PLOS Science Wednesday: Hi Reddit, we’re John and Yasser, and we found an unexpected mechanism of cell division in Chlamydia trachomatis unique to this human bacterial pathogen, creating implications for developing Chlamydia-specific therapies – Ask Us Anything!

Abstract

Hi Reddit,

I’m John Cox, a faculty member in the Department of Microbiology, Immunology, and Biochemistry at the University of Tennessee Health Science Center. And I am Yasser Abdelrahman a Senior Research Fellow in the Department of Restorative Dentistry at Oregon Health and Sciences University.

We have been interested in the molecular mechanisms that regulate the growth of Chlamydia trachomatis, an obligate intracellular pathogen that is the leading bacterial cause of sexually transmitted infections. The ultimate goal of these studies is to develop therapies to specifically prevent chlamydial growth. We recently published a manuscript, “Polarized Cell Division of Chlamydia trachomatis” in PLOS Pathogens. Although it was assumed that this organism divides by binary fission, we showed in this manuscript that Chlamydia trachomatis divides by a polarized cell division process that is similar to the budding process of a subset of the Planctomycetes. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments.

We’ll be answering your questions at 1pm ET -- Ask Us Anything!

Amazing work you guys are doing. Are you guys working on biologic therapy? Or pharmaceutical drug therapy to target the cell division process? I'm curious on how the targeting on the division works.

elleteehee

Hey guys, great work, and congrats on the PLOS Pathogens paper! I gave it a quick read-through, and I have a few questions.

My understanding is that recent work (from Liechti and others) suggests a model where Chlamydia still needs (and has) enough peptidoglycan (PG) to form a septum. The PBP enzymes that insert/act on this PG ring are coordinated by "bacterial actin" MreB (which normally organizes elongation, not division) rather than "bacterial tubulin" FtsZ. However, FtsQ (normally part of the FtsZ-coordinated division machinery) still localizes to the septum. This suggests a hybrid model of division, where components of different growth/division machinery work together in an unusual fashion (for example, MreB and FtsQ). Please correct me if I am wrong.

My questions are:

Do your results have any impact on this model, to either support or update it?



Your results seem to confirm that the major determinants of division in Chlamydia are mostly involved in necessary functions in other bacteria (e.g., MreB, FtsQ, etc.). Why, then, do you think that this asymmetric division suggests new drug targets or treatment paradigms? Have you discovered any new components necessary for division in Chlamydia? How significant is it that the MOMPs localize asymmetrically? Does this suggest they are involved in division/asymmetry generation?



Do you think that Chlamydia's asymmetric division is adaptive, or do you think it's a byproduct of a bug that lives in a fairly unique environment (intracellular) that has a stripped down "minimal machinery" for division, and that this set of minimal machinery happens to be disregulated enough that it now doesn't have much control over septum placement?

Thanks!

We have just started high-throughput screens to identify drugs that may be used to specifically prevent the chlamydial cell division process. The only other organisms that we are aware of that utilize a similar mechanism of cell division are members of the Planctomycetes, which are not human pathogens. While antibiotic resistance is not currently a problem with Chlamydia, there are major changes in the microbiome of the female genital tract when broad spectrum antibiotics are used to treat chlamydial genital tract infections. This change in the microbiome can lead to outgrowths of Candida (yeast infections) and bacterial vaginosis, both of which negatively impact the quality of life for women. It is our hope that the development of specific inhibitors will prevent these unwanted outcomes of broad spectrum antibiotic therapies for treating Chlamydia genital tract infections.


In the Nature Review piece that covers your work and that of Jutras, Scott et al. who looked at lyme disease, it seems there is a knowledge gap about a number of pathogens and how they divide. What is next on your list to look at? And what are the unique challenges in observing them divide?

firedrops

It is a challenge working with obligate intracellular organisms like Chlamydia. However, recent advances in chlamydial genetics make our goal of identifying potentially novel machinery that is necessary for chlamydial cell division feasible. From our work and the work of others, we are developing some understanding of how Chlamydia develops polarity and forms a septum between dividing cells, but we have yet to identify a unique target that is necessary chlamydial cell division. It is our hope that coupling a genetic approach with inhibitor studies will lead to the identification of one or more of these key components in this unique polarized cell division process.


