BPC Science Writing Competition 3rd Place: “AMPed and Dangerous”

By Kimberly Malecka

“Antimicrobial resistance poses a catastrophic threat,” declared Dame Sally Davies, Chief Medical Officer for England, in her 2013 annual report (1). She described a post-antibiotic dystopian era in which patients once again succumb to simple infections (1,2). Advanced medical options of routine surgeries, organ transplants and cancer treatments will be nonviable due to our inability to stop infection (1,3). So grim will be the prospects for human health that Davies ranks antimicrobial resistance next to terrorism and global warming as the greatest threats to our current safety (2).

tablets-193666_640

A year later, the World Health Organization (WHO) published its findings from a study of 114 countries worldwide (2). They report that antibiotics are currently failing to treat half of all pneumonia, gonorrhea and simple urinary tract E. coli infections (2). More concerning, however, is the realization that carbapenem, a powerful and “last-resort” antibiotic, is ineffective in more than 50% of patients in some countries (2). The numbers of resistant infectious bacteria and futile antibiotics are only expected to increase alarmingly over the next several decades (2).

A combination of factors is hastening the speed at which microbes grow resistant to medications (2,3,4): overprescription by doctors, failure of patients to finish the prescribed amount, and fast rate of microbe mutation. Unfortunately, development of new antibiotics has been stagnant of late (4) leading to what Davies refers to as the “discovery void (1).” She is imploring scientific researchers to once again focus on antibiotic development in new and innovative ways (1,3).

Researchers in the June 2015 issue of Nature Structural & Molecular Biology heard her rally cry (5,6). These scientists are looking inward at the inherent biology of animals and plants. Upon infection by pathogens, animals and plants induce antimicrobial activities, including expression of antimicrobial peptides (AMPs) (7). AMPs are ancient weapons; these peptides have been used since the beginning of life as part of host immunity and they have remained effective since their inception (8). Interestingly, this fact confounds the current mantra that microbes will eventually develop resistance to any antibiotic. So, the key questions these scientists asked were how do AMPs kill bacteria and why do bacteria not become resistant?

Broadly speaking, AMPs fall into one of four categories, but proline-rich AMPs (PrAMPs) are particularly interesting in terms of antibiotic development. The goal of any antibiotic is for it to get inside and kill a bacterial cell while leaving the host cells untouched. PrAMPs are actively brought inside Gram-negative bacteria, such as E. coli, through specialized proteins on their surface. Mammalian cells lack these specialized proteins so PrAMPs cannot enter (5). As to how the PrAMP kills the bacterial cell after getting inside? The PrAMP binds to the 70S ribosome and blocks the bacterial cell’s ability to make proteins. Proteins are essential to every function of a healthy cell; if protein synthesis is inhibited, then that cell will die almost immediately.

Protein synthesis is a highly choreographed event. It begins at the DNA. Specialized proteins transcribe a gene from our genome into a carrier molecule known as messenger RNA (mRNA). The mRNA then binds to a ribosome. Translation of the genetic material (mRNA) into a string of amino acids (protein) happens at the ribosome. This process is mediated by transfer RNA (tRNA). tRNAs bind to both the mRNA and the ribosome at specific spots, known as A, P and E. Using an analogy, in World War II, if the mRNA is an ecrypted message and protein is the translated message, then the ribosome is the deciphering Enigma machine. Functional ribosomes are essential to proper protein synthesis. Scientists know that ribosomes bound by PrAMPs cannot function, but they didn’t understand why until now.

Two laboratories independently solved the crystal structure of the PrAMP Onc112 bound to the 70s ribosome of Thermus thermophilus to atomic resolutions (5,6). Both structures are similar and together they describe the multi-headed attack by the PrAMP on the ribosome. The Onc112 peptide has four functions. First, residues 1-3 hinder the ability of an incoming tRNA to bind to the A site. Second, residues 5-8 disrupt proper tRNA binding to the P site. Third, residues 9-12 block the ribosomal exit tunnel where the translated protein leaves the ribosome. Fourth, residues 13-19 are necessary for the transport of Onc112 into a Gram-negative bacterium. Removal of those amino acids from Onc112 or the removal of SbmA from baceterial cells results in a peptide that lacks antimicrobial activity.

