Enhancing the antimicrobial potential of lanthipeptides by employing different engineering
strategies
Zhao, Xinghong
DOI:10.33612/diss.127409437
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Zhao, X. (2020). Enhancing the antimicrobial potential of lanthipeptides by employing different engineering strategies. University of Groningen. https://doi.org/10.33612/diss.127409437
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Chapter 6
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The research described in this thesis aimed to develop novel approaches to engineer lantibiotics, to control either planktonic pathogens or biofilm-infections. To expand the toolbox of engineering lantibiotics, this thesis furthermore aimed at generating a method for obtaining lanthipeptide synthetases with adapted substrate specificity by directed evolution.
In recent decades, more and more antibiotic resistance (AMR) has been encountered among bacterial pathogens. AMR causes more than half a million deaths each year. The UK government predicted that AMR will be the major cause of death in 2050 1. Despite the AMR situation being so urgent, the number of newly approved antibiotics has been decreasing over the past 50 years 2,3. Therefore, new antibiotics that circumvent resistance mechanisms are required to treat AMR infections. Lantibiotics are lanthionine ring-containing ribosomally synthesized and post-translationally modified peptides (RiPPs) 4. Many lantibiotics show potent antimicrobial activity against human pathogens and/or even against antibiotic-resistant human pathogens. Importantly, several lantibiotics, including duramycin, NVB-302, mutacin 1140 and NAI-107 that have been tested in the clinic or which are very close to the start of clinical trials, have been demonstrated to display potent antimicrobial activity in vivo 5– 7
. The ribosomal synthesis and low substrate specificity of some of the lantibiotic modification enzymes provide a great opportunity to engineer large numbers of novel antimicrobials. In this thesis, two strategies were employed to engineer lantibiotics by using the lanthipeptide biosynthetic machinery (Chapters 2 & 3), a high-throughput selection approach was developed to discover tailored lanthipeptide synthetases (Chapter 4), and a nano-technology based Ag@nisin NC was developed for biofilm-infection control (Chapter 5). Lipid II plays an essential role in the synthesis of the bacterial cell wall 8,9. The crucial role of lipid II in cell wall synthesis makes it an excellent target for many antibiotics, including vancomycin, ramoplanin, mannopeptimycins, teixobactin and a number of lantibiotics, e.g. nisin, NAI-107, gallidermin, nukacin ISK-1, mersacidin, haloduracin and lacticin A 8–17. In Chapter 2, we used the biosynthesis system of the lantibiotic nisin to synthesize a two lipid II binding motifs-containing lantibiotic, termed TL19, which contains the N-terminal lipid II binding motif of nisin and the distinct, internal, more C-terminally located
lipid II binding motif of haloduracin. Further characterization demonstrated that TL19 exerts 64-fold stronger antimicrobial activity against E. faecium than nisin(1-22), which has only one lipid II binding motif. Both the N- and C-terminal domains are essential for the potent antimicrobial activity of TL19 evidenced by mutagenesis of each single and double domains. The combined presence of two different lipid II binding sites within TL19 showed potent synergism in its antimicrobial activity (Fig. 1). Therefore, this study provides a new approach for biosynthesis of potent lantibiotics by combining two different lipid II binding motifs.
Fig. 1 Schematic presentation of the tentative antimicrobial mechanism of TL19. First, TL19
reaches the bacterial plasma membrane (a), where it binds to lipid II via one of the lipid II binding sites (b). This is then followed by TL19 reaching with its second lipid II binding site another lipid II (c), which leads to one TL19 binding to two lipid II molecules subsequently forming a carpet-like peptide-lipid II complex, thus potently inhibiting cell wall synthesis.
Non-ribosomally produced peptides (NRPs) constitute an important source of antibiotics. These peptide antibiotics include more than twenty marketed antibacterial drugs, such as vancomycin, penicillin and chloramphenicol 18. The synthesis of NRPs requires NRP synthetases (NRPSs) that act in a modular assembly-line logic 19,20. NRPSs are large proteins of about 220 kDa to 2.2 MDa mass range, with a high substrate specificity and encoded by very large gene clusters 21–24. Importantly, the combination of the large size of their gene
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clusters and the high substrate specificity of NRPSs make it difficult to efficiently engineer libraries of new NRP structural- and functional mimetics to screen for activity against antibiotic-resistant pathogens. In Chapter 3, we applied a completely novel strategy to synthesize mimetics of a recently discovered antimicrobial NRP, brevicidine. We mimicked the molecular structure of brevicidine by ribosomal synthesis and subsequent posttranslational modification. Such synthesis is typical of another class of peptides, RiPPs, ribosomally synthesized, post-translationally modified peptides. To this end, we employed a dehydration and circularization enzyme from a lantibiotic biosynthetic system to mimic the brevicidine lactone ring by a lanthionine ring. Along the peptide chain, we replaced positively charged ornithines by lysines, which are the closest resembling residues. Moreover, a fatty acid tail was mimicked by introducing 2 or 3 hydrophobic amino acid residues at the N-terminus. Following this strategy, peptides were engineered with potent antibacterial activity against Gram-negative pathogenic bacteria susceptible to brevicidine. This study demonstrates the feasibility of a strategy to mimic NRPs by employing the synthesis and posttranslational modifications typical for RiPPs (Fig. 2). This enables the future synthesis of large genetically-encoded peptide libraries of NRP-mimicking structures to screen for antimicrobial activity against relevant pathogens. These results demonstrate that structural and functional conversion of NRPs to RiPPs is possible and offers great opportunities for engineering a wide range of effective antibiotics.
