University of Groningen
Breaking walls: combined peptidic activities against Gram-negative human pathogens
Li, Qian
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2019
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Li, Q. (2019). Breaking walls: combined peptidic activities against Gram-negative human pathogens. University of Groningen.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
Chapter
6
CH APTER 6: G en era l Di sc us sio n
6
Antibiotic resistance has become one of the biggest threats to glob-al heglob-alth today [1, 2]. The development of antibiotic resistance is a natural ecological phenomenon, but the abuse and misuse of antibi-otics is accelerating the process [1–4]. It is estimated that more than 2 million people are infected with antibiotic-resistant bacteria, which causes 23,000 deaths and renders $20 billion per year in excess health care costs in USA annually [5–7]. In Europe, around 400,000 people were infected by multi-drug resistant bacteria, in 2007. The costs as-sociated with these infections in terms of extra hospital expense and productivity losses exceeds €1.5 billion annually [7]. Many of these infections are caused by Gram-negative pathogens, which have an additional protective outer-membrane. However, the pace of discovery and development of new antibiotics is much slower than the emergence and spreading of the antibiotic resistance [1]. There is an urgent need for discovering new antibiotics and for a better use of existing antibi-otics, in order to fight the increased resistance of bacteria to existing antibiotics. One group of novel compounds with a new mechanism of action in therapeutics is constituted by lantibitotics, which have been regarded as good candidates for clinical development.
Lantibiotics are ribosomally synthesized and post-translationally modified peptides, containing lanthionine (Lan) and methyllanthi-onine (MeLan) residues, which possess antimicrobial activity. In the model lantibiotic nisin, these (methyl)lanthionine bonds are intro-duced by the enzymes NisB and NisC, which dehydrate and cyclize the peptide, respectively, before the transporter NisT exports the peptide. Nisin becomes active when the protease NisP, at the outside, cleaves off the leader peptide. The two-component system NisRK constitutes the nisin inducible system (NICE), that controls the expression of these enzymes as well as of the immunity system. (Methyl)lanthionines are critical for lantibiotic activities [8, 9] as well as their thermostabili-ty, proteolytic resistance and are important features in pharmaceu-tical applications [10–12]. The NICE system has been reported to be extensively and successfully used for high expression of proteins from different origins for various applications, and the promiscuity of NisBTC enlarged the expression system achieving the introduction of Melan/Lan to clinically relevant peptides to improve their stability and pharmacodynamic properties [13–18].
158
CH APTER 6: G en era l Di sc us sio n6
Nisin is one of the oldest and most widely used antibiotics and is highly effective against Gram-positive bacteria, with its minimal inhibition concentration being at nanomolar concentrations [19, 20]. Nisin binds to the pyrophosphate moiety of lipid II via the N- terminal rings A and B that form a pyrophosphate cage. Then nisin bends, in-serts its C-terminus into the membrane and forms transmembrane pores [21]. Thus, nisin exerts two killing mechanisms: it permeabilizes the membrane and inhibits cell wall synthesis [19, 22, 23]. Since the pyrophosphate is essential and not prone to mutation and also facili-tates the transmembrane orientation of nisin, low levels of resistance to nisin can be expected [19, 24]. Like nisin, vancomycin also binds to lipid II and exerts antibiotic activity against Gram-positive bacteria
Figure 1. Schematic overview of the two strategies for nisin/vancomycin to penetrate the outer- membrane. Left: Vancomycin or nisin alone cannot penetrate the outer-membrane due
to their size and charge. Middle: in the fused peptide, the tail can help the nisin moiety to pass through the outer-membrane and reach the inner membrane. Right, in the presence of GNPs, a gap will be formed on the OM and vancomycin or nisin can reach the inner membrane. GNPs, anti-Gram-negative peptides.
CH APTER 6: G en era l Di sc us sio n
6
[19]. Its target is formed by D-Ala-D-Ala peptides in lipid II, blocking the glycan polymerization and cross-linking [25, 26].
Nevertheless, the activity of nisin or vancomycin against Gram- negative bacteria is much lower than that against Gram-positive bacteria. The main reason is that the protective outer-membrane of Gram-negative bacteria constitutes an efficient barrier to prevent ni-sin or vancomycin to reach its target, lipid II, at the inner membrane. Thus, the main bottleneck for nisin or vancomycin to be effective against Gram-negative pathogens is their capacity to penetrate the outer-membrane. To address this issue, two strategies were employed (Figure 1)(Chapter 3, Chapter 4 and Chapter 5).