Is there a benefit of this mechanism of division for the bacterium? Does it provide an advantage, especially in the context of obligate intracellular growth?

SpacemanSpiff958

Yasser:

As for the advantages offered by budding over binary fission, we can just speculate two points: 1) Offer a mechanism to localize the division septum in the absence of ftsZ 2) Offer the possibility of switching growth phases by regulating protein sharing between mother and daughter cells, for example switching from a differentiating EB to a fully growing RB, switching from an RB to an EB, switching from an RB to a PB.


Hey guys, great work, and congrats on the PLOS Pathogens paper! I gave it a quick read-through, and I have a few questions.

My understanding is that recent work (from Liechti and others) suggests a model where Chlamydia still needs (and has) enough peptidoglycan (PG) to form a septum. The PBP enzymes that insert/act on this PG ring are coordinated by "bacterial actin" MreB (which normally organizes elongation, not division) rather than "bacterial tubulin" FtsZ. However, FtsQ (normally part of the FtsZ-coordinated division machinery) still localizes to the septum. This suggests a hybrid model of division, where components of different growth/division machinery work together in an unusual fashion (for example, MreB and FtsQ). Please correct me if I am wrong.

My questions are:

  • Do your results have any impact on this model, to either support or update it?

  • Your results seem to confirm that the major determinants of division in Chlamydia are mostly involved in necessary functions in other bacteria (e.g., MreB, FtsQ, etc.). Why, then, do you think that this asymmetric division suggests new drug targets or treatment paradigms? Have you discovered any new components necessary for division in Chlamydia? How significant is it that the MOMPs localize asymmetrically? Does this suggest they are involved in division/asymmetry generation?

  • Do you think that Chlamydia's asymmetric division is adaptive, or do you think it's a byproduct of a bug that lives in a fairly unique environment (intracellular) that has a stripped down "minimal machinery" for division, and that this set of minimal machinery happens to be disregulated enough that it now doesn't have much control over septum placement?

Thanks!

subito_lucres

You are correct that Chlamydia, which lacks FtsZ, is dependent upon MreB for assembling peptidoglycan at the septal plane. This was elegantly shown recently by Liechti and his colleagues. We also know from our work that FtsQ accumulates in the septum between dividing cells. Whether FtsQ localization is dependent upon MreB is not clear at this time. We have not yet identified any new components necessary for this polarized division process. This is the goal of our current studies. MOMP localization serves as a marker for the division process but it does not direct asymmetric membrane expansion. Our recent studies have shown that Chlamydia trachomatis serovar B (a trachoma causing serovar) divides by the same polarized division process but MOMP accumulates in the septum not in the expanding membrane of the daughter cell. So MOMP isn’t driving the process. Our goal is to identify what is driving the process. The fact that division septum is localized to the base of the bud suggests that there is a specific mechanism involved. This mode of cell division seems to have evolved in free-living bacteria (Planctomycetes) and obligate intracellular bacteria (Chlamydia). As you point out both organisms lack FtsZ. Whether it is the absence of FtsZ that accounts the multi-budding phenotype we observe in Chlamydia remains to be established.


Thank you for your hard work.
Do you suspect that the chlamydia trachomatis division is the same throughout the chlamydia species?
Does the polarized cell division have some stages or steps that happen in the latent chlamydial stage?

HempInvader

Our recent studies have shown that Chlamydia trachomatis serovar B (a trachoma causing serovar) divides by the same polarized division process.


As a layperson in this field, is the following summary of my understanding correct?

  • Most of the time, C. trachomatis divides into two cells similar in spatial dimensions, but differentiated by one being MOMP-heavy and the other being Hsp60-heavy.

  • Sometimes, however, two or more smaller MOMP-heavy cells bud from the original larger Hsp60-heavy cell.

drsjsmith

Great summary, This uneven distribution of proteins disappears as soon as the division process is completed (two identical sized cells).


What sorts of inhibitors would be able to disrupt the division of cells in this process? Has it long been thought that Chlamydia contained tubulin or FtsZ and we just hadn't seen it, or has this been a mystery for a while? Are there other bacteria/cells that multiply this way, besides the planctomycetes?

