Don’t Forget About Fungi! A Rich Source for New Antibiotics…

Essig A et al. 2014. Copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis. Journal of Biological Chemistry. 289 (50): 34953-34964. doi: 10.1074/jbc.M114.599878

Antibiotics have proved fruitful in the clinic since the development of penicillin in the mid 1900’s. However, a growing problem is that bacteria have developed strategies of resistance to these antibiotics. Antimicrobial peptides (AMPs) secreted by fungi as a defensive strategy may be one avenue of developing new antibiotics that delay antibiotic resistance (Frey-Klett et al, 2011). Fungi and bacteria utilize defensive secretory strategies to guard their respective ecological niche in environments where they are co-localized (Scherlach). The secreted fungal AMPs can prove useful in the development of new antibiotics through mechanisms that may make it difficult for bacteria to develop resistance. In this study, the interaction of the fungal model organism Coprinopsis cineria with various bacteria was investigated in order to determine if the secreted AMPs could serve as useful antibiotics.

The researchers began their experimentation by co-incubating C. cineria with B. subtilis, P. aeruginosa, and E. coli and observed that the Gram-negative P. aeruginosa and E. coli demonstrated an inhibitory effect on the growth of C. cineria and C. cineria, in turn, demonstrated an inhibitory effect on the growth of the Gram-positive B. subtilis (Figure 1). In order to acquire a causative secretory agent of bacterial growth inhibition, C. cineria was grown in unchallenged minimal media. Proteins were extracted from the media and the CC1G_13813 protein (which they denoted copsin) was present in large concentration and produced a significant zone of inhibition in Gram-positive bacteria. The sequence and structure of copsin was determined through rt-PCR and NMR, and the structure revealed a highly stable architecture reminiscent of other known fungal defensins.

Figure 1: Co-incubation with C. cinerea and bacteria. A: White depicts fungal growth. After 48 hours of incubation, C. cinerea growth increased when incubated with B. subtitles (Gram-positive), but decreased when incubated with E. coli or P. aeruginosa (Gram-negative). B: Bacterial growth was quantified by OD 600 readings. These coincide with the qualitative results in A. C. cinerea inhibited the growth of B. subtilis, while the growth of C. cinerea was inhibited through co-incubated with both P. aeruginosa and E. coli.
Figure 1: Co-incubation with C. cinerea and bacteria. A: White depicts fungal growth. After 48 hours of incubation, C. cinerea growth increased when incubated with B. subtitles (Gram-positive), but decreased when incubated with E. coli or P. aeruginosa (Gram-negative). B: Bacterial growth was quantified by OD 600 readings. These coincide with the qualitative results in A. C. cinerea inhibited the growth of B. subtilis, while the growth of C. cinerea was inhibited through co-incubated with both P. aeruginosa and E. coli.

The activity of copsin was tested in another disk diffusion assay and a low minimum inhibitory concentration was observed in Gram-positive strains, implying that copsin was an effective agent for killing Gram-positive bacteria. Gram-negative bacteria were unaffected. The researchers then investigated a molecular target of copsin, and an obvious starting place was the bacterial cell wall. In a cellular localization assay, copsin was shown to bind extracellularly and was present in the same location as the known peptidoglycan inhibitor vancomycin (Figure 6). In order to determine a more specific target of copsin, binding assays with cell wall precursors were performed, and the results suggested a high affinity of copsin for both lipid I and lipid II.

Figure 6: Co-localization of copsin and vancomycin. The phenomenon that copsin and vancomycin were both found at the surface of B. subtilis and S. carnosus (both Gram-positive) suggests that the molecular targets are found in similar regions of the bacterial cell.
Figure 6: Co-localization of copsin and vancomycin. The phenomenon that copsin and vancomycin were both found at the surface of B. subtilis and S. carnosus (both Gram-positive) suggests that the molecular targets are found in similar regions of the bacterial cell.

This study highlights copsin as a potential future antibiotic. By extension, other fungal secretory elements could serve as additional therapeutic options, and this study promotes the promise that this investigation may hold. Additionally, the binding of copsin to lipid moieties (as opposed to a protein) reduces the likelihood of developing resistance in the short run. This is due to the fact that lipid moieties are synthesized by organic precursors as opposed to proteins, which are susceptible to mutation (Wright).