Chapter 3 describes the bioengineering of nisin to increase its
activ-ity against Gram-negative pathogens. Several tails with transmembrane activity were attached to various nisin moieties. And then, rational design of nisin, tails or both was performed. After expression and purification of sufficient amounts, the activities of nisin, the tails and fusions were tested both on Gram-negative and Gram-positive bac-teria. The results revealed that the tails of the fusions changed both the activity and spectrum of activity of nisin, but the fusion was still bactericidal. Variant T16m2 was proved to be up to 12-fold better than nisin against Gram-negative bacteria. These data support that rational design is a potential way to develop highly potent lantibiotic- derivatives with modified activities.
For this strategy, fusions were bioengineered combining the capacity of penetrating outer-membrane and binding to lipid II. The tails work as a Trojan horse and enable the nisin moieties to reach the inner membrane. Nonetheless, there are three disadvantages of these fusion peptides, namely the big size, low production yield and purity. The big size of the hybrid molecules might impact the stability, bioavailabil-ity and even the route of administration. The low production yield can make this production costly and unpractical. In order to obtain peptides with high purity, without a mixture of different dehydration statuses, several steps of purification should be performed and it can be time-consuming and difficult. One possible venue to facilitate the development of the most successful candidates is the directed evolution of enzymes (i.e. NisB or even NisC), that can recognize them as their ideal cognate peptide, thereby reducing heterogeneity and increasing
160
CH APTER 6: G en era l Di sc us sio n6
the production yield. Semisynthesis of nisin (or nisin parts) with im-proved properties has been accomplished using commercial nisin, treating it with appropriate proteases for prolonged time if necessary, and then attaching the second molecule with chemical methods [27, 28]. This approach is of particular interest when nonpeptidic moieties are going to be tested. In any case, further preclinical tests on toxicity and pharmacokinetic and pharmacodynamics characterization are still needed for these heterologous peptides.
A more direct way to expand the activity of lantibiotics, and similarly other anti-Gram-positive antibiotics, against Gram-negative bacteria is their combination with independent molecules that can facilitate their access to the periplasmic space. Following this reasoning, synergism between membrane perturbing peptides and lipid II-targeting anti-biotics is employed in both Chapter 4 and Chapter 5. In Chapter 4, the synergism between several anti-Gram-negative peptides (GNPs) (L-peptides) with modest activity against Gram-negative bacteria and nisin or vancomycin was tested. These peptides were synthesized and tested either alone or in combination with vancomycin/nisin against five selected Gram-negative pathogens. We observed that GNP-6 was very efficient alone and showed a modest synergism when combined with either vancomycin or nisin against the Gram-negative pathogens tested. In contrast, GNP-8 exhibited an astonishing synergism with vancomycin or nisin in spite of its poor activity when tested alone. Highly synergistic activity of GNP-8 and vancomycin or nisin leads to dramatically reduced minimal inhibitory concentration values (up to 32-fold). Considering the size, charge and potency of the L-GNPs, GNP-6 and GNP-8 were found to be the best candidates for further investigation in combination with either vancomycin or nisin. A mech-anism of gateway activity of membrane disrupting peptides is proposed. Subsequently in Chapter 5, some rational mutants were design and synthesized for GNP-6 and GNP-8, and the MIC of them alone and combined with vancomycin/nisin were tested. Overall, engineering and sequence reversal did not provide better molecules with the ex-ception of the D-GNPs that retain the activity and are less prone to proteolytic degradation. Thus, no specific receptors are involved in the actions of the selected peptides GNP-D6 or GNP-D8. LPS and Mg2+ were added in the activity assays, and the activity of vancomycin,
CH APTER 6: G en era l Di sc us sio n
6
GNP-D6 and GNP-D8 were significantly compromised either when they were tested alone or in combination. These results further suggest that the GNPs, especially GNP-D8, interact with LPS on the outer- membrane and improve the perturbation capacity of outer-membrane, although a detailed mechanism has not been proved yet. Thus, in the presence of these GNPs, nisin/vancomycin can penetrate the outer-membrane and reach lipid II then.