EDIT: I'd like to add another, more important question, so if you can only answer one, answer this one: What is the timeline for new potential treatments that might come out of this?

tendorphin

Yasser: We tried several inhibitors in the paper, and every inhibitor appears to have a slightly different effect. Chlamydia is known to lack ftsZ gene, in fact Chlamydia is one of very few bacteria that lacks ftsZ. if you scan the literature you will find several budding bacteria, but looking at the pictures and comparing them to budding yeast or to our pictures it becomes hard morphologically to group budding bacteria in a single group.


Hi guys, thanks so much for doing this AMA. I'm a sophomore microbiology student and one of the professors at my school also works on C. trachomatis. What do you see as the next step towards using this discovery to treat chlamydia? Do you think it will be possible to find drugs that inhibit this cell division mechanism specifically? How does the cell regulate this process and are the other steps of cell replication (like DNA replication) regulated similarly in this process to cell fission?

I_LIKE_POTATOE

This is John How these processes are regulated in Chlamydia is not known at this time. Our studies suggest lipid asymmetry is associated with the cell division process. I am hopeful that our inhibitor screens will identify factors necessary for establishing and maintaining these specialized membrane domains, which are likely necessary for the polarized division process.


Why is Chlamydia often associated with Gonorrhea?

Truthplease5

Yasser: Chlamydia trachomais and Gonococcus are both sexually transmitted diseases.


How did you observe the replication?

icarus14

Yasser: late at night after 6 hours of confocal use, I saw these weird structures that looked like a lady's bag!!! :-)


Are Chlamydia that grow in this form of division able to switch back and forth between the budding process and more standard cell division? If so, what circumstances govern switching to the budding process (for example - is it more advantageous in pathogenic situations where the Chlamydia is trying to actively kill its host cell)?

iTyrosinekinase

This is John We have not been able to document Chlamydia undergoing standard (binary) cell division. While it may occur at later stages of growth within infected cells, these stages are much more difficult to image using immunofluorescent techniques. Based on our studies that examined the first few divisions within infected cells, all of the Chlamydia divide by the budding process.


What evolutionary process or factor lead to the short-list of bacteria that divide by polarized cell division? Chlamydia and Planctomycetes appear very different (obligate intracellular vs aquatic) so why would they converge on this trait?

Infectious_Pen

Yasser: Both were grouped together based on 16S sequences. "The PVC superphylum is a grouping of distinct phyla of the domain bacteria proposed initially on the basis of 16S rRNA gene sequence analysis. It consists of a core of phyla Planctomycetes, Verrucomicrobia and Chlamydiae" https://www.ncbi.nlm.nih.gov/pubmed/23912444


How would you compare this division to something like a budding yeast? I ask because these cells stain similar to Candida with different dyes of course, calcofluor white for one.

iamnomoney

It is unclear the advantage this mode of division offers to the organism. Again, only organisms lacking the bacterial homologue of tubulin, FtsZ, seem to employ it. We were initially struck by the similarity between chlamydial division and the division of budding yeast. How polarity is established in yeast is well characterized and we have referred to that literature in thinking about the chlamydial cell division process.


How would you compare this division to something like a budding yeast? I ask because these cells stain similar to Candida with different dyes of course, calcofluor white for one.

iamnomoney

Yasser:

Morphologically they are very similar


I was just looking at the genome (from ensembl genomes) and the geneome doesn't look very big (less than 1000 genes). Does that make research easier or harder?

jbsinger

Yasser: Easier if you do bioinformatics, harder if you can only grow it in cell lines (not agar). And harder if making any genetics on them was impossible till last year (still very very hard).


Great work! Many relevant questions have already been asked, but I did have two additional:

I see that you performed this study in serovar L2. Have you observed this in any other serovars or is there any reason to expect a different result in the others?

Given the efficacy, low cost, and clinical ease (1 dose) of erythromycin (or doxy with multiple doses), what niche would a new targeted therapy fill? I assume a large motivating factor is antibiotic stewardship and preventing formation of ery-resistance, so do you see this as a potential replacement for erythromycin therapy that wouldn't lead to Ery resistance in other "non-target" bacteria, or are there other specific advantages of this type of therapy that you anticipate?