References:

Frey-Klett K et al. 2011. Bacterial-fungal interactions: hyphens between agricultural, clinical, environmental, and food microbiologists. Microbiol. Mol. Biol. Rev. 75: 583-609. doi: 10.1128/MMBR.00020-11

Scherlack K et al. 2013. Molecular bacteria-fungi interactions: effects on environment, food, and medicine. Ann. Rev. Microbial. 67: 375-397. doi: 0.1146/annurev-micro-092412-155702

Wright, Gerard. 2015. An irresistible newcomer. Nature News and Views. 517: 442-444. doi: 10.1038/nature14193

15 Replies to “Don’t Forget About Fungi! A Rich Source for New Antibiotics…”

  1. Hi Zach! Ineresting article! It was cool to read an antibiotic article that tied in with the vancomycin story we looked at in class. I thought it was interesting to see that copsin also targets lipid I and II. I also thought it was interesting that copsin is derived from fungi, perhaps an often overlooked source! Do you think that copsin would be a more or less effective future antiobiotic than vancomycin? It seems that they both target lipids instead of proteins, already giving them a leg up on other antibiotics. It also seems that they both have two targets, implying that it will take longer to develop resistance to them than antibiotics with protein targets. I see in the discussion of the paper the authors state that, “Binding studies of copsin with truncated versions of lipid I revealed that, instead of D-Ala and D-Glu, the third amino acid in the pentapeptide side chain is crucial for binding to copsin, independently of whether L-Lys or diaminopimelic acid is located at this position”, which highlights one crucial difference between the targets and mechanisms of the two drugs. It’s amazing that shifting the target one amino acid over can make such a change! Do you think that copsin would just be used for patients who are vancomycin resistant or would this prove to be a more effective first line treatment even in patients who are not vancomycin resistant? Thanks!

    1. Great thoughts! Thanks for your input. Based on the fact that vancomycin and copsin operate through similar molecular mechanisms, I would predict that they would be of similar effectiveness. This is also assuming that copsin actually proves useful as an antibiotic in trials in higher microorganisms (mice, monkeys, humans, etc). To answer your second question, I don’t see why copsin could not be an effective antibiotic against non-vancomycin resistant patients. I think that the statement in the discussion sentence that you quoted was just to highlight the fact that copsin could be an effective alternative for patients who are vancomycin-resistant. Hopefully copsin will prove to be a useful antibiotic in the future. Thank you for your thoughts!

  2. Hi Zach! As I was reading, I was wondering if the authors knew the potential antibiotic would be a peptide beforehand because of the route they took in discovering the specific antimicrobial agent. It seemed like the authors had a hunch that the antibiotic would be a peptide because they immediately extracted proteins from the minimal media. Do you know if its common for fungi to produce antimicrobial peptides as opposed to small molecules? Or did the authors just happen to look for a protein target first because it is an easier experiment? Does growing in unchallenged minimal media have anything to do with this?
    I was also wondering if you think that this antibiotic would be an even better antibiotic than Teixobactin, which also targets functional lipids in bacterial cell membranes, because it is made by fungi. Because of this, it would be less likely to see bacteria, even in small populations, resistant to copsin because a bacteria did not produce it. This would mean that a resistance gene does not exist and therefore it cannot be transferred to other bacteria through horizontal gene transfer. What are your thoughts on this?

    1. Hey Matt. I do think that the authors knew that the potential antibiotic would be a peptide antibiotic. It was known in the literature that antimicrobial peptides were used by fungi as defense mechanisms. I think that this study was novel because these researchers actually extracted a peptide that could prove to be a useful antibiotic. So yes, I believe that it is common for fungi to produce AMPs as opposed to small molecules. I was also wondering why the authors used unchallenged media to extract the proteins. From my understanding, I thought that fungi secreted AMPs in response to bacteria occupying a similar environment in order to protect/acquire their environmental niche. Perhaps the secreted AMPs are substances that are naturally secreted by fungi even without a stimulus (maybe as a preventative mechanisms for the possibility of other competing microorganisms). If this is so, it would make sense for the authors to simply extract the secreted AMPs from unchallenged media. This point is controversial, which is also why I decided to include that qualification in my spotlight summary. Your last thought is very interesting. Honestly, I do not know if teixobactin would be better or worse than copsin. I think that more studies would need to be performed in order for this comparison to be made. They work through similar mechanisms, but I wonder whether the affinities of copsin and teixobactin would be different for the lipids. This information would probably suggest which of the two antibiotics would better. I hope I have answered all of your questions. Thanks for your thoughts, Matt!