Notably, vancomycin is already a clinically used antibiotic, while nisin is not. Therefore, the antimicrobial activity towards MDR Gram-negative pathogens, the cytotoxicity, hemolytic effect were test-ed for vancomycin, GNP-D6, GNP-D8 and the combinations thereof. Vancomycin and GNP-D8 neither caused lysis of hRBCs nor showed
significant toxicity against the human cell line HEK-239 when tested alone or in combination. The concentration of GNP-D6 to cause hemo-lysis and toxicity was high compared to the concentration required to inhibit the growth of Gram-negative pathogens, including MDR strains. Thus, vancomycin, GNP-D8 and GNP-D6 are potential candidates for therapeutic use although further clinical characterization is required. Remarkably, the synergism between two compounds highly increas-es the activity of nisin against Gram-negative pathogens (more than 30-fold, higher than fusions, which is 12-fold). Lower amounts of both membrane perturbing peptides or vancomycin/nisin are desirable, which can lower the chance of adverse effects appearing. In addition, each compound possesses a different mechanism of action, therefore reducing the chances to resistance happening simultaneously to both compounds. Moreover, synergism overcomes the disadvantages of fusion peptides. For this strategy, the membrane perturbing peptides, as well as nisin and vancomycin, can be commercial synthesized or purchased. We do not need to worry about the production yield and purity. Nisin, vancomycin and the membrane perturbing peptides are separated molecular entities, which decrease the size of each molecule a bit. The size of our membrane perturbing peptides is around 1.2 kDa. This may help the administration of drugs. Especially vancomycin is
already a clinical used antibiotic. Nisin is safe and has been used as food preservative for decades and nisin has been reported to be safe in preclinical tests (cell toxicity and hemolysis) [29]. Therefore, nisin in synergy with D8 might fond topical or GI-tract (gastrointestinal tract)
162
CH APTER 6: G en era l Di sc us sio n6
applications. D-peptides are also more stable in vivo than L-peptides against proteolysis. In vivo experiments are ongoing for vancomycin and GNP-D8 at the moment and further pharmacokinetic/pharma-codynamics tests will be provided to probe how far this combination can go into therapeutics. Combined therapy is already in use against, for instance, tuberculosis and many authors point at this strategy as an alternative to deal with MDR infections [30–33].
The research work described in this thesis focuses on expanding the use of the lantibiotic engineering and nisin/vancomycin (Figure 2).
Chapter 3, Chapter 4 and Chapter 5 address work expanding the
spectrum and use of nisin or vancomycin to inhibit the growth of Gram-negative pathogens. The bioengineering strategy, described in
Chapter 3, proved the feasibility of the discovery of new compounds
and new activities by engineering. But the inefficient production yield for engineered peptides limits its use and only peptide compounds are attachable in this system. The synergistic strategy performed in
Chapter 4 and Chapter 5 provides a quick assay of known available
compounds (not only peptides) and it is an easy screening in high throughput systems for synergy. It also overcomes the low yield and
Figure 2. Overview scheme of this thesis showing the relation between different chapters.
The nisin biosynthesis machinery was successfully applied to produce fused peptides which were against Gram-negative pathogens as well as introduce a lanthionine ring into vasopressin. In the battle against Gram-negative pathogens, nisin can be either bioengineeried or combined with membrane perturbing peptides, and then to be active against Gram-negative pathogens. Vanco-mycin also exhibited high synergism with membrane perturbing peptides against Gram-negative pathogens.
CH APTER 6: G en era l Di sc us sio n
6
purity problem when using the bioengineering process. However, in this system, only a pair of compounds can be detected at one time.
In Chapter 2, the NICE system and nisin modification machinery was applied to introducing a lanthionine bridge into vasopressin. In order to do that, the first cysteine of wild type vasopressin was changed to serine, and then expressed in the NICE system and isolated, purified and characterized. The formation of a lanthionine ring has been con-firmed by the LC-MS/MS results. It has been reported that cyclization of therapeutic peptides is a successful method to produce peptide ana-logs with improved stability and biological properties [10]. Thioether bridges can render peptide analogues more stable when compared to their linear or disulfide bond-cyclized counterparts due to the higher stability of the thioether linkage against oxidation and proteolysis compared to the disulfide bond [34]. Lantionine-containing clinically relevant peptides, including somatostain, angiotensin, and enkephalin, have been reported to exerted improve stability and pharmacodynamic properties [17, 18, 35]. Still, further purification and characterization of both in vitro and in vivo tests are needed for this work. As in
Chap-ter 3, production of sufficient amounts of highly pure compound has
been the main bottleneck for a thorough characterization of this novel vasopressin derivative. Further engineering e.g. by directed evolution of the already promiscuous NisB and NisC remains a challenge that can boost the use of this system for the production of relevant bioactive or antimicrobial (e.g. nisin hybrids, exotic lantibiotics) compounds [36]. In summary, the nisin biosynthesis machinery was successfully ap-plied and produced functional fused peptides against Gram-negative pathogens as well as introduced a lanthionine ring into vasopressin. Moreover, two strategies were employed to increase the activity of nisin against selected Gram-negative pathogens. Even though further in vivo tests (e.g. pharmacokinetic/pharmacodynamics tests) are needed, the fused peptides and combinations of two compounds described here constitute new candidate therapeutic approaches to deal with (MDR) Gram-negative pathogens.
