be thinking this is some sort of ESBL you know, and neoporin loss or something. I don't think I would ever have thought that it was a penicillin binding protein free point mutation. How common is that?

Jame: 28:57

Me neither, and well it's not very common in gram nex, it's much more common in gram pos as a resistance mechanism, but, with increasing beta lactam resistance we will see this more and more, I feel. And then the other thing that they can do is they can just hyper produce PBP2. And that will lead to a sort of moderately increased MIC to to penicillins. And then the last thing we got to talk about Calum is

Callum: 29:26

do people want more Pseudomonas content?

Jame: 29:29

I know, Well, I mean, whether or not they want it, it's coming back. I mean, you can always stop listening, loyal listeners. In which case you will be flipped to non loyal listeners in my heart. But anyway, Pseudomonas has eight PBPs. The class A's are 1A the class B's are 2 and 3. But oh, there's another one called 3X. But the important thing is that PBP3 is essential for Pseudomonas growth. You cannot get around it, and for this reason, PBP3 is the main target of anti pseudomonal beta lactams, and that is why Keftazidime targets Pseudomonas, but Keftriaxone does not, because Keftriaxone doesn't target PBP3 in Pseudomonas, but Pepericillin, Keftazidime, Kefapim, Astrinam, the carbapenems, they do. So if they're going to get around it, because PBP3 is essential for growth, they can't use a substitute. You know, like PBP2A in Staph aureus. They have to mutate or modify the PPP3 and still continue to use it. So they can do this. You can get reduced binding, to say papericillin with PPP3 mutants. And so sometimes you might see something that's, papericillin R, but everything else sensitive. That might be quite a weird thing. Looking pattern. You might think that the, the reaction has failed in your antibiotic sensitivity testing. But it could be a genuine result. And the cause of it could be a PPP three mutation in pseudomonas

Callum: 31:09

So that's some different examples of penicillin binding protein and I guess what we've gone through today is we've talked about, well we've gone back to basics and said what is a cell wall and then looked at peptidoglycan in more detail than you've probably ever done before or ever wanted to and talked that down to the NAGs and the NAMs and how that they're cross linked and how that differs between gram positive and negative and how the, how the. Way in which things are cross-linked influences the shape of the bacteria, which I did not know, so that's quite cool. Then we talked about what penicillin binding proteins are. So those are the, the the enzymes that undertake

Jame: 31:48

transept and trans glycosylation.

Callum: 31:52

And so we talked about that and then all the different penicillin binding proteins and briefly, which organisms use which penicillin binding proteins and gave you some quick examples of where this can lead to resistance. So we hope that's been useful. Check out the show notes that Jame has produced because they've got a really useful summary. And don't forget about penicillin binding proteins when you're next seeing a unusual resistance pattern of your bacteria. Thanks.