University of Groningen Breaking walls: combined peptidic activities against Gram-negative human pathogens Li, Qian

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University of Groningen

Breaking walls: combined peptidic activities against Gram-negative human pathogens

Li, Qian

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Publication date: 2019

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Li, Q. (2019). Breaking walls: combined peptidic activities against Gram-negative human pathogens. University of Groningen.

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Breaking walls:

combined peptidic activities

against Gram-negative

human pathogens

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The work described in this thesis was carried out in Molecular Genetics Group of the Groningen Biomolecular Sciences and Biotechnology Institute (GBB) of the University of Groningen, the Netherlands. The candidate was financially supported by the China Scholarship Council(CSC). Printing of this thesis was financially supported by the Graduate School of Science and Engi-neering and the University of Groningen.

Cover: Qian Li & Lovebird design. Layout: Lovebird design. www.lovebird-design.com Printing: Eikon +

ISBN: 978-94-034-1343-3 (printed book) ISBN: 978-94-034-1342-6 (ebook)

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Breaking walls: combined peptidic

activities against Gram-negative

human pathogens

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus prof. E. Sterken

and in accordance with the decision by the College of Deans. This thesis will be defended in public on Monday 18 February 2019 at 9.00 hours

by

Qian Li

born on 10 April 1988 in Wuhan, China

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Supervisor

Prof. O.P. Kuipers

Co-supervisor

Dr. M. Montalban -Lopez

Assessment Committee

Prof. A.J.M. Driessen Prof. G.N. Moll Prof. W. Bitter

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To my family

致我亲爱的家人

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Chapter

1

General Introduction

Qian Li1, Manuel Montalban-Lopez1,2, Oscar P. Kuipers1

1Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands 2 Department of Microbiology, Faculty of Sciences, University of Granada, Spain

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CH APTER 1: O ver vie w o f l an thi pep tides

1. Overview of lanthipeptides

Ribosomally synthesized and post-translationally modified peptides (RiPPs) have been identified in the 21st century as the fifth major group of natural products, besides terpenoids, alkaloids, polyketides and non-ribosomal peptides [1]. Lanthipeptides are a class of polycyclic RiPPs containing meso-lanthionine (Lan) and 3-methyllanthionine (MeLan) residues [1, 2]. Lanthipeptides can display diverse activities, including morphogenetic [3], antiviral [4], antimicrobial [5] or antial-lodynic effect [6]. Lanthipeptides possessing antimicrobial activity are called lantibiotics (lanthionine-containing antibiotics). Lanthionine (Lan) is composed of two alanine residues whose beta carbons are crosslinked via a thioether bridge; while methyllanthionine (MeLan) contains one additional methyl group compared to lanthionine [2]. These (methyl)lanthionine bonds are critical for their activities [7, 8] as well as their thermostability, proteolytic resistance and are important features in pharmaceutical applications [9–11].

1.1. Classification of lanthipeptides

Natural lanthipeptides are ribosomally synthesized as precursor pep-tides and the linear precursor peptide contains a leader peptide and a core peptide. The core peptide can become the mature compound through the insertion of posttranslational modifications (PTMs) car-ried out by PTM enzymes and their transport and activation by the specific leader protease. These processes are mainly guided by the leader peptide [5, 12, 13]. The Lan and MeLan residues are introduced to the precursor peptides by two enzymatic steps mediated by one or more enzymes. Serine and threonine are dehydrated to become dehydroal-anine (Dha) and dehydrobutyrine (Dhb), respectively, which then can be coupled to a cysteine via a Michael-type addition to form a thioether link. Based on the PTM enzymes involved in the maturation process of core peptides, lanthipeptides can be divided into four distinct classes (Class I, II, III and IV) [2] (Figure 1).

In class I lanthipeptides (e.g. nisin, gallidermin), the (methyl) lan-thionine residues are formed by a dehydratase (LanB) and a cyclase (LanC) (Figure 1). Subsequently, the fully modified peptides are ex-ported by a transmembrane ATP-binding cassette (ABC) transporter

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CH APTER 1: G en era l I nt ro duc tio n

1

(LanT) and the leader peptides can be cleaved by a protease (generally

LanP) [14]. The elongated and flexible secondary structure of class I lanthipeptides plays a key role in the antimicrobial effect of them to bind to lipid II and/or form pores in most cases [15, 16].

In class II lanthipeptides (e.g. mersacidin, lacticin 481, halodura-cin), both the dehydration and cyclization reactions are catalyzed by a bifunctional modification enzyme, called LanM. The N-terminal dehydratase domain of LanM does not share similarities with Lan B [17] but the C-terminus shows about 25 % sequence homology to LanC, including the conserved zinc-binding residues [2, 18] (Figure 1). A single, multifunctional protein LanT, with a conserved N-terminal protease domain, is responsible for secretion and leader processing in class II lanthipeptides [2, 14]. It is notable that there are various two-component lantibiotics within class II lanthipeptides, including lacticin 3147 [19], haloduracin [20], lichenicidin [21], plantaricin W [22] and some others. The two peptides work synergistically to exert antimicrobial activity. They are encoded by their own structural genes and modified by individual LanM enzymes but transported by a single LanT, which will cleave off the leader peptides.

Class III lanthipeptides (e.g. SapB, SapT, labyrinthopeptins), which perform morphogenetic and signal functions instead of antimicrobial activity, are modified by a single trifunctional enzyme termed LanKC. LanKC contains an N-terminal lyase domain, a central kinase domain and a putative C-terminal cyclase domain [23]. The cyclase domain bears limited homology to LanC and LanM, but is lacking the con-served zinc ligands [24] (Figure 1). In addition, labyrinthopeptins, known as a class III lanthipeptides, can also form a so-called labionin structure [6]. The labionin (Lab) structure is synthesized from two serine residues and one cysteine residue. It refers to a carbocyclic structure formed by two steps, including 1) the generation of an eno-late intermediate by the addition of a cysteine thiol to Dha and 2) the addition of a second Dha to the intermediate [25, 26].

Class IV lanthipeptides have been established in 2010 after the inden-tification of venezuelin, in Streptomyces venezuelae [27]. The synthetase, LanL, resembles the LanKC, but differs at the C-terminal domain. The C-terminal cyclase domain of LanL shows homology to LanC and contains the characteristic zinc-binding motif [27, 28] (Figure 1).

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1.2. Activity of lanthipeptides

The mechanism by which lantibiotics exert their antimicrobial activities has been fully investigated only in a few cases, showing that the modes of action of lantibiotics are mainly based on the inhibition of cell wall biosynthesis, disruption of membrane integrity through pore forma-tion or a combinaforma-tion of both [2]. In some cases lipid II, the essential precursor for cell wall biosynthesis, serves as the target of lantibiotics to inhibit the growth of bacteria (Figure 2). Nisin binds to the pyro-phosphate moiety of lipid II via the N-terminal ring A and ring B and forms a pyrophosphate cage. Then nisin bends, inserts its C-terminus into the membrane and forms transmembrane pores [29]. Thus, nisin exerts two killing mechanisms: it permeabilizes the membrane and inhibits cell wall synthesis [16, 30, 31].