164
CH APTER 6: G en era l Di sc us sio n6
[1] Ferri M, Ranucci E, Romagnoli P, Giaccone V. Antimicrobial resistance: a global emerg-ing threat to public health systems. Criti-cal reviews in food science and nutrition 2017;57:2857–76.
[2] Laxminarayan R, Duse A, Wattal C, Zaidi AK, Wertheim HF, Sumpradit N, et al. An-tibiotic resistance-the need for global solutions. The lancet infectious diseases 2013;13:1057–98.
[3] Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges and respons-es. Nature medicine 2004;10:S122-S9. [4] D’Costa VM, King CE, Kalan L, Morar M, Sung
WW, Schwarz C, et al. Antibiotic resistance is ancient. Nature 2011;477:457. [5] Hampton T. Report reveals scope of US antibiotic
resistance threat. Jama 2013;310:1661–3. [6] Roberts RR, Hota B, Ahmad I, Scott RD, Foster
SD, Abbasi F, et al. Hospital and societal costs of antimicrobial-resistant infections in a Chicago teaching hospital: implications for antibiotic stewardship. Clinical infec-tious diseases 2009;49:1175–84. [7] Bush K, Courvalin P, Dantas G, Davies J,
Eisen-stein B, Huovinen P, et al. Tackling antibiot-ic resistance. Nature reviews mantibiot-icrobiology 2011;9:894–6.
[8] Lubelski J, Overkamp W, Kluskens LD, Moll GN, Kuipers OP. Influence of shifting positions of Ser, Thr and Cys residues in prenisin on the efficiency of modification reactions and on the antimicrobial activities of the modified prepeptides. Applied and envi-ronmental microbiology 2008;74:4680–5. [9] Bierbaum G, Szekat C, Josten M, Heidrich C,
Kempter C, Jung G, et al. Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5. Applied and environmental microbiology 1996;62:385–92.
[10] Kluskens LD, Nelemans SA, Rink R, de Vries L, Meter-Arkema A, Wang Y, et al. Angioten-sin-(1–7) with thioether bridge: an angio-tensin-converting enzyme-resistant, potent angiotensin-(1–7) analog. The journal of pharmacology and experimental therapeu-tics 2009;328:849–54.
[11] Suda S, Westerbeek A, O’Connor PM, Ross RP, Hill C, Cotter PD. Effect of bioengi-neering lacticin 3147 lanthionine bridg-es on specific activity and rbridg-esistance to heat and proteases. Chemistry & biology 2010;17:1151–60.
[12] Rink R, Arkema-Meter A, Baudoin I, Post E, Kuipers A, Nelemans S, et al. To protect peptide pharmaceuticals against peptidases.
Journal of pharmacological and toxicologi-cal methods 2010;61:210–8.
[13] Kunji ER, Slotboom D-J, Poolman B.
Lacto-coccus lactis as host for overproduction of
functional membrane proteins. Biochimica et biophysica acta 2003;1610:97–108. [14] Kunji ER, Chan KW, Slotboom DJ, Floyd S,
O’Connor R, Monne M. Eukaryotic mem-brane protein overproduction in
Lactococ-cus lactis. Current opinion in biotechnology
2005;16:546–51.
[15] Mierau I, Kleerebezem M. 10 years of the ni-sin-controlled gene expression system (NICE) in Lactococcus lactis. Applied micro-biology and biotechnology 2005;68:705–17. [16] Moll GN, Kuipers A, Rink R. Microbial en-gineering of dehydro-amino acids and lanthionines in non-lantibiotic peptides. Antonie van leeuwenhoek 2010;97:319–33. [17] Rew Y, Malkmus S, Svensson C, Yaksh TL,
Chung NN, Schiller PW, et al. Synthesis and biological activities of cyclic lanthio-nine enkephalin analogues: δ-opioid recep-tor selective ligands. Journal of medicinal chemistry 2002;45:3746–54.
