A brief overview of some antibacterial peptides belonging to a highly diverse class of ribosomal natural products.

Kevin Lyons

RiPPs

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly growing class of small (< 10 kDa) peptides, which are synthesized by cellular ribosomes and then subjected to some degree of enzymatic post-translational modification. This combination of initial synthesis and subsequent modification is known as ‘post-ribosomal peptide synthesis’ (PRPS). RiPPs are produced by a highly diverse range of organisms – including prokaryotes, eukaryotes and archaea – and have a highly diverse range of functions. It would be impossible to capture the full range and diversity of RiPPs in a single, short blog-post. Hence, the focus of this article will be on a select number of RiPPs which are produced by bacteria, and which possess antibacterial activity. The antibacterial RiPPs described in this article fall within a range of different molecule classes – including lanthipeptides, thiopeptides, sactipeptides, microcins and bottromycins. Several other classes of antibacterial RiPPs have been omitted due to space limitations. Readers in search of a comprehensive review of RiPPs would do well to consult the 2013 review by Arnison et al., which describes – in depth – the structures, functions and classification of a wide variety of RiPPs.

Nisin

Discovered almost 90 years ago, nisin is a 34-amino-acid polycyclic antibacterial lanthipeptide – classified as such due to the fact that it contains the uncommon amino acids meso-lanthionine and 3-methyllanthionine. These, as well as several other unusual amino acids, are introduced into the molecule during the post-translational modification of a 57-amino-acid precursor. Nisin is produced by the Gram-positive bacterium Lactococcus lactis and is used as a food preservative to prevent the growth of spoilage bacteria (such as other lactic acid bacteria) and/or disease-causing bacteria (such as Listeria monocytogenes) in or on food products.

L. monocytogenes is an important target in raw meats and soft cheeses, as this Gram-positive bacterium can cause a severe disease known as listeriosis, which although relatively rare, has a high fatality rate (~30%) in young, old, pregnant and immunocompromised (YOPI) individuals. Nisin binds to Lipid II, an essential intermediate involved in peptidoglycan biosynthesis, thereby inhibiting cell wall formation and inducing pore formation. Nisin has broad-spectrum antibacterial activity against many other Gram-positive bacterial species (including many drug-resistant and biofilm-forming strains): some examples include Staphylococcus aureus, Streptococcus pneumoniae, Bacillus cereus, Clostridium botulinum and Clostridium difficile.

Nisin1.png

Nisin is used as a food preservative, particularly in the production of meats and cheeses, and is active against a wide range of Gram-positive bacteria, including L. monocytogenes.

Micrococcin P1

Micrococcin P1 is a macrocyclic thiopeptide secreted by certain Gram-positive bacteria, including Micrococcus spp. and Bacillus pumilis (Arnison et al., 2013). Like nisin, micrococcin P1 inhibits the growth of L. monocytogenes and other Gram-positive bacteria on meats and soft cheeses. However, unlike nisin, micrococcin P1 does not target bacterial cell wall peptidoglycan biosynthesis; rather it binds specifically to the acceptor site of bacterial 50S ribosomal subunits, thereby inhibiting both ribosomal translocation and aminoacyl-tRNA binding (Otaka and Kaji, 1974). Micrococcin P1 is ineffective against Gram-negative bacteria, presumably because it cannot cross the Gram-negative outer membrane.

Micrococcin P1 was recently found to be secreted by a Staphylococcus equorum strain growing on the surface of a semi-hard traditional French raclette cheese (Carnio et al., 2000). When the macrocyclic thiopeptide was purified to homogeneity and applied to cheese contaminated with L. monocytogenes, a remarkable reduction in L. monocytogenes growth was observed. This reduction in growth can be directly attributed to secreted micrococcin P1, as the secretions of a micrococcin P1-deficient mutant of S. equorum did not induce the same reduction in L. monocytogenes growth.

Micrococcin P1.png

Micrococcin P1, produced by a S. equorum strain growing on the surface of a semi-hard traditional French raclette cheese inhibited the growth of the Gram-positive pathogen L. monocytogenes.

Sporulation Killing Factor (SKF)

Bacillus spp. are known for their ability to form dormant endospores as a way of surviving periods of nutrient limitation. If and when nutrients become available again, these endospores can germinate to form regular, vegetative cells which have the ability to grow and replicate. Upon induction of the endosporulation process, the cells quickly become committed to entering the dormant state, which – although it promotes long-term survival – means that they can no longer grow and replicate in the short-term. Hence, if the period of nutrient limitation proves to be fleeting, cells that form endospores are at an inherent fitness disadvantage compared to non-endosporulating cells, as they cannot immediately exploit any newly emerging nutrient source(s). Perhaps unsurprisingly, then, B. subtilis has developed a strategy to postpone endosporulation until it becomes absolutely necessary. That strategy is bacterial cannibalism.

B. subtilis cells living under limited nutrient conditions secrete a so-called sporulation killing factor (SKF), which is a 26-amino-acid disulfide-containing cyclic sactipeptide, with a thioether crosslink of a cysteine sulfur atom to the α-carbon of a methionine. Another factor, sporulation delaying protein (SDP), a 42-amino-acid peptide with one disulfide bridge, is also produced. The production of these factors causes neighbouring B. subtilis cells to lyse; although there has been some debate as to which molecule – SKF or SDP – is the principal cause of this (González-Pastor et al., 2003; Liu et al., 2010). Either way, lysis of neighbouring cells enables the SKF/SDP-secreting cell to keep growing and avoid sporulation by feeding on the nutrients released from lysed neighbours.