Since the ring pattern of rings A and B are quite conserved in some lantibiotics other than nisin, including microbisporicin, mutacin 1140, gallidermin and epidermin, the same binding motif can probably also be formed for these antibiotics [2, 32, 33]. However, unlike nisin, the

Figure 1. Schematic representation of the four classes of lanthipeptides, based on the lanthi-onine introducing modification enzymes (adapted from Knerr et al. 2012 [2]). The dark lines

in LanC and LanC-like cyclase domain represent the conserved Zn-ligands. LanB, lanthipeptide dehydratase; LanC, lanthipeptide cyclase; LanM, class II lanthipeptide synthetase; LanKC, class III lanthipeptide synthetase; LanL, class IV lanthipeptide synthetase.

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C-terminal part of epidermin and gallidermin is shorter and thus the compound is unable to translocate over the cell membrane to form pores [32]. Lipid II is also a target for class II lantibiotics such as mersacidin, which inhibits transglycosylation, but does not form pores [34, 35]. Ring C is essential for this interaction and it is conserved in mersacidin-like peptides, which suggests a similar reaction for those peptides [35, 36]. For the two-component lantibiotics, the two peptides work synergisti-cally, in such a way that the α peptide binds to lipid II resembling the mersacidin-binding motif (Figure 2) and the β peptide is involved in

Figure 2. Structures of representative lanthipeptides (adapted from Knerr et.al. 2012 [2] and Dischinger et.al. 2014 [14]). The canonical nisin- and mersacidin-lipid II binding motifs

are highlighted with green or red dashed circles, respectively. The rings of nisin, haloduracin, SapT are labelled. Dehydrated amino acids are shown in green. Dha denotes dehydroalanine and Dhb is dehydrobutyrine. Lan are shown in pink, MeLan are shown in blue. Disulfide bridges are shown is yellow.

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pore formation upon binding to the complex lipid II-α-peptide [37, 38]. Some other activities were also reported, such as for Pep5 and epilancin K7, which do not use lipid II as target, but still form pores [15].

Cinnamycin-like peptides were found to inhibit phospholipase A2 [39]. They bind to phosphatidylethanolamine in the cell wall, induce transbilayer lipid movement and may confer toxic effects [40].

1.3. Additional post-translational modifications (PTMs) of lantibiotics

Lan/MeLan residues define and play vital roles in the biological activity and stability of the lanthipeptides. At present, many other different post-translational modifications (PTMs) have been documented in lantibiotics [2, 41, 42]. The PTMs have greatly enhanced the diversity of the lantibiotics, which initially is limited to 20 canonical amino acids [43]. During the process of Lan/MeLan formation, cysteine residues are involved and bound to unsaturated amino acids dehydroalanine (Dha) and dehydrobutyrine (Dhb). Moreover, the C-terminal cysteine residues can be enzymatically oxidized and decarboxylated render-ing S-aminovinyl-D-cysteine (AviCys) or S-aminovinyl-3-methyl-D- cysteine (AviMeCys) structures, as found in epidermin and mersacidin, respectively [42]. These structures are introduced by EpiD and MrsD, both of which reveal a conserved Rossman fold typically found in flavo-doxin-like proteins [44, 45]. AviCys and AviMeCys can protect the pep-tide from carboxypeptidases and contribute to the full activity [46, 47]. Spontaneous hydrolysis of N-terminally exposed Dha and Dhb resi-dues has also been reported during the maturation of Pep5, epicidin 280 and epilancin 15X [48–50]. Dha and Dhb residues become exposed after leader processing and are subsequently hydrolyzed to yield 2-oxopro-pionyl (OPr) and 2-oxobutyryl (OBu) [51]. OPr can be further reduced by a LanO enzyme to form a 2-hydroxypropionyl (Hop) residue [49, 50]. Acetylation of the N-terminus of mature lantibiotics is observed in paenibacillin, isolated from Paenibacillus polymyxa OSY-DF [52, 53]. This N-terminal capping is likely to protect the compound from

aminopeptidases [42].

Moreover, LanJ can convert L-serine (L-Ser) to D-alanine (D-Ala) with Dha as an intermediate in lactocin S, carnolysin and lacticin 3147 [54–56]. The mechanism by which L-Ser is converted to D-Ala

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residues in lacticin 3147 is a two-step process involving dehydration

by the enzyme LtnM and stereospecific hydrogenation by LtnJ [57]. Cinnamycin and duramycin contain a lysinoalanine bridge (catalyzed by Cinorf7) and a hydroxylated aspartic acid (catalyzed by Cinx) [58, 59]. Microbisporicin exhibits two unique PTMs, a chlorinated trypto-phan (Cl-Trp) and a hydroxylated proline (HPro) [2, 60]. The disulfide ring presented in haloduracin and plantaricin W can protect the com-pounds from proteolytic degradation by proteases [61].

2. Nisin

Nisin, produced by Lactococcus lactis, is one of the oldest and most widely used antimicrobials and was first reported in 1928 [62]. Nisin is a cationic, amphipathic peptide, which can effectively kill Gram- positive bacteria including Bacillus cereus, Listeria monocytogenes, Staphylococci and Streptococci [12, 63, 64]. It is generally recognized as a safe additive in food preservation and recent studies show that it could also be applied as pharmaceutical [30, 64–66].

2.1. Biosynthesis of nisin

A two-component system, involving a histidine protein kinase NisK and a response regulator protein NisR, is required for the nisin biosyn-thesis regulation [12, 67] (Figure 3). In response to the external signal, which is fully mature nisin, the sensor kinase NisK phosphorylates itself and transfers a phosphoryl group to a conserved aspartic acid of NisR [12, 67]. NisR triggers the binding of the response regulator to nisA and nisF operators and then activates the transcription of the operons nisABTCIP and nisFEG [63, 68, 69]. The nisA gene encodes precursor nisin that consists of a leader peptide and a core peptide part. After ribosomal synthesis, prenisin can be dehydrated by NisB and the dehydrated residues are coupled to cysteine by NisC to form (methyl)lanthionine rings [12, 70]. Subsequently, the modified peptide is transported out of the cell by the ABC-transporter NisT and then the protease NisP can cut off the N-terminal leader peptide and liber-ate active nisin [71–74]. NisI and NisFEG are immunity proteins that protect the host from the antimicrobial action of nisin [12, 67, 75, 76].

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CH

APTER 1: N

isin

The dehydration, cyclization and transport, which are performed by NisB, NisC and NisT, respectively, are three crucial steps during nisin biosynthesis. It has been reported that a wide range of clinical relevant non-lantibiotic peptides (e.g. enkephalin, angiotensin-(1–7) and an erythropoietin-mimicking-peptide) can be successfully de-hydrated and secreted by a L. lactis strain containing nisBTC genes [77]. In vivo experiments with NisB, NisC and NisT were performed and the results showed that nisin modification enzymes have very relaxed substrate specificities [71, 77, 78]. Therefore, it suggests that the nisin modification system is very useful for efficient biotechno-logical production of various non-lantibiotic peptides with enhanced stability and/or modulated bioactivities.

Figure 3. Mode of biosynthesis of nisin in Lactococcus lactis (based on Oscar P. Kuipers,

et al, 1995 [67], Chan-Ick Cheigh and Yu-Ryang Pyun, 2005 [63]). The extracellular mature

nisin can act as an antimicrobial and the producing cells are protected against the nisin activity via the immunity system consisting of NisI and NisFEG. Mature nisin can also activate the biosynthesis of prenisin via NisR and NisK. Promoters marked with star (P*) are controlled by the two-component system NisRK.