  3. Hi Zach,
    Great article! It’s nice to see something that’s both on the bleeding edge of science, and has relations to major problems in medicine as well. In light of the different mechanism of action compared to beta-lactam antibiotics, the authors hypothesize that classical methods of resistance to beta-lactam antibiotics bacterial populations undergo would be ineffective, leading to comparative lack of acquired resistances in the clinical setting.

    While there is certainly evidence to support the slower rise of resistance within the context of lipid-targeted antibiotics, there still exists the possibility for horizontal gene transfer-derived resistance as indicated in the case of Vancomycin (1).

    Given the authors claims that similarly discovered antibiotics could forestall the looming threat of antibiotic resistance, how do you think that this potential threat of acquired resistance should be approached? How important do you think it is for current research to effectively determine ways to inhibit the already low rate of cell wall-focused antibiotic resistance acquired in target organisms? Are there any methods you feel the authors of this paper will further/should further explore?

    Thank you again for a clear and interesting article!

    1. Marshall CG, Lessard IAD, Park I-S, Wright GD. Glycopeptide Antibiotic Resistance Genes in Glycopeptide-Producing Organisms. Antimicrobial Agents and Chemotherapy. 1998;42(9):2215-2220.

      http://www.ncbi.nlm.nih.gov/pmc/articles/PMC105782/

    1. Thanks for your thoughts, Anthony. I do believe that the authors were very excited about this AMP because of its lipid target. I also feel as though it is difficult to predict how fast bacteria will develop resistance to an antibiotic. I’m really not sure how scientists could predict this due to the fact that bacteria intrinsically have much higher rates of mutation than eukaryotes. I feel as though the authors are addressing the concept of acquired resistance appropriately by highlighting the lipid target component. In my opinion, this is the best bet for developing new antibiotics. So how intensely should scientists be looking at ways to limit horizontal gene transfer of lipid-targeted antibiotics? I think that it should be relaxed. Developing new antibiotics is already a huge task, and trying to predict targets of resistance will prove futile until we know how bacteria acquire resistance to lipid-targeted antibiotics.

  4. Hey Zach! Nice job on this article. The information is fresh as we prepare for tomorrow and you explained the significance of these findings very well. I was hoping you could give me a little more depth on the last sentence of your summary… I might be forgetting some background, but why does the binding of copsin to lipid moeities reduce resistance development when compared to proteins? You say they’re susceptible to mutations, but could you draw this out a little more for me?

    My other question is, where do we go from here? We need to get these antibiotics in the clinic, there are lives to save! We are neither organic chemists nor pharmacologists, but how would you propose moving forward with an antibiotic like this? Is it worth the time to continue with in situ synthesis and glass beds or iChips or whatever, or do we want to look at offshoots of this compound. Can this alpha helix and double beta strand be slapped with some biologically active molecules until another effective antibiotic is found? …… I guess it’s more like 2 questions.. 1) possibility of genetic engineering for mass production… and…. 2) chance of synthetic modification to create other equally as effective drugs?

    Sorry for the many questions and slight confusion about the resistance mechanism… No need to answer them all. Any response will be helpful I’m sure! Thanks a bunch (y)

    1. No problem – thanks for your thoughts. To address your first question, bacteria have a higher rate of mutation than eukaryotes and divide very rapidly. Mutations are also random (for the most part). It is more likely that bacteria will develop resistance to a peptide-based antibiotic because these mutations are heritable – they are encoded in the DNA sequence of the bacteria and can, therefore, be passed along as the bacteria divide. Lipids are synthesized from small organic molecules (as we know), and the synthesis of lipids is not heritable, but the synthesis of proteins is heritable. To answer your other questions – yes, I think more experiments should be performed on copsin to test its efficacy. If shown to be very efficacious, it should be tested in higher order organisms for stability and safety. I do think that copsin could be a good candidate for mass production due to the intrinsic stability of its structure described in the paper. As for synthetic modifications, I honestly don’t really know. The authors explain that the fact that copsin has a lipid target allows it to evade short-run resistance, but I believe that synthetic modifications could have an impact on copsin’s mechanism of action both structurally and electronically. Perhaps moieties could be added onto copsin that allow it to bind to other lipid targets? Not sure. But thanks for you thoughts!

