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New Antimicrobials

Final Report Summary - NAM (New antimicrobials)

The overall objectives of the NAM project were to:

1. develop an active collaboration between industry and academia in the area of new antimicrobials, encouraging transfer of personnel, know-how, ideas and molecules;
2. explore new sources and approaches for generating novel antimicrobials;
3. produce understanding of the mechanism of action of these antimicrobials;
4. combine the strengths of antimicrobial agents from different sources to develop more robust anti-infective agents.

The search for new antimicrobial compounds, and the gaining of a comprehensive understanding of the molecular mechanisms of action underlying their microbicidal activities, is urgently needed to identify future therapeutics that will help to overcome the problem of bacterial resistance. Amongst the plethora of currently used drugs, antibiotics have had a tremendous impact on the life expectancy of mankind. Starting in the late 30s and 40s, the golden era of antibiotic chemotherapy has now lasted almost 70 years, but is seriously threatened. It has become increasingly evident that the capacity of microbes to develop resistance is almost unlimited, and multi-drug resistance has become a very common and dangerous characteristic of many human pathogens, some of which are now susceptible to only one antibiotic. There is therefore an urgent, acute and continuous need for novel antimicrobials.

NAM was set up as a multidisciplinary consortium, consisting of groups active in biology, peptide chemistry, biophysics, microbiology and biotechnology. It comprised of two companies (one small and medium-sized enterprise (SME) and a large biotech company), and three university research laboratories in four different countries within the European Union (EU). Our common interest was the assessment of as yet unused classes of antimicrobials, such as the endogenous antimicrobial peptide (AMP)s produced by all higher organisms, and the secondary products of plant endophytes, internal plant microbes. Exchange of staff, especially young researchers, took place between the university groups and two companies, resulting in innovative joint projects between the partners. In the NAM project, new antimicrobials were not only searched for, but also extensively studied with respect to their mechanism of action.

One of the new sources of potentially useful antimicrobial agents that were extensively investigated was plant endophytes. These are mostly unidentified microbes that are symbiotic to plants, although it is know that their functions also involve protection of the plant host against various types of pathogens, by production of bioactive compounds. They therefore represent an extremely rich, untapped source of bioactive compounds that could potentially be useful in antibiotic therapy. During the NAM project, we have developed methods for isolating and screening endophytic fungi from northern plants, and found the highest percentage of bioactive fungi towards Staphylococcus aureus in grasses (38 - 100 %), spruce (27 %) and pine (25 %). However, most of these endophytic microorganisms were not easy to culture and maintain in vitro. We therefore studied metatranscriptomic and metagenomic tools to better access active compounds from such unculturable sources from the plant interior, and identified one antibacterial protein and several AMPs from the endophytic fungi and bacteria present in Empetrum nigrum (crowberry), Rhododendron tomentosum (northern Labrador tea), and Pinus sylvestris (Scots pine), by using these methods.

The NAM project had a strong focus on AMPs, not only from endophytic sources, but also from other bacteria and fungi, as well as the endogenous molecules produced as part of animal host defence. These endogenous AMPs are considered a promising source of new antimicrobials, often with a rapid and broad defence activity. Apart from the capacity to directly inactivate pathogens, many cationic AMPs of animal origin have been shown to have interesting immunomodulary activities, i.e. they also function by alerting and fortifying the immune system. They are thus multifunctional antibiotics, capable of directly inactivating pathogens using multiple mechanisms, as well as stimulating the host's immune capacities. This is considered an important reason why these ubiquitous endogenous antibiotics have managed to remain active throughout evolution, while the highly potent man-made antibiotic drugs encounter resistance problems within a few years of their introduction.

In the NAM project, several different classes of AMPs were considered from among the animal cathelicidins and ß-defensins, fungal defensins (plectasin) and bacterial lantibiotics. Cathelicidins are an important family of AMPs present in all vertebrate animals. They are produced as small proteins with a common and conserved pro-region, to which AMPs of different structural types are attached. When released, these peptides are capable of efficiently inactivating pathogens using distinct mechanisms. We have concentrated on helical cathelicidins; cationic, amphipathic helical peptides with a broad spectrum activity directed to the microbial membrane, and the Proline-rich cathelicidins, or PRAMPs, with a selective activity against Gram-negative bacteria, and having internal targets. Beta-defensins are another ubiquitous class of vertebrate animal AMPs, with activity also directed to the microbial membrane, but in a different manner to the helical peptides, and in some cases an interesting convergence in the mechanism was fund with the fungal plectasin and bacterial lantibiotics.