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The nisin inducible system (NICE), which employs the auto-

induction mechanism of nisin for gene expression, was developed by Kuipers et al. [67, 79]. This induction system has been used to pro-duce a set of lanthionine-containing designed substrates that helped the characterization of the enzyme promiscuity and provided novel antimicrobial molecules. Initially, a pNZ-based vector where nisin mutants or fusions of peptides linked to the nisin leader peptide, un-der the control of PnisA, were constructed and expressed in a ΔnisA derivative of NZ9700. This renders mature molecules without the leader peptide in the culture supernatant, which eventually could give immunity problems. Thus, a two plasmids system was created to overcome the immunity issue when the designed peptides have antimicrobial activity. One contains nisB/C/T (or variants thereof) and the other one contains a polylinker, by which easy cloning of diverse structural genes fused to the nisin leader peptide's sequence can be achieved. Both the enzymes and the polylinker are under the control of the inducible promoter PnisA. In any case, when a gene of interest is placed behind the promoter PnisA on a plasmid [80] or on the chromosome [81–83], the expression of the gene can be triggered by nisin and the modification will be inserted. The modified precursor peptides can be isolated from the supernatant or cytoplasm, depending on whether NisT is included in the expression system or not. The NICE system has been extensively and successfully used for high expression of proteins from different origins for various applications [77, 84–88]. 2.2. Protein engineering of nisin

Five natural nisin variants have been described so far: nisin A, nisin Z, nisin Q, nisin U1 and nisin U2 [12]. Among them, both nisin A and nisin Z are produced by L. lactis and have only one amino acid differ-ence in position 27 (histidine in Nisin A and asparagine in Nisin Z) [42, 63]. The biological activities of nisin A and nisin Z are reported to be similar [89]. These variants highlight the tolerance of certain residues and domains within the molecule to change.

The engineering of nisin can help us to understand the mode of action, substrate specificity, biosynthesis regulation and biosynthesis machinery as well as to obtain new variants of nisin with altered bio-logical activities [12].

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A variety of nisin mutants have been created and reported (Table 1) since the first mutation made for nisin Z in 1992 [90]. Another expres-sion system was reported recently [91], via which nisin can produced with leader and then be processed in vitro later on for downstream applications. This well-established approach enabled the generation of interesting and improved mutants [71, 92].

Among all the mutants made so far, only two mutants with alteration of amino acids connected to the sulfur atoms forming lanthionine bonds were reported (S3T and T13C) [93, 94]. This resulted in the replace-ment of lanthionine by methyllanthionine and vice versa. Both of them strongly reduced the activity of nisin Z against Gram-positive bacteria and their activity on liposomes is also decreased. Thus, the specific ring structure of nisin is vital to the activity of nisin. However, the change of other amino acid residues within the (methyl)lanthionine ring had less impact on the activity (I4, S5, G10, M17 and G18) [90, 92–95]. The mutation I4K/S5F/L6I can even increase the activity of nisin and the stability of S5T was improved compared to that of wide type nisin Z. The lysine at position 12, which is between ring B and ring C, was proved to be a quite tolerant position for substitutions. K12A, K12S, K12P, K12V and K12T displayed slightly enhanced antimicrobial activities relative to nisin and K12A was 2–4 fold more active than nisin against all the nine strains tested [96]. The hinge region between rings A/B/C and rings D/E, which is postulated to be vital to confer flexibility for pore formation, has been quite intensively investigated by amino acids alterations [93, 97–99]. It has been reported that the introduction of aromatic residues or negatively charged residues at any position in the hinge had a negative impact on nisin bioactivity [97]. The introduction of positively charged residues is preferred and generally tolerated. The mutants display an activity similar to wild type nisin, but there are still structural considerations (the bulkier arginine residue showed the most reduced activity) [97]. Mutants N20P, M21V, M21G, M21A, K22G, K22A, K22T and K22S exerted enhanced bioactivity against Gram-positive bacteria including L. lactis, Listeria monocytogenes and/ or Staphylococcus aureus [97]. Mutants N20K and M21K have a higher solubility than wide type nisin Z and displayed some antimicrobial activity against Gram- negative bacteria including Shigella flexneri, Pseudomonas aeruginosa and Salmonella enterica [98]. These and other

CH

APTER 1: N

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results indicated a preference for small, positively charged and chiral

amino acids within the hinge region. The deletion of amino acids at position 20 and 21, which showed strongly reduced activity, illustrated that the length is also important for the hinge region functionality [93]. In 2012, Des Field and his co-authors reported a series of variants with changes at the position 29 of nisin A [100]. S29G and S29A were found to have enhanced efficacy against Staphylococcus aureus SA113, as well as Escherichia coli, Cronobacter sakazakii and Salmonella enterica [100]. Mutants with truncated nisin A/Z were also reported, and the results illustrated the importance of the ring structures for the activity [92, 93, 101, 102]. The mutant of nisin A with 32 amino acids of nisin A and an amidated C-terminus, kept a similar activity compared to nisin A, while the other mutants lost most of their antimicrobial activity against L. lactis and Micrococcus luteus or other strains (e.g. NisA1-31, NisA1-29, NisA1-20, NisA1-22 and NisA1-29) [92, 101, 102]. Moreover, the introduc-tion of fluorescent labels and tryptophan or its analogues all increase the fluorescent properties of nisin but decrease the activity of the mutants [95, 102, 103]. When a tail, which can facilitate the compounds to pass the outer-membrane of Gram-negative species, was added to nisin or truncated nisin, the activity of fusions against Gram-negative pathogens can be improved [104, 105]. These noticeable achievements by engi-neering nisin encourage the further investigation and application of lantibiotic compounds and provides a novel technology for molecular improvement.

3. Status of antibiotic use

‘Antibiotic’ was firstly used as a designation by Selman Waksman in 1941 to describe any small molecule which was made by a microbe to antago-nize the growth of other microbes [109]. Antibiotic discovery and clinical use is undoubtedly one of the landmark medical advances of modern medicine. Since Alexander Fleming found penicillin in 1928, the earliest use of antibiotics had a dramatic impact on the decrease of mortality of life-threatening bacterial infections. The period from 1945 to 1955, with the development of penicillin, streptomycin, chloramphenicol, and tetracycline, can be regarded as the golden age for antibiotics [109]. The industrial production made antibiotics available for common treatments.