[18] Osapay G, Prokai L, Kim H-S, Medzihradsz-ky KF, Coy DH, Liapakis G, et al. Lan-thionine-somatostatin analogs: synthesis, characterization, biological activity and enzymatic stability studies. Journal of me-dicinal chemistry 1997;40:2241–51. [19] Breukink E, de Kruijff B. Lipid II as a target for
antibiotics. Nature reviews drug discovery 2006;5:321–32.
[20] Li B, Yu JP, Brunzelle JS, Moll GN, van der Donk WA, Nair SK. Structure and mechanism of the lantibiotic cyclase involved in nisin biosynthesis. Science 2006;311:1464–7. [21] Hsu S-TD, Breukink E, Tischenko E, Lutters MA,
de Kruijff B, Kaptein R, et al. The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel anti-biotics. Nature structural and molecular biology 2004;11:963.
[22] Breukink E, van Heusden HE, Vollmerhaus PJ, Swiezewska E, Brunner L, Walker S, et al. Lipid II is an intrinsic component of the pore induced by nisin in bacterial mem-branes. The journal of biological chemistry 2003;278:19898–903.
[23] Breukink E, Wiedemann I, Van Kraaij C, Kuipers O, Sahl H-G, De Kruijff B. Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic. Science 1999;286:2361–4.
[24] Hasper HE, Kramer NE, Smith JL, Hillman J, Zachariah C, Kuipers OP, et al. An
CH APTER 6: G en era l Di sc us sio n
6
alternative bactericidal mechanism of ac-tion for lantibiotic peptides that target lipid II. Science 2006;313:1636–7.
[25] Kahne D, Leimkuhler C, Lu W, Walsh C. Gly-copeptide and lipoglyGly-copeptide antibiotics. Chemical reviews 2005;105:425–48. [26] Brötz H, Sahl H-G. New insights into the
mechanism of action of lantibiotics-diverse biological effects by binding to the same molecular target. Journal of antimicrobial chemotherapy 2000;46:1–6.
[27] Koopmans T, Wood TM, ’t Hart P, Kleijn LH, Hendrickx AP, Willems RJ, et al. Semi-synthetic lipopeptides derived from nisin display antibacterial activity and lipid II binding on par with that of the parent com-pound. Journal of the american chemical society 2015;137:9382–9.
[28] Arnusch CJ, Bonvin AM, Verel AM, Jansen WT, Liskamp RM, de Kruijff B, et al. The vanco-mycin-nisin (1–12) hybrid restores activity against vancomycin resistant Enterococci. Biochemistry 2008;47:12661–3. [29] Maher S, McClean S. Investigation of the
cyto-toxicity of eukaryotic and prokaryotic an-timicrobial peptides in intestinal epithelial cells in vitro. Biochemical pharmacology 2006;71:1289–98.
[30] Naghmouchi K, Baah J, Hober D, Jouy E, Ru-brecht C, Sané F, et al. Synergistic effect between colistin and bacteriocins in con-trolling Gram-negative pathogens and their potential to reduce antibiotic toxicity in mammalian epithelial cells. Antimicrobial agents and chemotherapy 2013;57:2719–25. [31] Cavera VL, Arthur TD, Kashtanov D, Chikindas ML. Bacteriocins and their position in the next wave of conventional antibiotics. In-ternational journal of antimicrobial agents 2015;46:494–501.
[32] Zayyad H, Eliakim-Raz N, Leibovici L, Paul M. Revival of old antibiotics: needs, the state of evidence and expectations. Inter-national journal of antimicrobial agents 2017;49:536–41.
[33] Cassir N, Rolain J-M, Brouqui P. A new strategy to fight antimicrobial resistance: the revival of old antibiotics. Frontiers in microbiology 2014;5:551.
[34] Tugyi R, Mezö G, Fellinger E, Andreu D, Hudecz F. The effect of cyclization on the enzymatic degradation of herpes simplex virus glyco-protein D derived epitope peptide. Journal of peptide science 2005;11:642–9. [35] Durik M, van Veghel R, Kuipers A, Rink R,
Haas Jimoh Akanbi M, Moll G, et al. The effect of the thioether-bridged, stabilized
angiotensin-(1–7) analogue cyclic ang-(1– 7) on cardiac remodeling and endothelial function in rats with myocardial infarc-tion. International journal of hypertension 2011;2012:1–8.
[36] van Heel AJ, Kloosterman TG, Montalban-Lopez M, Deng J, Plat A, Baudu B, et al. Discovery, production and modification of five novel lantibiotics using the promiscuous nisin modification machinery. ACS synthetic biology 2016;5:1146–54.