Fig. 2 Schematic diagram of mimicking NRPs into RiPPs.
opportunities to design and engineer a large variety of novel antimicrobial compounds 25,26. However, a limitation in the heterologous production of novel or engineered lanthipeptides is the inefficiency of dehydration due to enzyme-unfavorable amino acid residues that may flank Ser/Thr 27–29. To fully exploit the potential of RiPPs as antimicrobial candidates, tailored enzymes for the dehydration of unsuitable substrates in a lantibiotic are required. In Chapter 4, we showed that cell surface display of a cyclic HPQF-containing streptavidin ligand is suitable for de novo selection of mutant NisB dehydratases capable of dehydrating a Ser preceded by an Asp residue in the strep-ligand context. Following the critical dehydration step, the cyclic strep-ligand was formed either by the cyclase NisC or spontaneously, and subsequently displayed on the cell surface. The bacteria that displayed cyclic HPQF showed a much higher affinity to streptavidin than linear ligand containing bacteria. This allowed the selection of cyclic strep-ligand displaying Lactococci containing mutant NisB by using streptavidin-coated magnetic beads. These results indicate that cell surface display of cyclic strep-ligand is an excellent method for the mining of novel genetically encoded libraries of lanthipeptide synthetases (Fig. 3).
Fig. 3 Schematic diagram of engineering of NisB with adapted dehydration capacity. Cyclic
strep-ligand has higher affinity to streptavidin than linear one, allowing streptavidin-coupled magnetic bead to fish out the bacterial with NisBmut that can dehydrate Ser preceded by Asp.
Wound infection is a serious threat to patients, in particular septic wound
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infections, which result in high mortality rates 30,31. Moreover, the treatment of wound infections with biofilm-forming pathogens can be challenging. Nisin is a green natural antimicrobial peptide. It shows potent antimicrobial activity against Gram-positive pathogens, but shows very limited antimicrobial activity against Gram-negative pathogens 32,33. However, Most of the clinical wound infections are mixed Gram-negative and Gram-positive pathogens infections 30,31,34–37
. This situation limited the application of nisin as an antimicrobial agent to treat wound infections. Silver has a potent bactericidal effect against both Gram-positive and Gram-negative pathogens 38,39, but it’s difficult to balance antimicrobial activity with cytotoxicity. In Chapter 5, we show that silver@nisin nanoclusters, with an average diameter of 60 nm, can be synthesized by microwave-assisted molecular self-assembly. The synthesized Ag@nisin NC showed higher antimicrobial activity than either silver nitrate or nisin alone. Notably, Ag@nisin NC showed potent anti-biofilm activity against S. aureus, P.
aeruginosa, A. baumannii, K. pneumoniae, and E. coli, which are pathogens
responsible for wound infections. Moreover, due to the nisin peridium, Ag@nisin NC showed much lower cytotoxicity than silver nitrate to a human kidney epithelium cell line. This work shows that microwave-assisted molecular self-assembly is a powerful and novel strategy to synthesize metal@antimicrobial peptide nanocomposites for biofilm-infected wound control.
Outlook
In Chapter 2, our results showed that a combination of two different lipid II binding sites within one peptide showed potent synergism in its antimicrobial activity especially against E. faecium, suggesting future work may focus on constructing more two different lipid II binding sites-containing peptides. Furthermore, synthesis of hybrid compounds with different antimicrobial mechanisms in one molecule would be interesting. Our results in Chapter 3 showed that mimicking NRPs by ribosomal synthesis is a novel approach to engineer antimicrobials, and enables the future synthesis of large genetically-encoded peptide libraries of NRP-mimicking structures to screen for antimicrobial activity against relevant pathogens. Future studies may focus on
mimicking more NRPs by ribosomal synthesis, followed by screening of more active antimicrobials from large genetically-encoded peptide libraries based on the NRPs-mimicked active molecules. Our results of Chapter 4 demonstrated that the selection of mutant modification enzymes from genetically encoded libraries can be obtained by cell-surface display of mutant-enzyme-modifiable products. In the future, this concept can be applied to other enzymes to generate desired properties, e.g. to LanMs and LanCs. In Chapter 5, our results showed that Ag@nisin NC was synthesized by microwave-assisted molecular self-assembly, and the synthesized Ag@nisin NC showed potent anti-biofilm activity against bacterial pathogens which are responsible for wound infections. Moreover, due to the nisin peridium, Ag@nisin NC showed much lower cytotoxicity than silver nitrate to a human kidney epithelium cell line. In the future, more metal@antimicrobial peptide nanocomposites could be synthesized for biofilm-infection control.
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