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CH APTER 1: S ta tu s o f a nt ib io tic u se Ta bl e 1. N isin A/Z m uta nts a nd the ir cha rac te ris tics. ID M ut ati on G ene _name Bi ol og ic al ac tiv ity (r el at iv e t o the w ild t yp e) Ph ys ic al p ro pe rt ies (r el at iv e to the w ild t yp e) Cha rac te ris tics Ref er en ce Pa rt I V ari an ts w ith fu ll l en gth o f nis in A/Z 1 I1W ni sZ Simi la r ac tiv ity a ND Fl uo res cen t l ab el [103] 2 I1W ni sA Re duce d ac tiv ity b ND In tro ducin g t ryp to ph an [95] 3 I1–5FW ni sA Re duce d ac tiv ity b ND In tro ducin g t ryp to ph an a na logue [95] 4 I1–5HW ni sA St ro ng ly r ed uce d ac tiv ity b ND In tro ducin g t ryp to ph an a na logue [95] 5 T2S ni sZ Incr eas ed ac tiv ity a, c Re duce d ac tiv ity o n li pos om e D ha p res en t in t he fin al p ro duc t in ste ad o f D hb [93] 6 T2A ni sZ Simi la r ac tiv ity a, c Re duce d ac tiv ity o n li pos om e A lter in g de hy dra te d r esid ues [93] 7 T2V ni sZ Simi la r ac tiv ity a, c Re duce d ac tiv ity o n li pos om e A lter in g de hy dra te d r esid ues [93] 8 S3T ni sZ St ro ng ly r ed uce d ac tiv ity a, c Re duce d ac tiv ity o n li pos om e D hb p res en t in t he fin al p ro duc t in ste ad o f D ha [93] 9 I4–5FW ni sA Re duce d ac tiv ity b ND In tro ducin g t ryp to ph an a na logue [95] 10 I4K/L6I ni sA Simi la r ac tiv ity d,e ND A lter in g r esid ues in r in g A o f ni sin A [92] 11 I4K/S5F/L6I ni sA Incr eas ed ac tiv ity d,e ND A lter in g r esid ues in r in g A o f ni sin A [92] 12 I4V/S5F/L6G ni sA re duce d ac tiv ity d,e ND A lter in g r esid ues in r in g A o f ni sin A [92] 13 S5C ni sZ St ro ng ly r ed uce d ac tiv ity b, c ND A ltera tio n o f de hy dra ta ble r esid ue w hic h t ak es p ar t in t he r in g for m at ion [94] 14 S5T ni sZ Re duce d ac tiv ity a, c In cr ea se s ta bi lit y D hb p res en t in t he fin al p ro duc t in ste ad o f D ha [90] 15 Dh a5 Dh b ni sZ St ro ng ly r ed uce d ac tiv ity a, c In cr ea se s ta bi lit y D hb p res en t in t he fin al p ro duc t in ste ad o f D ha [89] 16 S5A ni sZ Re duce d ac tiv ity ND A lter in g de hy dra te d r esid ues [106] 17 S5A/S33A ni sA St ro ng ly r ed uce d ac tiv ity ND A lter in g de hy dra te d r esid ues [106] 18 G10T ni sA St ro ng ly r ed uce d ac tiv ity d,e ND A lter in g r esid ues t o de hy dra te d r esid ue in r in g B o f ni sin A [92] 19 K12H ni sA Re duce d ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 20 K12R ni sA Re duce d ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 21 K12T ni sA Incr eas ed ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96]

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ID M ut ati on G ene na me Bi ol og ic al ac tiv ity (r el at iv e t o the w ild t yp e) Ph ys ic al p ro pe rt ies (r el at iv e to the w ild t yp e) Cha rac te ris tics Ref er en ce Pa rt I V ari an ts w ith fu ll l en gth o f nis in A/Z 22 K12S ni sA Incr eas ed ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 23 K12N ni sA Simi la r ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 24 K12Q ni sA Simi la r ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 25 K12Y ni sA Re duce d ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 26 K12A ni sA Incr eas ed ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 27 K12P ni sA Incr eas ed ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 28 K12V ni sA Incr eas ed ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 29 K12M ni sA Simi la r ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 30 K12C ni sA Simi la r ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 31 K12L ni sA Simi la r ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 32 K12I ni sA Simi la r ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 33 K12G ni sA Re duce d ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 34 K12W ni sA Re duce d ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 35 K12F ni sA Re duce d ac tiv ity b ND A lter in g r esid ue b et w een r in g A a nd r in g B/C [96] 36 K12P ni sZ Simi la r ac tiv ity a ND Posi tiv e c ha rg e r ed uc tio n [103] 37 T13C ni sZ St ro ng ly r ed uce d ac tiv ity a, c St ro ng ly r ed uce d ac tiv ity o n lip os om e A ltera tio n o f de hy dra ta ble r esid ue w hic h t ak es p ar t in t he r in g for m at ion [93] 38 M17W ni sA St ro ng ly r ed uce d ac tiv ity b ND In tro ducin g t ryp to ph an [95] 39 M17–5HW ni sA St ro ng ly r ed uce d ac tiv ity b ND In tro ducin g t ryp to ph an a na logue [95] 40 M17W ni sZ Re duce d ac tiv ity a, c Simi la r ac tiv ity o n li pos om e Fl uo res cen t l ab el [107] 41 M17K ni sZ Re duce d ac tiv ity a, c In cr ea se so lu bi lit y Posi tiv e c ha rg e in tro duc tio n [93] 42 M17C ni sZ St ro ng ly r ed uce d ac tiv ity b, c ND In tro duc tio n o f de hy dra ta ble r esid ue w hic h t ak es p ar t in t he rin g f or m at io n [94] 43 M17Q/G18T ni sZ Simi la r ac tiv ity a, c ND A lter in g r esid ues in r in g C o f ni sin Z [90]

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ID M ut ati on G ene _name Bi ol og ic al ac tiv ity (r el at iv e t o the w ild t yp e) Ph ys ic al p ro pe rt ies (r el at iv e to the w ild t yp e) Cha rac te ris tics Ref er en ce Pa rt I V ari an ts w ith fu ll l en gth o f nis in A/Z 44 M17Q/G18D hb ni sZ Simi la r ac tiv ity a, c ND A lter in g r esid ues in r in g C o f ni sin Z [90] 45 N20C ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 46 N20A ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 47 N20S ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 48 N20T ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 49 N20V ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 50 N20L ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 51 N20I ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 52 N20P ni sA Incr eas ed ac tiv ity f ND A lter in g r esid ues in hin ge r eg io n [97] St ro ng ly r ed uce d ac tiv ity g 53 N20F ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 54 N20Y ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 55 N20W ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 56 N20D ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 57 N20R ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 58 N20H ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 59 M21N ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 60 M21Q ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 61 M21C ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 62 M21G ni sA Incr eas ed ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 63 M21A ni sA Incr eas ed ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 64 M21S ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 65 M21T ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] CH APTER 1: S ta tu s o f a nt ib io tic u se

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ID M ut ati on G ene na me Bi ol og ic al ac tiv ity (r el at iv e t o the w ild t yp e) Ph ys ic al p ro pe rt ies (r el at iv e to the w ild t yp e) Cha rac te ris tics Ref er en ce Pa rt I V ari an ts w ith fu ll l en gth o f nis in A/Z 66 M21V ni sA Incr eas ed ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 67 M21L ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 68 M21I ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 69 M21P ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 70 M21F ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 71 M21Y ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 72 M21W ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 73 M21E ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 74 M21R ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 75 M21K ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 76 K22Q ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 77 K22G ni sA Incr eas ed ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 78 K22A ni sA Incr eas ed ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 79 K22S ni sA Incr eas ed ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 80 K22T ni sA Incr eas ed ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 81 K22V ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 82 K22L ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 83 K22P ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 84 K22M ni sA Re duce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 85 K22F ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 86 K22W ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 87 K22D ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 88 K22E ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97]

