Blog Spotlight #2

Blog Spotlight #2

Article: Human commensals producing a novel antibiotic impair pathogen colonization

In a recent study by Zipperer et al., scientists discovered a new antibiotic called lugdunin that is derived from Staphylococcus lugdunensis. The peculiar and exciting part of this discovery is that Staphylococcus lugdunensis is found in human noses and would be one of the first examples of human commensal bacteria producing antibiotics (Zipperer et al. 2016). The pressure for new antibiotics is higher than ever because of the presence of antibiotic resistant bacteria such as MRSA. The scary part of MRSA is its ability to resist many different antibiotics with mechanisms to resist penicillin, methicillin, vancomycin and many more (Lowy, 2003).  In an interview with Dr. Margaret Chan, (director general of the UN’s World Health Organization) she was quoted saying “Antimicrobial resistance poses a fundamental threat to human health, development and security” (NBCnews). With many health officials worrying about the prevalence and possible spread of antibiotic resistant infections, humanity needs new antibiotics that bacteria can not resist. In this study the scientists isolate, sequence and determine the effectiveness of a new novel antibiotic called lugdunin.

 

In the beginning of this study the scientists tested a variety of bacteria to see if any had antimicrobial activity against Staphylococcus aureus. In their testing only one bacteria, Staphylococcus lugdunensis (strain IVK28), had noticeable antimicrobial activity. To test this the group plated S. lugduensis on an agar plate surrounded by S. aureus. In this plating there was a noticeable area around the S. lugduensis colony where no S. aureus bacteria survived. To see what caused this antimicrobial activity, the scientists performed a transposon mutagenesis on the S. lugduensis and found its antimicrobial function no longer existed once mutagenesis occurred (M1 mutated strain). Analysis of the transposon insertion site found an unknown gene that coded for a non-ribosomal peptide synthetase. This gene encoded an operon that controlled the activity of multiple antibiotic biosynthesis related genes. The operon of interest named lug controlled the expression of four genes named lug A, B, C, D. To further test whether this gene lead to the antimicrobial effects of S. lugduensis the group removed the lug D domain and found that once again its microbial effects went away.

 

Each of these four genes code for enzymes that contain adenylation, peptidyl carrier protein, condensation, epimerization or reductase protein domains. This type of protein domain combination within enzymes is very similar to the one we studied called DEBS which used multiple domains to make a different polypeptide sequence. To study this new novel antibiotic of interest, the group used reverse-phase high performance liquid chromatography (HPLC) on IVK28 wild-type and M1 mutant cells to isolate the antimicrobial molecule of interest (lugdunin). They then used ultraviolet/mass spectroscopy to determine the composition of lugdunin. The composition of lugdunin differed between the IVK28 and M1 forms of the enzyme (this further proved that lugdunin was the antimicrobial agent that kills S. aureus). The mass spectroscopy revealed an ionized mass of 783.45858 which did not match the mass of any other known antibiotic, which meant they had discovered a new, novel antibiotic. One problem with this method though was that the bacteria did not produce enough lugdunin to study its physical characteristics and biological properties.

 

To solve this Zipperer et al. removed the lug R operon repressor of the lug operon and replaced it with a xylAB promoter upstream to the lug operon. The xylAB promoter was used in tandem with a gene that coded for a xylose-sensitive repressor (XylR) of the xylAB promoter. In the presence of xylose, the repressor comes off the xylAB promoter and leads to the activation of the lug operon. This allowed the scientists to produce a viable amount of lugdunin to be further studied. First the group put the lugdunin products into through NMR, electrospray ionization high-resolution mass spectrometry and Marfey’s analysis. The electrospray ionization helps to determine the sequence of atoms in the molecule by measuring small bits of the molecule with mass spectroscopy. In tandem with this, Markey’s analysis and NMR were performed which help to show the 3-D structure of the molecule.

Lugdunin:

Proposed molecular structure of lugdunin (provided by Zipperer et al. 2016)

 

The one interesting part of this molecule’s structure is the presence of a thiazolidine group, which according to Zipperer et al. is not usually present in macrocyclic molecules. To make sure the group isolated the correct antimicrobial molecule of interest, the proposed lugdunin was tested on bacteria and was able to effectively kill them proving they isolated the correct molecule.