As a side note, an interesting paper was published in FEBS Letters in 2007 showing that in mixed cultures – containing B. subtilis cells and the cells of one other bacterial species, either Escherichia coli, Acinetobacter lwoffii, Xanthomonas campestris or Pseudomonas aeruginosa B. subtilis favoured predation (i.e. killing the non-B. subtilis cells) over cannibalism in every case. However, this predation mechanism appears to rely on a factor or factors other than SKF and SDP.

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Chemical structures of sporulation killing factor (SKF) and sporulation delaying protein (SDP) from B. subtilis (Liu et al., 2010)

Microcin B17 and Microcin J25

Microcins are small antibacterial RiPPs which are produced by members of the Gram-negative Enterobacteriaceae family. These RiPPs have potent antibacterial activity against other Gram-negative bacteria and are resistant to various stresses, such as proteolysis, extreme pH and extreme temperature.

Microcin B17 is a linear azol(in)e-containing peptide (LAP) which can inhibit DNA gyrase (Arnison et al., 2013). Microcin J25 is a 21-amino acid lasso peptide which can inhibit RNA polymerase (Duquesne et al., 2007). The antibacterial activities of microcins are enhanced by various receptor-mediated uptake mechanisms. Specifically, they hijack receptors on sensitive bacterial cells which are involved in the uptake of essential nutrients such as iron. For example, many microcins parasitize iron siderophore receptors or porins. In order to enter a target cell, microcin J25 exploits FhuA (an outer membrane receptor for the siderophore ferrichrome), whereas microcin B17 utilizes the porin OmpF. After crossing the outer membrane, microcins often require an inner membrane protein to exert their toxic activity. In the case of both microcins B17 and J25, this protein is SbmA (Arnison et al., 2013).

Microcins.png

Chemical structures of microcin B17 and microcin J25 from E. coli (Oman and van der Donk, 2009)

Bottromycin A2

The original bottromycin was isolated from the fermentation broth of Streptomyces bottropensis DSM 40262 in 1957. Since then, a number of related compounds have been discovered, including bottromycin A2, which is a 7-amino-acid macrocyclic RiPP (Gouda et al., 2012). Bottromycin A2 contains four unusual amino acid residues, possesses a 12-membered cyclic skeleton and, like all known bottromycins, displays potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The mechanism of action of bottromycins is similar to that of nisin, in that it involves the blocking of aminoacyl-tRNA binding to the acceptor site of bacterial 50S ribosomal subunits. Unlike nisin, however, bottromycin A2 does not prevent peptide bond formation or ribosome translocation.

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Chemical structure of bottromycin A2, and artificially-colourised micrographs of Staphylococcus aureus (centre) and Enterococcus faecalis (right)

Conclusion

Given the potent antibacterial properties of these (and other) RiPPs, there is – it seems – great potential in this class of molecules in terms of developing effective antibacterial agents for food production, and novel antibiotics for clinical use. Research is currently underway to decipher the biosynthetic pathways which lead to the production of these compounds, so that we may continue to benefit from their usage into the future.

References:

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  2. Carnio, M.C., Höltzel, A., Rudolf, M., Henle, T., Jung, G. and Scherer, S. (2000) The macrocyclic peptide antibiotic micrococcin P1 is secreted by the food-borne bacterium Staphylococcus equorum WS 2733 and inhibits Listeria monocytogenes on soft cheese. Applied and Environmental Microbiology. 66(6), 2378–84.
  3. Otaka, T. and Kaji, A. (1974) Micrococcin: acceptor-site-specific inhibitor of protein synthesis. European Journal of Biochemistry. 50(1), 101–6.
  4. González-Pastor, J.E., Hobbs, E.C. and Losick, R. (2003) Cannibalism by sporulating bacteria. Science. 401(5632), 510–3.
  5. Liu, W.T., Yang, Y.L., Xu, Y., Lamsa, A., Haste, N.M., Yang, J.Y., Ng, J., Gonzalez, D., Ellermeier, C.D., Straight, P.D., Pevzner, P.A., Pogliano, J., Nizet, V., Pogliano, K. and Dorrestein, P.C. (2010) Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis. Proceedings of the National Academy of Sciences of the United States of America. 107(37), 16286–90.
  6. Duquesne, S., Destoumieux-Garzón, D., Peduzzi, J. and Rebuffat, S. (2007) Microcins, gene-encoded antibacterial peptides from enterobacteria. Natural Product Reports. 24(4), 708–34.
  7. Oman, T.J. and van der Donk, W.A. (2009) Follow the leader: the use of leader peptides to guide natural product biosynthesis. Nature Chemical Biology. 6(1), 9–18.
  8. Gouda, H., Kobayashi, Y., Yamada, T., Ideguchi, T., Sugawara, A., Hirose, T., Omura, S., Sunazuka, T., Hirono, S. (2012) Three-dimensional solution structure of bottromycin A2: a potent antibiotic active against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. Chemical and Pharmaceutical Bulletin (Tokyo). 60(2), 169–71.
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