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ID M ut ati on G ene _name Bi ol og ic al ac tiv ity (r el at iv e t o the w ild t yp e) Ph ys ic al p ro pe rt ies (r el at iv e to the w ild t yp e) Cha rac te ris tics Ref er en ce Pa rt I V ari an ts w ith fu ll l en gth o f nis in A/Z 89 K22R ni sA St ro ng ly r ed uce d ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 90 K22H ni sA Simi la r ac tiv ity f,g ND A lter in g r esid ues in hin ge r eg io n [97] 91 N20A/M21A/K22A ni sA Simi la r ac tiv ity b ND A lter in g r esid ues in hin ge r eg io n [99] St ro ng ly r ed uce d ac tiv ity f 92 N20A/M21A ni sA St ro ng ly r ed uce d ac tiv ity b, f ND A lter in g r esid ues in hin ge r eg io n [99] 93 N20S/M21A/K22A ni sA St ro ng ly r ed uce d ac tiv ity b, f ND A lter in g r esid ues in hin ge r eg io n [99] 94 N20S/M21L/K22S ni sA St ro ng ly r ed uce d ac tiv ity b, f ND A lter in g r esid ues in hin ge r eg io n [99] 95 M21A/K22I ni sA Simi la r ac tiv ity b St ro ng ly r ed uce d ac tiv ity f ND A lter in g r esid ues in hin ge r eg io n [99] 96 N20E ni sZ St ro ng ly r ed uce d ac tiv ity a, c ND A lter in g r esid ues in hin ge r eg io n [98] 97 N20F ni sZ Simi la r ac tiv ity a, c Simi la r s ta bi lit y A lter in g r esid ues in hin ge r eg io n [98] 98 N20H ni sZ Simi la r ac tiv ity a, c Simi la r s ta bi lit y A lter in g r esid ues in hin ge r eg io n [98] 99 N20K ni sZ Incr eas ed ac tiv ity h,i In cr ea se d s olu bi lit y A lter in g r esid ues in hin ge r eg io n b y in tro ducin g p osi tiv e ch arge [98] Simi la r ac tiv ity a, c 100 N20Q ni sZ Simi la r ac tiv ity a, c Im pr ov ed s ta bi lit y in hig her tem pera tur e a nd pH A lter in g r esid ues in hin ge r eg io n [98] 101 N20V ni sZ St ro ng ly r ed uce d ac tiv ity a, c Simi la r s ta bi lit y A lter in g r esid ues in hin ge r eg io n [98] 102 M21E ni sZ St ro ng ly r ed uce d ac tiv ity a, c ND A lter in g r esid ues in hin ge r eg io n [98] 103 M21G ni sZ Simi la r ac tiv ity a, c Im pr ov ed s ta bi lit y in hig her tem pera tur e a nd pH A lter in g r esid ues in hin ge r eg io n [98] 104 M21H ni sZ Simi la r ac tiv ity a, c Simi la r s ta bi lit y A lter in g r esid ues in hin ge r eg io n [98] 105 M21K ni sZ Incr eas ed ac tiv ity h,i In cr ea se d s olu bi lit y A lter in g r esid ues in hin ge r eg io n b y in tro ducin g p osi tiv e ch arge [98] Simi la r ac tiv ity a, c 106 K22G ni sZ Simi la r ac tiv ity a, c Simi la r s ta bi lit y A lter in g r esid ues in hin ge r eg io n [98] 107 K22H ni sZ Simi la r ac tiv ity a, c Simi la r s ta bi lit y A lter in g r esid ues in hin ge r eg io n [98] CH APTER 1: S ta tu s o f a nt ib io tic u se

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ID M ut ati on G ene na me Bi ol og ic al ac tiv ity (r el at iv e t o the w ild t yp e) Ph ys ic al p ro pe rt ies (r el at iv e to the w ild t yp e) Cha rac te ris tics Ref er en ce Pa rt I V ari an ts w ith fu ll l en gth o f nis in A/Z 108 N20K/M21K ni sZ Simi la r ac tiv ity a, c Simi la r s ta bi lit y D ou ble m ut at io n o f a sp ara gin e 20 an d m et hio nin e 21 t o l ysin es [98] 109 N20F/M21L/K22Q ni sZ Simi la r ac tiv ity a, c Simi la r s ta bi lit y H in ge r eg io n o f ni sinZ t o hin ge reg io n o f s ub tilin [98] 110 N20A/M21K/D hb/ K22G ni sZ St ro ng ly r ed uce d ac tiv ity a, c Simi la r s ta bi lit y H in ge r eg io n o f ni sinZ t o hin ge reg io n o f ep ider min [98] 111 N20P/M21P ni sZ St ro ng ly r ed uce d ac tiv ity a, c St ro ng ly r ed uce d ac tiv ity o n lip os om e A lter in g r esid ues in hin ge r eg io n [93] 112 M21G ni sZ St ro ng ly r ed uce d ac tiv ity a , c St ro ng ly r ed uce d ac tiv ity o n lip os om e A lter in g r esid ues in hin ge r eg io n [93] 113 N27K ni sZ Simi la r ac tiv ity a,b , c In cr ea se d s olu bi lit y Ch ar ge a ltera tio n [89] 114 S29T ni sA Re duce d ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 115 S29Q ni sA Simi la r ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 116 S29N ni sA Simi la r ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 117 S29Y ni sA Re duce d ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 118 S29D ni sA Incr eas ed ac tiv ity b, k ND A lter in g t he r esid ue a t p osi tio n 29 [100] Simi la r ac tiv ity f, j 119 S29E ni sA Incr eas ed ac tiv ity b,h , j,l ND A lter in g t he r esid ue a t p osi tio n 29 [100] Simi la r ac tiv ity f 120 S29R ni sA Re duce d ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 121 S29H ni sA Re duce d ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 122 S29K ni sA Re duce d ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 123 S29A ni sA Incr eas ed ac tiv ity b, f,h, j, k,l ND A lter in g t he r esid ue a t p osi tio n 29 [100] 124 S29V ni sA Re duce d ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 125 S29G ni sA Incr eas ed ac tiv ity b, f,h, j, k,l ND A lter in g t he r esid ue a t p osi tio n 29 [100] 126 S29C ni sA Re duce d ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100]