  5. Hey Zach,

    So as I was reading this paper, and specifically the conclusion, I kept thinking that it was interesting that copsin also interacted with lipid 2, as do many antibiotics, and this got me thinking about the mechanism of antibiotic resistance development. Lets say, for example, that like vancomycin, a given drug binds a certain portion on lipid two that eventually induces the bacteria to change the composition of lipid two to reduce the binding affinity of the drug. The canonical solution to this would simply be to find a new antibiotic, but this appears to be a less than sustainable system. I wonder, if there would be some way to synthesize a new antibiotic with the objective to cause the bacteria to revert its lipid composition back to the original composition so that vancomycin could again be used? In other words, do you think its possible to create a “resistance loop” of sorts, so that two antibiotics could effectively treat a given bacteria forever?

    1. That’s an interesting thought. Designing an antibiotic that modified the lipid back to it’s original form is something that I have not thought about before. I’m not sure if this would be possible. I would assume that bacteria could modify the lipid II motif by many different amino acid substitutions for the D-Ala-D-Ala. I know that we talked about a D-Ala-D-Ser modification, but I’m not sure how an antibiotic could actually alter the chemical composition of one amino acid residue. I would predict that methods such as steric blocking of an enzyme would be more likely. I don’t really think that an antibiotic has the capability of modifying one amino acid residue.

  6. Zack – How did Week 14 get here so fast?! Great job closing out the spotlight project this semester. It only seems appropriate that I ask you why this article was only published in the JBC. Specifically, I am thinking about it in juxtaposition with the Nature antibiotic article from class. The first thing that came to my mind was that the article from class featured a novel culturing technique. But in this paper, the authors did do something interesting by growing on glass beads in media (admittedly not as exciting as the iChip, but still pretty interesting, no?!) Besides that, they have the sequence and NMR spectrum, and in the discussion they tout how Copsin has “a distinct antibacterial profile differentiating it from other fungal defensins such as plectasin or micasin”. So where’s the rub? Obviously they don’t have the extensive resistance studies, but why else is this paper relegated to the JBC? In the current antibiotic-resistance climate, shouldn’t a discovery of a vancomycin molecule be higher impact? What is holding this paper back?

    1. These are good questions, Zach. One thing that came to mind was that copsin exhibits a mechanism of action that is similar to vancomycin, so maybe the scientific community is thinking that they have seen it before. I do think that the development of the iChip really bolstered the impact of the teixobactin paper, and also the fact that teixobactin was isolated from uncultured bacteria, in contrast to copsin which was extracted from a fungus. I also think that the methods of this paper were very chemistry-oriented, which could be a reason for its location in JBC. The purification and cDNA synthesis are very specifically laid out in the methods section (classic JBC), as well as structural and NMR identification (all very chemistry-based). And as you said, some more testing on the efficacy of copsin probably would have boosted its impact.

  7. Hey Zach,
    Good choice of article, and I think this paralleled the teixobactin article very well, which was pretty cool. After a cursory reading of the Wikipedia page on penicillin, it seems that (at least at one point), mass-production of penicillin was achieved by growing huge volumes of fungi (“mold”) that synthesize the drug. It seems, then, that while they knew both bacteria and fungi produced penicillin, they found fungi more suitable to synthesize it for mass-production. Contrast that to copsin, which is only fungi-produced. Do you think there’s anything that might be a drawback to copsin (as a potentially marketable drug that may one day need to be mass-produced), given this difference? Or is the fact that they grew penicillin in fungi (and NOT bacteria) evidence enough that fungi mass-production is the way to go, and mass-production of copsin shouldn’t be much different? Thanks!

    1. Great thoughts, Besher. I think that the primary means of answering these questions stems from the fact that copsin is originally found as an antimicrobial peptide against bacteria. The authors outline a discussion of bacteria/fungi competitive relationships, and I think that fungal production of copsin stems from that, as opposed to bacteria producing copsin to combat other bacteria. As for mass production, I don’t see why bacteria would not be able to mass-produce copsin. It may be possible that copsin could be incorporated into a plasmid and inserted into E. coli for mass production, especially given the fact that E. coli are Gram-negative bacteria.

Comments are closed.