TooBusyToLive

This is John Yes we have now observed this polarized mode of division in Chlamydia trachomatis serovar B and Chlamydia muridarum. Although the changes in morphology during the division process are very similar during cell division in these organisms, the localization of the markers (MOMP and LPS) we have used to visualize the division process vary substantially. We hope that the differences observed among the different Chlamydia will aid us in identifying factors responsible for establishing and maintaining polarity in these cells.

Yes an advantage of developing specific therapies is the prevention of resistance in non-target bacteria, such as Gonorrhea.


Great work! Many relevant questions have already been asked, but I did have two additional:

I see that you performed this study in serovar L2. Have you observed this in any other serovars or is there any reason to expect a different result in the others?

Given the efficacy, low cost, and clinical ease (1 dose) of erythromycin (or doxy with multiple doses), what niche would a new targeted therapy fill? I assume a large motivating factor is antibiotic stewardship and preventing formation of ery-resistance, so do you see this as a potential replacement for erythromycin therapy that wouldn't lead to Ery resistance in other "non-target" bacteria, or are there other specific advantages of this type of therapy that you anticipate?

TooBusyToLive

Yasser: Our recent studies have shown that Chlamydia trachomatis serovar B (a trachoma causing serovar) divides by the same polarized division process but MOMP accumulates in the septum not in the expanding membrane of the daughter cell.

While antibiotic resistance is not currently a problem with Chlamydia, there are major changes in the microbiome of the female genital tract when broad spectrum antibiotics are used to treat chlamydial genital tract infections. This change in the microbiome can lead to outgrowths of Candida (yeast infections) and bacterial vaginosis, both of which negatively impact the quality of life for women. It is our hope that the development of specific inhibitors will prevent these unwanted outcomes of broad spectrum antibiotic therapies for treating Chlamydia genital tract infections.


Also for Yasser, how does this work relate back to your research in restorative dentistry (or is this from a previous focus of yours)?

TooBusyToLive

Yasser: Thanks for your Question, I just moved to OHSU, and I am still studying microbiology. I did all the practical work for this paper in University of Tennessee with John


Long ago, bacteria phage were considered as a solution for treating infections. As a technology, phage lost out to antibiotics in large part because each phage was very narrowly applicable. The same antibiotic that can treat a blood infection by one infectious species can also treat an ear infection by an unrelated infectious species. That broad spectrum quality made antibiotics a better business case for R&D since:

  1. Doctors and hospitals would not need to carry a separate medication for every potential infectious disease.

  2. Treatment could begin prior to or even without precise identification of the infectious species.

  3. Because the antibiotics are broadly applicable, they can be produced at scale for pennies due to economies of scale.

Now, here's the thing: Antibiotics are, despite the advantages listed above, notoriously a poor investment for R&D... that's why the resistance problem exists... nobody wants to risk the excruciating cost of bringing a new drug to market for something as unprofitable as an antibiotic.

Why is a drug that only works on Chlamydia worth developing? Or to bring it back to the beginning... in what way would developing a novel anti-chlamydial drug be better than using an anti-chlamydial phage (something we know is a losing business case)?

Lucretius

Yasser: While antibiotic resistance is not currently a problem with Chlamydia, there are major changes in the microbiome of the female genital tract when broad spectrum antibiotics are used to treat chlamydial genital tract infections. This change in the microbiome can lead to outgrowths of Candida (yeast infections) and bacterial vaginosis, both of which negatively impact the quality of life for women. It is our hope that the development of specific inhibitors will prevent these unwanted outcomes of broad spectrum antibiotic therapies for treating Chlamydia genital tract infections.


Why would you need a Chlamydia specific therapy?

I'll phrase that question another way. Some antibiotics can cause harm. Colistin, for instance, is a fantastic antibiotic but absolutely destroys your kidneys and is infrequently used. Vancomycin and aminoglycosides can wreck your inner ear. Testing these drugs on humans could potentially harm humans even after careful animal studies (hence the reason for needing a phase I trial). I forget what the drug was but there was a recent phase I trial in Europe for a drug that inadvertently that led to a high rate of hemorrhagic strokes, several people died and it was quite tragic.