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ID M ut ati on G ene _name Bi ol og ic al ac tiv ity (r el at iv e t o the w ild t yp e) Ph ys ic al p ro pe rt ies (r el at iv e to the w ild t yp e) Cha rac te ris tics Ref er en ce Pa rt I V ari an ts w ith fu ll l en gth o f nis in A/Z 127 S29L ni sA Simi la r ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 128 S29I ni sA Re duce d ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 129 S29W ni sA Simi la r ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 130 S29F ni sA Re duce d ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 131 S29M ni sA Simi la r ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 132 S29P ni sA Simi la r ac tiv ity b ND A lter in g t he r esid ue a t p osi tio n 29 [100] 133 I30W ni sA Simi la r ac tiv ity b ND Fl uo res cen t l ab el [108] 134 H31K ni sZ Simi la r ac tiv ity a, c In cr ea se d s olu bi lit y Ch ar ge a ltera tio n [89] St ro ng ly r ed uce d ac tiv ity b 135 V32W ni sZ Re duce d ac tiv ity a, c ND Fl uo res cen t l ab el [102] 136 V32K ni sZ Re duce d ac tiv ity a, c ND Posi tiv e c ha rg e in tro duc tio n [102] 137 V32E ni sZ St ro ng ly r ed uce d ac tiv ity a, c ND N ega tiv e c ha rg e in tro duc tio n [102] 138 S33A ni sA St ro ng ly r ed uce d ac tiv ity ND A lter in g de hy dra te d r esid ues [106] Pa rt II V ari an ts w ith t runc at ed nis in A/Z 139 ΔN20/ΔM21 ni sZ St ro ng ly r ed uce d ac tiv ity a, c St ro ng ly r ed uce d ac tiv ity o n lip os om e A lter in g hin ge r eg io n [93] 140 Ni sZ 1-32 V32E ni sZ St ro ng ly r ed uce d ac tiv ity a , c ND Infl uen ce o f t he C-t er min al [102] 141 Ni sA 1-32 amide ni sA Simi la r ac tiv ity b,m ND Pr ot eo lyt ic al ly c le av ed , a ll l an thio nin e r in g p res en t [101] 142 Ni sA 1-31 ni sA St ro ng ly r ed uce d ac tiv ity b ND Pr ot eo lyt ic al ly c le av ed , a ll l an thio nin e r in g p res en t [101] 143 Ni sA 1-29 ni sA St ro ng ly r ed uce d ac tiv ity b,m ND Pr ot eo lyt ic al ly c le av ed , a ll l an thio nin e r in g p res en t [101] 144 Ni sA 1-20 ni sA St ro ng ly r ed uce d ac tiv ity b,m ND Pr ot eo lyt ic al ly c le av ed , r in g D a nd E r em ov ed [101] 145 Ni sA 1-12 ni sA St ro ng ly r ed uce d ac tiv ity b,m ND Pr ot eo lyt ic al ly c le av ed , r in g C, D a nd E r em ov ed [101] 146 Ni sA 1-22 ni sA St ro ng ly r ed uce d ac tiv ity b ND rin g D a nd E r em ov ed [92] 147 Ni sA 1-22 G10T ni sA St ro ng ly r ed uce d ac tiv ity b ND rin g D a nd E r em ov ed , A lter in g r esid ues in r in g B o f ni sin A [92]

CHAPTER 1: Status of antibiotic use

CH APTER 1: S ta tu s o f a nt ib io tic u se

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ID M ut ati on G ene na me Bi ol og ic al ac tiv ity (r el at iv e t o the w ild t yp e) Ph ys ic al p ro pe rt ies (r el at iv e to the w ild t yp e) Cha rac te ris tics Ref er en ce Pa rt II V ari an ts w ith t runc at ed nis in A/Z 148 Ni sA 1-22 I4K/L6I ni sA St ro ng ly r ed uce d ac tiv ity b ND rin g D a nd E r em ov ed , A lter in g r esid ues in r in g A o f ni sin A [92] 149 Ni sA 1-22 I4K/S5F/L6I ni sA St ro ng ly r ed uce d ac tiv ity b ND rin g D a nd E r em ov ed , A lter in g r esid ues in r in g A o f ni sin A [92] 150 Ni sA 1-22 I4V/S5F/L6G ni sA St ro ng ly r ed uce d ac tiv ity b ND rin g D a nd E r em ov ed , A lter in g r esid ues in r in g A o f ni sin A [92] Pa rt III V ari an ts w ith nis in A a nd ta ils 151 Ni sA 1-34 PR PPH PR L ni sA Incr eas ed ac tiv ity l ND “P RP PHP RL ” w er e adde d a fter ni sin A [105] St ro ng ly r ed uce d ac tiv ity b 152 Ni sA 1-34 N GV Q PKY ni sA Incr eas ed ac tiv ity l,i ,n, o ND “N GV Q PKY ” w er e adde d a fter ni sin A [104] St ro ng ly r ed uce d ac tiv ity b 153 Ni sA 1-28 SVN GV Q PKYK ni sA Incr eas ed ac tiv ity l,i ,n, p ND “SVN GV Q PKYK ” w er e adde d a fter r in g AB CD E o f ni sin A [104] St ro ng ly r ed uce d ac tiv ity b 154 Ni sA 1-28 SVKI AKV ALK ALK ni sA Incr eas ed ac tiv ity l,i ,n, o, p ND “SVKI AKV ALK ALK ” w er e adde d a fter r in g AB CD E o f ni sin A [104] St ro ng ly r ed uce d ac tiv ity b 155 Ni sA 1-28 SVP RP PHP RLK ni sA Incr eas ed ac tiv ity l,i ,n, o, p ND “SVP RP PHP RLK ” w er e adde d a fter r in g AB CD E o f ni sin A [104] St ro ng ly r ed uce d ac tiv ity b N ot e: ND , n ot det er min ed . (1) I ncr ea se d ac tiv ity , >120 % co m pa re d t o t he ac tiv ity o f w ild t yp e ni sin A/Z; S imi la r ac tiv ity , 80 %-100 %; R ed uce d ac tiv ity , 20–80 %; S tro ng ly r ed uce d ac tiv ity , <20 %. (2) C ol umn s a re l ab el le d acco rdin g t o t he b io log ic al ac tiv ities o f t he m ut an ts. M ut an ts w ith in cr ea se d b io log ic al ac tiv ity a re l ab el le d a s b old a nd d ar k g re y; m ut an ts w ith simi la r bio log ic al ac tiv ity a re l ab el le d a s lig ht g ra y. (3) 5FW , 5-fl uo ro tr yp to ph an; 5HW , 5-h ydr oxyt ryp to ph an. H in ge r eg io n, a min o acid r esid ues b et w een r in g A/B/C a nd r in g D/E; ΔN20/ΔM21, de let io n o f a sp ara gin e in p osi tio n 20 an d m et hio nin e in p osi tio n21; N um ber in s up er scr ip t, a min o acid p osi tio n. (4) B io log ic al ac tiv ity (r el at iv e t o t he w ild t yp e), let ter in s up er scr ip t f or in dic at or s tra in s: a, M icr oc oc cu s fla vu s; b , L ac to co cc us lac tis ; c, St re pt oco cc us the rmo ph ile s; d , P ed ioc oc cu s pen to sa ceu s; e, Leu co no sto c m es en ter oi de s; f, St ap hy loc oc cu s a ur eu s; g , S trep toc oc cu s a ga la ct ia e, h, Sa lmo ne lla en te rica ; i , P se ud omo na s ae ru gi no sa ; j , B aci llu s c er eu s; k , C ro no ba ct er sa kaz ak ii; l, Es ch er ich ia c ol i; m, M icr oc oc cu s l ut eu s; n, Kle bs iel la p ne um on ia e; o , A cin et ob ac te r b aum an nii ; p , E nt er ob ac ter a er ogen es .

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CH APTER 1: B ac ter ia l ce ll en ve lo pe a nd a nt ib io tics ac tin g a t t he ce ll en ve lo pe

According to a report for 71 selected countries, between 2000 and 2010, the consumption of antibiotic drugs substantially increased by 35 % (from 52,057,163,835 standard units in 2000 to 70,440,786,553 standard units in 2010, where standard unit means a single dose unit, i.e. pill/cap-sule/or ampoule). Among these selected countries, India was revealed to be the biggest consumer of antibiotics in 2010 with the consumption of 12.9 × 109 units in total and 10.7 units per person, followed by China (approximate 10.0 × 109 units in total, 7.5 units per person) and the USA (6.8 × 109 units in total, 22.0 units per person, with a moderate decrease from 2000 to 2010 actually) [110]. Moderately high consumption of an-tibiotics was also reported for Australia and New Zealand. The antibiotic consumption increased substantially in developing countries, and the highest rates are found in BRICS countries (Brazil, Russia, India, China, and South Africa) and French West Africa. An increased consumption of glycopeptides, carbapenems, polymixins, and monobactams was observed and reported in many countries [110].