 

The scientists also discussed a few unique characteristics of the protein domains that make up the enzymes produced from genes lug A, B, C and D. In the second adenylation site of lug A, it has a specificity that is usually seen in binding threonine but in this case, it binds to tryptophan. This leads to the potential for a new form of specificity in adenylation protein domains. The other surprising finding came in gene lug C where one adenylation site activates three valines in a row. This use of an adenylation domain has only been discovered recently and not used in many other macromolecule mechanisms. These unique findings lead to the possibility that this mechanism is not derived from any known mechanisms.

 

The next focus of this study was to see the effectiveness of this drug against multiple pathogens. These bacteria included vancomycin resistant Enterococcus faecalis, MRSA and Streptococcus pneumoniae with an MIC ranging from 1.5 to 12 ug/ml. MIC stands for minimum inhibitory concentration and it is defined as the minimum concentration of the drug that can effectively prevent growth of targeted bacteria in human hosts. The MIC range mentioned before is very promising because it has a low concentration needed to start killing bacteria. The one very promising finding from this study showed that MRSA strains could be fully killed at levels 10x its MIC (1.5 ug/ml) without harming human host neutrophils or erythrocytes. The other benefit of this new antibiotic is that no where within the study did the scientists see evidence of resistance appearing even after 30 days of continuous exposure. This means bacteria targeted by lugdunin will have a very hard time developing resistance to this drug even after long term exposure. The exact mechanism of action for lugdunin was undetermined, but bacterial cells exposed to lugdunin stopped producing DNA, RNA, protein and cell-wall substrates. This most likely means it targets an enzyme or substrate within a prominent metabolic pathway.

 

To further test the effectiveness of the drug in live hosts, mice skin was injured using adhesive tape to take off multiple layers of skin. These wounds were then infected with S. aureus and mice were treated with lugdunin 24, 30 or 42 hours after the infection. In almost all cases except two (these two cases were most likely due to mice licking off the lugdunin), the S. aureus infection had a significant decrease in bacteria and was almost fully gone.

 

The last major portion of this study tested whether S. lugduensis could out compete S. aureus and if those findings reflected similarly in humans. IVK28 wild-type S. lugduensis and mutant Δlug D (missing the lug D gene and therefore cannot produce lugdunin) were grown on agar plates with S. aureus. All forms of IVK28 wild-type were able to out grow S. aureus even when inoculum had 10 times more S. aureus than S. lugduensis. On the other hand, the mutant Δlug D form was not able to outgrow S. aureus no matter what concentration it competed against. To test whether these findings happened in humans too the group acquired 187 nasal swabs from hospitalized patients colonized by S. lugdunensis, S. aureus or both. Using a chi-squared test the group found that when both S. lugdunensis and S. aureus existed in the same nasal passage, S. aureus levels were 5.9 times lower than people with just S. aureus in their nasal passages.

 

This article has many ground-breaking implications for future work. It has presented a new novel antibiotic that is extremely hard for bacteria to develop antibiotics to. It is one of the first antibiotics discovered that originate from bacteria that are commensal with humans. It is the first time that humanity has seen a multicyclic antibiotic that contains a thiazolidine group. Lastly, its second adenylation domain from lug A has shown a unique specificity for tryptophan. Overall, this study paves the way for new research in many different areas of study.

 

This connects to what we learned in class very recently. A couple days ago we discussed the mechanisms by which penicillin and vancomycin work and how certain forms of S. aureus resist them. In class we discussed the need for newer and better ways to fight certain bacterial infections and this article presents a perfect example of a new way to fight these infections.

 

references:

Lowy, F. D. (2003). Antimicrobial resistance: the example of Staphylococcus aureus. Journal of Clinical Investigation, 111(9), 1265–1273. https://doi.org/10.1172/JCI200318535
Zipperer, A., Konnerth, M. C., Laux, C., Berscheid, A., Janek, D., Weidenmaier, C., … Krismer, B. (2016). Human commensals producing a novel antibiotic impair pathogen colonization. Nature, 535(7613), 511–516. https://doi.org/10.1038/nature18634
Drug-resistant superbugs are a “fundamental threat,” WHO says. (n.d.). Retrieved April 25, 2018, from https://www.nbcnews.com/health/health-news/who-labels-drug-resistant-superbugs-fundamental-threat-humans-n651981

10 thoughts on “Blog Spotlight #2

  1. Hi Brandon! This is a interesting spotlight with many connections to our recent lessons on antibiotic resistance. As someone interested in drug design, it’s especially intriguing to see how multifaceted the lug gene is and how it can be harnessed to learn more about bacterial resistance and design more effective antibiotics. You mention that lug A has a specificity for tryptophan. Why do you think this is? Is this based on structure?