Ethically, should we be doing multiple phase I trials on antibiotics specific to each microorganism or should we just do less phase I trials on a shotgun approach broad spectrum antibiotic?

drseus127

While antibiotic resistance is not currently a problem with Chlamydia, there are major changes in the microbiome of the female genital tract when broad spectrum antibiotics are used to treat chlamydial genital tract infections. This change in the microbiome can lead to outgrowths of Candida (yeast infections) and bacterial vaginosis, both of which negatively impact the quality of life for women. It is our hope that the development of specific inhibitors will prevent these unwanted outcomes of broad spectrum antibiotic therapies for treating Chlamydia genital tract infections.


Why is it important to have Chlamydia specific therapies?

dregan

While antibiotic resistance is not currently a problem with Chlamydia, there are major changes in the microbiome of the female genital tract when broad spectrum antibiotics are used to treat chlamydial genital tract infections. This change in the microbiome can lead to outgrowths of Candida (yeast infections) and bacterial vaginosis, both of which negatively impact the quality of life for women. It is our hope that the development of specific inhibitors will prevent these unwanted outcomes of broad spectrum antibiotic therapies for treating Chlamydia genital tract infections.


I swear I thought that said you were Yasser of the PLO for a second.

How is your research funded and are there any conditions, restrictions or obligations tied to the funding?

I_LikeToReddit

Yasser: you are not far off, I was named after him!!!!.

The whole project costed us pennies (price for secondary antibiotics, time on the confocal, some common reagents, lots of gifted antibodies and inhibitors)


Any evidence that Chlamydia trachomatis synthesizes sterols, like its Planctomycetes cousin, Gemmata obscuriglobus, does?

If it does, there's a potential target for pharmaceutical action right there.

drdrewross

Yasser: Chlamydia gets lots for Cholesterol from the Host. I am not sure if they require Cholesterol for any unique biological use.


Very cool, I love potential treatments which target something so specific. My dad is an entomologist who works on biological control of invasive plants and insects, probably 90% of his work is making sure everything they release will attack only the target species (or even subspecies).

As far as I know, chlamydia is easily and effectively treated with antibiotics; why are you working on these bacteria in particular?

Davecasa

While antibiotic resistance is not currently a problem with Chlamydia, there are major changes in the microbiome of the female genital tract when broad spectrum antibiotics are used to treat chlamydial genital tract infections. This change in the microbiome can lead to outgrowths of Candida (yeast infections) and bacterial vaginosis, both of which negatively impact the quality of life for women. It is our hope that the development of specific inhibitors will prevent these unwanted outcomes of broad spectrum antibiotic therapies for treating Chlamydia genital tract infections.


how about a round of "applause"

BurpingHamster

lots of tears


Is this akin to fighting fire with fire? What are some examples of said therapies?

elkturd

While antibiotic resistance is not currently a problem with Chlamydia, there are major changes in the microbiome of the female genital tract when broad spectrum antibiotics are used to treat chlamydial genital tract infections. This change in the microbiome can lead to outgrowths of Candida (yeast infections) and bacterial vaginosis, both of which negatively impact the quality of life for women. It is our hope that the development of specific inhibitors will prevent these unwanted outcomes of broad spectrum antibiotic therapies for treating Chlamydia genital tract infections.


How do you think your prospective drugs will compare to the standard, single dose, 1g Azithromycin?

Is resistance to azithro growing, leading to a need for unique mechanisms?

UnclePetey

While antibiotic resistance is not currently a problem with Chlamydia, there are major changes in the microbiome of the female genital tract when broad spectrum antibiotics are used to treat chlamydial genital tract infections. This change in the microbiome can lead to outgrowths of Candida (yeast infections) and bacterial vaginosis, both of which negatively impact the quality of life for women. It is our hope that the development of specific inhibitors will prevent these unwanted outcomes of broad spectrum antibiotic therapies for treating Chlamydia genital tract infections.


What is polarized cell division?

What are you goals for your research?

icarus14

Understanding how Chlamydia divide while laking ftsZ


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