The widespread excessive or sometimes abusive use of the antibiot-ics in agriculture, veterinary and human medicine is one of the main reasons for the dramatic increase of bacterial resistance [111, 112]. In order to fight the increased resistance of bacteria to existing antibiotics, there is a rather urgent need for discovering and developing new anti-biotics. However, the time-consuming and costly clinical development process and unpredictable economic benefit of antibiotics at the generic market has lost its attractiveness to pharmaceutical industry [113]. As a consequence, only a few antibiotics reached the market in recent decades [114–116]. Figure 4 shows the novel antibiotics and the total number of molecules approved by US Food and Drug Administration (FDA) for each year from 2003 to 2017 [114].

4. Bacterial cell envelope and antibiotics

acting at the cell envelope

4.1. Bacterial cell envelope

Bacteria face various environments, which are usually unpredictable and hostile. To survive, bacteria have evolved a sophisticated and complex cell envelop that acts as a barrier to the environment and

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protects them [117, 118]. The cell envelope comprises membrane(s)

and other structures that surround and protect the cytoplasm. Besides protection, the cell envelope allows the selective transport of nutrients from the outside and waste products from the inside of the cells, and it is also indispensable for division, growth, and morphogenesis [117]. The cell envelope can fall in two major categories, Gram-negative and Gram-positive (Figure 5), distinguished by Gram staining [117]. There are two membranes in Gram-negative bacteria, the outer membrane (OM), a thin peptidoglycan layer in between, and the cytoplasmic or inner membrane (IM). The OM is composed of glycolipids, princi-pally lipopolysaccharide (LPS) [119] and it is responsible for the low penetrability and high resistance to some antibiotics [120, 121]. LPS is critical to the barrier function of OM and is responsible for the endo-toxic shock associated with the septicemia caused by Gram-negative organisms [117, 122]. The proteins of the OM can be divided into two categories, lipoproteins (LP) and β-barrel proteins, which are also called outer membrane proteins (OMP). The thin peptidoglycan layer cannot retain the crystal violet stain upon decoloration with ethanol during Gram staining. The Gram-positive bacteria lack a defined periplasmic space and OM and the cytoplasmic membrane is surrounded by a very thick peptidoglycan (PG) layer (30–100 nm) with other molecules, e.g. teichoic acids, attached to it [117]. The PG is essential for morphol-ogy and responsible for the retention of the crystal violet dye during Gram staining procedure [123]. The PG is composed of a disaccharide- peptide repeat coupled through glycosidic bonds to form linear glycan strands and peptide bonds to link the glycan strands. It differs among different Gram-positive bacteria [117, 118, 124].

4.2. Outer membrane (OM) permeability

The outer-membrane (OM) of Gram-negative bacteria is crucial for bacterial survival in harsh environment and serves as a selective and low penetrable barrier for the exchange of materials [125, 126]. The OM is mostly an asymmetric and highly hydrophobic bilayer com-posed of glycerol phospholipids and LPS, as well as pore-forming proteins of specific size-exclusion properties. A typical LPS molecule consists of three parts: 1) lipid A, a glucosamine-based phospholipid; 2) a core oligosaccharide and 3) a distal polysaccharide (O-antigen)

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Figure 4. Number of novel FDA-approved drugs by year (Based on Stefan et al. 2018 [114]).

Figure 5. Schematic overview of the Gram-positive and Gram-negative cell envelope (adapted from Thomas J. Silhavy et al. 2010 [117] and Samuel I. Miller 2016 [125]). Peptidoglycan

(PG) layer is much thinner in Gram-negative bacteria than in Gram-positive bacteria. WTA, wall teichoic acid; CAP, covalently attached protein; LTA, lipoteichoic acid; IMP, inner membrane protein; LPS, lipopolysaccharide; LP, lipoprotein; OMP, outer membrane protein.

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[122]. The length of the core oligosaccharide and O-antigen varies in

different strains of E. coli since these structures are not essential for the species growth. Some of the core oligosaccharide and O-antigen sugars contain phosphate groups that mediate the interaction with divalent

metal ions, e.g. Mg2+_{, and this contributes to the tightly assembled}

structure of LPS. This well-packed structure creates an extremely or-dered network with sugar chains on the cell face. This well-assembled and low fluidity surface, hydrophobicity of LPS, as well as the diverse and widely distributed efflux pumps [127], are directly responsible for the low penetration of OM for some compounds [126, 128].

Although the composition and tightly-packed surface of the OM prevent the access of antibiotics and other molecules to the cytoplasm, this barrier also presents opportunities for the uptake of some com-ponents [125, 129]. There are two pathways for antibiotics to traverse the OM other than disruption and permeabilization of the OM barri-er with polymyxins and othbarri-er cationic antimicrobial peptides. Some small hydrophobic antibiotics, such as chloramphenicol, macrolides (erythromycin), rifamycins, novobiocin, fusidic acid and aminoglyco-sides (gentamycin, kanamycin), are able to diffuse through the lipid components of the OM. Specific β-barrel proteins can form porins or selective channels and allow hydrophilic compounds, e.g. penicillin and other β-lactam-based antibiotics, to pass through the OM [128, 130]. Notably, it was reported that both the presence of porins (OmpF) and the manipulations that disrupt the OM can sensitize drug flux and susceptibility of quinolones [131, 132]. What is more, there is an equilibrium of charged and uncharged species of quinolones de-pending on the pH. The quinolone molecules with negative charge are prone to pass through porin channels, while the uncharged quinolone molecules prefer the lipid-mediated pathway [133]. Porin-deficient mutants of E. coli were more resistant to tetracycline than the wild-type (increased minimal inhibitory concentration of tetracycline against E. coli) [133–135] and uncharged tetracycline was observed to enter the cell via diffusion through the lipid layer of OM [133]. Thus, quinolones and tetracycline can utilize both pathways to pass

through the OM depending on their protonation status.

P. aeruginosa is less susceptible to most antibiotics than other Gram-negative microorganisms and this phenomenon was initially

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believed to be due to active efflux pumps [128, 129, 136, 137]. It was recently shown that the triclosan resistance of P. aeruginosa PAO1 is due to the carriage of an insensitive allele of fabI which encodes an enoyl-ACP reductase enzyme (the target for triclosan in sensitive species) [130, 138]. S. typhimurium was found to rapidly regulate membrane permeability via alteration of OM porins in peroxide treatment [139].

Daptomycin and vancomycin are both active against Gram-positive bacteria, but not effective against Gram-negative bacteria. However, the causes of their inefficiency against Gram-negatives are different.

The antibacterial mechanism of action of daptomycin is the Ca2+_-

mediated insertion into the cytoplasmic membrane causing depolar-ization and the loss of intracellular contents [140, 141]. Nevertheless, the lower proportion of anionic phospholipids in the cytoplasmic membrane in Gram-negative bacteria reduced the efficiency of dap-tomycin insertion [140]. As for vancomycin, its target is D-Ala-D-Ala peptides in lipid II and then inhibits the crosslinking of peptidoglycan. However, vancomycin cannot pass through the OM and reach its target in the periplasm [130, 142].