    1. The authors gave no indication on why they were surprised that the adenylation site targeted tryptophan instead of threonine. Although I do not have an answer as to why this change in specification I can lead you to a paper that might be able to answer this. Weber, T. et al. antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters. This is also reference 36 in the paper.

  2. Hi Brendan!
    Thank you for this interesting review! This discovery is truly remarkable especially at a time when humans are in dire need for new antibiotics against multi-drug resistant organisms. As I was reading about lugdunin and its potential to combat different types of bacteria, I thought about the differences between Gram-positive and Gram-negative bacteria. I realize that the authors do not have a proposed mechanism of action for the antibiotic yet, but they do mention that it has potent antimicrobial activity against a wide range of Gram-positive bacteria. My question is, do they at any point test its activity against Gram-negative bacteria as well? Would it beneficial to compare the efficacy of this antibiotic between both types, which might allow them to determine if it penetrates the lipopolysaccharide outer membrane for example?

    1. Throughout the test they did not test this antibiotic against any forms of gram-negative bacteria. I believe they did this because they saw the antibiotic worked on peptidoglycan substrate or cross-link forming enzyme. Most gram-negative bacteria have the ability to inherently resist peptidoglycan targeting antibiotics because they have a membrane in between peptidoglycan and the exterior environment. I do not believe it would be beneficial to compare these results, but I do think this has potential to work on gram-negative bacteria if it was used in tandem with something like pentamidine.

  3. Hi Brendan,
    Thanks for a great review! It’s interesting how the authors thought to look towards our commensal bacteria and the ways in which they’re able to interfere with pathogen colonization as a source for new antibiotics. However, I wonder whether modifying commensal bacteria might lead to issues in maintaining a healthy balance. Although infections with S. lugdenensis are rare, they still occur, meaning that increasing its chances of survival may therefore increase risk. As a solution, they authors suggest using a mutant lacking potential virulence factors or introducing the lug genes into an exclusively commensal species. Yet these methods still consist in using a bacteria to produce ludgunin. Is there a reason they can’t administer the antibiotic alone?

  4. Neat article Brendan! Antibiotic resistance is one of the main health concerns today, and it was interesting to see the process of how antibiotics are discovered from the first observation of resistance to isolating and identifying the molecule with HPLC and mass spec. The authors discovered a lot in their paper, but they did not explore the exact mechanism of lugdunin. Since you mentioned that cell wall substrates were not produced when exposed to lugdunin, do you think lugdunin exerts its effects by inhibiting a substrate for peptidoglycan formation or cross-linking since Staph aureus is gram positive?

    1. I tried looking at how other similar antibiotics work (watasemycins) but could not find the mechanism to them. Still, I think this antibiotic would work by binding to peptidoglycan (substrate) because the S. aureus was not able to form a resistance to the antibiotic even when the scientists attempted to put selective pressure on S. aureus to form resistance. As we learned in class it is usually harder for bacteria to resist substrate binding antibiotic rather than enzyme inhibiting antibiotics. So I assume it is a substrate binding antibiotic.

  5. Hey Brendan, thank you for your review. This paper is very topical for our last module in BCM441 and very important! I thought it was very interesting how the authors used HPLC, UV-Vis, NMR, MS, and other biological techniques to find lugdunin. With this being said, do you know of any labs that were able to recreate this antibiotic from putting a plasmid of this antibiotic in another organism? If so, do you think we could genetic engineer people who are highly susceptible people like nurses to resistant bacteria like MRSA?

  6. Hi Brendan, great spotlight! Laura Kiessling, the keynote speaker at the FCBIS conference on Saturday, explored the ways in which we are able to choose which bacteria are commensal and which are infectious. Lectins, molecules that selectively bind carbohydrates, may be key to this process. Do the authors say anything about why Staphylococcus lugdunensis itself is commensal, or if it’s able to selectively target non-commensal bacteria rather than indiscriminately kill off all the bacteria in your nose? Differentiation between friend and foe is critical.

  7. This was a very interesting find. The idea that human commensal bacteria are capable of creating antibiotics. The implications of using commensalistic bacteria for future drug discovery is ground-breaking. These bacteria must by design make effective antibiotics without causing sufficient harm to the host organism. I wonder if we are host to other similar strains within our microbiota that are capable of similar products. These would make an excellent source of relatively safe antibiotic agents.

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