Regarding the helical peptides, studies on the mode of action (MoA) focused on the sole human cathelicidin LL-37 and its orthologues in other mammals. This AMP functions by interacting with, and inserting into microbial membranes, which it then permeabilises, killing the pathogen. It appears that interaction with host cell membranes also underlies its potent immunomodulatory activities. Its primary structure determines a strong tendency to oligomerise, with important consequences on membrane interactions, affecting both the direct antimicrobial and immunomodulatory activities. Our studies have provided interesting new information on the oligomerisation process and the possibility of controlling it so as to potentially optimise these activities. New members of the helical cathelicidins have also been identified.

Proline-rich cathelicidins were determined to function in with a different mechanism, involving the translocation into the cytoplasm of Gram-negative bacteria, using an active transport system, and subsequent interaction with cytoplasmic targets. Using a PRAMP of bovine origin, we were able to identify some of the different components of putative translocation machinery, in both the inner and outer membranes, as well as the power source for translocation. We also increased our understanding of the internal targets, which seem to involve both protein synthesis and folding. This type of peptide are of considerable interest, not only because they represent a new class of potent, selective antibiotics for Gram-negative pathogens, but because they reveal new potential targets for the rational design of new anti-infective agents, and also the translocation machinery which may be used to internalise antibiotic cargo.

With respect to the beta-defensins, we have selected the beta-defensin 3 produced by human epithelial cells and neutrophils for a detailed study of its mode of antibacterial action. hBD3 has the most potent and robust antimicrobial activity amongst the characterised human defensins. Its activity was thought to be due to its compact, highly cationic and amphipathic structure, which enables binding and disruption of microbial cytoplasmic membranes. However, the transcriptional response pattern of hBD3-treated staphylococcal cells indicated that inhibition of cell wall biosynthesis could be a major component of the killing process. We found that hBD3 binds to defined, lipid II-rich sites of cell wall biosynthesis, which may lead to perturbation of the biosynthesis machinery and results in localised lesions in the bacterial cell wall, possibly adding to membranolytic effects of the peptide. In this respect, dissection of the structure using a minimalised analog allowed to determine how the peptide can anchor efficiently and selectively to bacterial membranes.

A wide range of genetic and biochemical approaches were also used to identify cell-wall biosynthesis as the pathway targeted by plectasin. This fungal defensin also acts by directly binding the bacterial cell-wall precursor Lipid II as the specific cellular target, but with an even higher affinity than hBD3. This mechanism, common also to bacterial lantibiotics, may thus be an important general feature in the MoA of different classes of AMPs, ranging in origin from microbes to animals. Furthermore, as reported in the well-reputed Science journal, key residues in plectasin involved in complex formation were identified using nuclear magnetic resonance spectroscopy and computational modelling, opening the way for rational design of molecules with improved activity.

The mechanism of action of many known antibacterials is still elusive, although this information is important for their use as drug compounds. The problem is amplified in large-molecule antibiotics, such as most AMPs, that can have several different activities concurrently and that do not necessarily conform to having only one target and one MoA. By using a highly multidisciplinary approach, pooling knowledge and resources, the NAM project has nevertheless led us to the realisation that natural antimicrobial substances of quite different origin, such as the lantibiotics nisin and mersacidin, the fungal defensin plectasin, and the human beta-defensin hBD3, all interfere with the same target, Lipid II, inhibiting the same cell-wall biosynthesis pathway in pathogen Staphylocuccus aureus. A detailed scheme for this pathway's components and their interactions has been built. This knowledge will be invaluable for the future development of novel antibiotics and identifying new targets for them. Studies carried out on PRAMP variants to characterise the structural factors affecting their uptake, or to better understand the mode of membrane-directed action of helical cathelicidins, has allowed functional characterisation of other target pathways that will also serve the development of novel antimicrobials functioning with different mechanisms. It is hoped that the combined rational use of these substances may contribute to resolving the resistance problem.