Moreover, the OM of Gram-negative bacteria is hard to penetrate, but there are still some reports of peptides which can pass through the membrane and inhibit the growth of the bacteria [104, 143, 144]. 4.3. Antibiotics acting at peptidoglycan (PG)

The cell envelope is one of the main targets for numerous antibiotics, including some with high clinical relevance [30, 145, 146]. Antibiot-ics either inhibit the activity of enzymes or sequester the substrates [118, 146]. The first committed step of peptidoglycan (PG) synthesis is inhibited by fosfomycin, of which MurA is the target. Fosfomycin inactivates the MurA-catalyzed reaction acting as a structural analog of the cosubstrate of the reaction [118, 147]. D-Cycloserine can in-hibit both D-alanine racemase and D-alanine/D-alanine ligase, which finally prevents the crosslinking of the peptidoglycan network [148, 149]. Lipid II has been recognized as the target for lots of antibiotics including lantibiotics, ramoplanin, vancomycin or bacitracin [2, 12, 30, 66]. Nisin links to the pyrophosphate and forms a pyrophosphate cage [12, 30]. Vancomycin and other glycopeptide antibiotics, such as

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teicoplanin, also bind to lipid II, but to the D-alanine dipeptide

termi-nus. Thus, they will block the glycan polymerization and cross-linking [150, 151]. Ramoplanin is produced by non-ribosomal peptide syn-thesis, and binds to lipid II on the external surface of the membrane as well [152, 153]. Bacitracin, a cyclic nonribosomally synthesized do-decylepeptide antibiotic, binds tightly to undecaprenyl pyrophosphate and then prevents the cycling of the lipid carrier by dephosphorylation [154, 155]. Penicillin and some other β-lactams inhibit the formation of peptidoglycan cross links through covalently modify the active site of transpeptidases, which are also called penicillin-binding proteins (PBPs). The β-lactam antibiotics are analogues of the D-alanyl-D- alanine terminus of the pentapeptide side chain, and the amount of PBPs and the affinities of PBPs binding β-lactams vary among bacterial species [118, 145, 151].

5. Antibiotic resistance (AR)

Antibiotic resistance (AR) refers to the ability of microorganisms to resist the effect of an antibiotic, which was once successfully used to fight the microbe [156, 157]. AR has been an issue since the introduc-tion of the first agents into clinical use in the 1940s and became one of the most serious global public health threats in this century [111, 157]. The development of AR is a natural ecological phenomenon and AR has been found in the microorganism from pristine sites, e.g. isolated caves and permafrost [158, 159]. AR has brought enormous damages to human health and economy throughout the world [160]. What is even worse, in recent times, the development of bacterial resistance to several antibiotic classes has resulted in quite dangerous multidrug- resistant (MDR) bacterial strains such as methicillin-resistant Staph-ylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) [112], carbapenem-resistant Enterobacteriaceae (CRE) [161], multidrug-resistant Acinetobacter baumannii (MRAB) [162], third generation penicillin-resistant Enterobacter aerogenes and Klebsiella pneumoniae strains [112, 161], or MDR Salmonella typhimurium phage type DT10 [157]. It was reported in Europe in 2007 that the number of infections by MDR bacteria was 400,000. The cost associated with these

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CH APTER 1: A nt ib io tic r esi sta nce (AR)

infections in terms of extra hospital expense and productivity losses exceed €1.5 billion annually [163]. In the United States, antibiotic- resistant infections render $20 billion per year in excess health care costs per year and 23,000 deaths as a direct result [163, 164].

5.1. Intrinsic resistances

Bacteria can be intrinsically resistant to certain antibiotics, which can be explained by their inherent structural or functional characteristics. As discussed in section 4.2, the protective OM of Gram-negative bacte-ria acts as an efficient barrier to prevent several antibiotics (vancomy-cin, teicoplanin, nisin, gallidermin, epidermin, mersacidin and other lantibiotics) from reaching their targets at the cytoplasmic membrane and/or the cytoplasm, which complicates treatments towards (multi-drug-resistance (MDR)) Gram-negative pathogens [165, 166]. The intrinsic difference of the cytoplasmic membrane of Gram-negative

bacteria and Gram-positive bacteria affects the Ca2+_mediated

inser-tion of daptomycin as well as the antibiotic efficiency of daptomycin [140]. Like OM, biofilms in P. aeruginosa, E. coli and S. epidermidis behave as an impenetratable barrier to the diffusion of antibiotics and reducing the efficiency of antibiotics [167, 168]. Besides, efflux pumps are capable of transporting antibiotics out of the bacterial cell and then exhibit resistance to certain compounds [127, 169]. In Enterobacteri-aceae, Pseudomonas spp. and Acinetobacter spp., reduction of porin expression was proved to contribute to the resistance to carbapenems and cephalosporins [170–172]. These approaches for AR are all a re-sult of the absence of susceptible targets of specific antibiotics or the difficulty to reach them.

Recently, many genes have been identified to be responsible for in-trinsic resistance to antibiotics [141, 173]. Isolates of Gram-negative bacteria such as K. pneumoniae, E. coli, P. aeruginosa and A. baumannii have emerged to be resistant to all β-lactam antibiotics as a result of β-lactamases production in the strains [174–176]. It was reported that various phenotypes of E. coli can be generated from genes knockouts and the susceptibility of these strains displayed significantly increased sensitivity to at least one of the antibiotics (e.g. triclosan, rifampin, nitrofurantoin, aminoglycosides and β-lactams) [173].

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5.2. Acquired resistances

One of the main intrinsic capacities for Gram-negative bacteria to protect themselves against antibiotics is prevention of access to the antibiotic target. However, it can also be achieved by a new acquisition. The exposure to carbapenems exerted a selective pressure and accu-mulated emergence of mutations in porin genes as well as the genes that regulate porin expression in Enterobacter spp.. The alteration in porin expression, including the shift of porin expression and lack of porins, contributed to the reduced permeability and the strain’s adaptive response to carbapenems treatment [171, 177, 178]. It was reported that an IncH1 plasmid, isolated from a Citrobacter freundii strain, was shown to carry genes coding a tripartite resistance nodu-lation division (RND) pump [179]. Thus, this resistance mechanism became transmissible.

The second strategy for bacterial acquisition of AR is the alteration or modification of the targets. AR can be acquired by alteration of target proteins, e.g. methylation of the ribosome [180, 181], or genetic exchange of the targets, including mutations of one or more genes [182], transformation by plasmids [183], transduction of plasmids [157], conjugation of plasmids [184], transposons [185] and integrons [157], both between and within species [157]. The chloramphenicol- florfenicol resistance (cfr) methyltransferase can specifically methyl-ate A2503 in the 23S rRNA, which then confer resistance to various antibiotics that have targets near this site [186]. Uptake of DNA from the environment leads to the formation of mosaic genes and confers antibiotic resistance by target protein modification. A mosaic penA, which encodes a penicillin-binding protein in N. gonorrhoeae, was found to exhibit high-level resistance to cefixime and ceftriaxone [187]. In methicillin-resistant S. aureus (MRSA), mecC and mecA are two allele genes, which encode the β-lactam-insensitive protein. The isolates of MRSA carrying mecC are more sensitive to oxacillin than the ones carrying mecA [188].

In addition, bacteria also exhibit resistance to antibiotics via inac-tivation of antibiotics via hydrolysis or transfer of a chemical group that blocks their action. Diverse enzymes have been identified that can degrade and modify antibiotics since the discovery of penicilli-nase in 1940 [176, 189–191]. Gram-negative bacteria carrying diverse

University of Groningen Breaking walls: combined peptidic activities against Gram-negative human pathogens Li, Qian