The areas of contact for the Onc112 peptide within the ribosome are not surprising. Many antibiotics on the market also target these areas individually (5,6). Clindamycin (acne, taxoplasmosis) and thiostrepton (veterinary mastitis) both prevent tRNA accommodation at the A site. Chloramphenicol (eye infections, swimmer’s ear) binds to a similar area as residues 5-8 of Onc112. Erythromycin (acne, chlamydia, syphilis) and azithromycin (tonsillitis, gonorrhea) are part of the macrolide group, which bind within the ribosomal exit tunnel. Thus, while the majority of available antibiotics have uni-modal mechanisms, Onc112 has a tetra-modal (four-moded) mechanism. The first three parts of the peptide bind to and completely shut down a ribosome’s ability to translate proteins; the fourth part ensures that such a powerful peptide is only taken up by infectious bacterial cells. The benefits of a tetra-modal peptide over a uni-modal small molecule are the differences between a short road to antimicrobial resistance and a long road.

These two published structures display why AMPs have been efficacious since the beginning of time: their success is protected by redundancy. A single mutation within the peptide or the ribosome is unlikely to destroy the peptide’s antimicrobial activity. Hurt one area of interaction and there are still others available! This protection is not present for small molecule antibiotics. One nucleotide change within the ribosome can be enough to destabilize the binding of the antibiotic to the ribosome. Bacteria evolve fast. If we show them the same antibiotic enough through over-prescription, then we are hastening the selection of viable bacteria that harbor that key nucleotide change.

Scientists are now trying to capitalize on the secret of AMPs by tweaking current antibiotics to bind mutated or modified ribosomes (11). However, a more effective path is now being pursued based on the research presented here. Scientists are buildling chimeric antibiotics, such as macrolide-chloramphenicol, that show promising results in the clinic (11). Scientists can also successfully replace L-amino acids with D-amino acids in these peptides without disrupting their antibiotic potency (8). Peptides of D-amino acids cannot be broken down mammalian cells. This result can pave the way for peptide-based antibiotics, as well. Seefeldt summarizes that “…it appears likely that [AMPs] represent a vast, untapped resource for the development of new therapeutics.” The past can be our best teacher when it comes to guiding the development of new antimicrobials to assuage the fears of Sally Davies, the WHO, and to protect our future generations.

REFERENCES

  1. Antimicrobial resistance poses ‘catastrophic threat’, says Chief Medical Officer. (2015). <http://bsac.org.uk/news/antimicrobial-resistance-poses-catastrophic-threat-says-chief-medical-officer/&gt;.
  2. Stephens, P. Antibiotic resistance now ‘global threat’, WHO warns. (2014). <http://www.bbc.com/news/health-27204988&gt;.
  3. Hampton, T. Novel Programs and Discoveries Aim to Combat Antibiotic Resistance. The Journal of the American Medical Association 313 (2015).
  4. Hampton, T. Novel Programs and Discoveries Aim to Combat Antibiotic Resistance. The Journal of the American Medical Association 313 (2015).
  5. Roy, R., Lomakin, I., Gagnon, M. & Steitz, T. The mechanism of inhibition of protein synthesis by the proline-rich peptide oncocin. Nature structural & molecular biology 22, 466 – 469 (2015).
  6. Seefeldt, A. et al. The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex. Nature structural & molecular biology 22, 470 – 475 (2015).
  7. de Souza Candido, E. et al. The use of versatile plant antimicrobial peptides in agribusiness and human health. Peptides 55, 65 – 78 (2014).
  8. Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 15, 389 – 395 (2002).
  9. Li, W. et al. Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria. Amino Acids 46, 2287 – 2294 (2014).
  10. Krizsan, A. et al. Insect-derived proline-rich antimicrobial peptides kill bacteria by inhibiting bacterial protein translation at the 70S ribosome. Angewandte Chemie 53, 12236 – 12239 (2014).

11.   Wilson, D. Ribosome-targeting antibiotics and mechanisms of bacteria resistance. Nature reviews. Microbiolo

Advertisements

Tags:

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: