Lichenicidin biosynthesis: LicP protease specificity

Universidade de Aveiro 2011

Departamento de Biologia

Raquel Cristina

Laranjeira Faria

Lichenicidin biosynthesis: LicP protease

specificity

Especificidade da protease LicP na

biossíntese da lichenicidina

Universidade de Aveiro 2011

Departamento de Biologia

Raquel Cristina

Laranjeira Faria

Lichenicidin biosynthesis: LicP protease

specificity

Especificidade da protease LicP na

biossíntese da lichenicidina

Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Microbiologia, realizada sob a orientação científica da Doutora Sónia Mendo, Professora Auxiliar do Departamento de Biologia da Universidade de Aveiro

o júri

Presidente Prof. Doutor António Carlos Matias Correia

professor catedrático da Universidade de Aveiro

Doutor Artur Jorge da Costa Peixoto Alves

investigador auxiliar da Universidade de Aveiro

Prof. Doutora Sónia Alexandra Leite Velho Mendo Barroso

agradecimentos Gostaria de agradecer à Prof. Sónia ter-me aceitado como

sua aluna e o modo como me recebeu no seu laboratório. Muito obrigada pela sua orientação e dedicação. Bem haja pela sua humanidade e simplicidade.

Agradeço aos meus colegas do LBM a simpatia com que me receberam e a permanente disponibilidade para ajudar. Foi muito bom conhecer-vos e poder usufruir da vossa companhia e boa disposição.

Agradeço, em particular, à Tânia, por todo o apoio. A sua paciência, os seus ensinamentos e sugestões foram preciosos para a conclusão deste trabalho.

palavras-chave Lantibiótico, lichenicidina, protease, especificidade.

resumo Os lantibióticos são péptidos com actividade antibiótica ou

morfogénica, sintetizados nos ribossomas de células Gram-positivas. Após tradução, os lantibióticos sofrem modificações que resultam na formação de aminoácidos pouco comuns (lantionina e metil-lantionina) e na formação de ligações químicas que lhes conferem uma estrutura policíclica. Após estas modificações químicas, a sequência líder do pré-lantibiótico é removida tornando o péptido activo. Este processo pode envolver uma única reacção de proteólise ou duas reacções sucessivas.

Os lantibióticos são divididos em diferentes classes consoante o seu mecanismo de biosíntese e a sua actividade biológica. Uma destas sub-classes inclui os lantibióticos de dois componentes, que são formados por dois péptidos, α e β, que, em conjunto, lhes conferem total actividade.

A lichenicidina pertence a esta classe de lantibióticos. É produzida por Bacillus licheniformis, um microrganismo vulgarmente

encontrado no solo. A remoção da sequência líder de um dos péptidos deste lantibiótico (péptido α) é removida numa única reacção proteolítica enquanto que, a formação do segundo péptido (o péptido β) implica a ocorrência de duas reacções de proteólise, presumivelmente efectuadas por acção sucessiva de duas enzimas, LicT e LicP, codificadas no genoma de B. licheniformis.

Constituiu o objectivo do presente trabalho, avaliar a especificidade da protease LicP. Para tal, foram produzidos mutantes do péptido β nos quais, os aminoácidos da sequência líder foram substituídos por alanina.

Os resultados mostraram que a funcionalidade da protease LicP não foi afectada pelas substituições de um único aminoácido. Os resultados mostraram, ainda, que a protease LicT deve actuar antes da protease LicP uma vez que, a introdução de uma mutação no local de corte da enzima LicT impediu a produção do péptido β.

keywords Lantibiotic, lichenicidin, protease, specificity.

abstract The lantibiotics are ribosomally synthesized peptides produced by

Gram-positive cells that have antibiotic or morphogenic activity. After translation, the lantibiotics undergo chemical changes that result in the formation of unusual amino acids (lanthionine and methylanthionine), as well as the establishment of chemical bonds that confer a polycyclic structure to the peptide chain. After modifications occur, the leader sequence of the prelantibiotic is removed to yield the active lantibiotic, a process that may involve a single proteolitical reaction or two consecutive reactions. The lantibiotics are divided into classes according to their mechanism of biosynthesis and its biological activity. A particular sub-class of such compounds consists of two-component lantibiotics, in which each lantibiotic consists of two peptides, α and β, which act in synergy to confer it full activity. Lichenicidin is a two component lantibiotic produced by a microorganism commonly found in soil, Bacillus licheniformis. The

leader sequence of one of the lichenicidin peptides (α-peptide) is totally removed through a single proteolytic reaction while the formation of the second peptide (β-peptide) implies the occurrence of two reactions of proteolysis, presumably made by the successive action of two enzymes, LicT and LicP, encoded in B. licheniformis

genome. In this work, we evaluated the specificity of the LicP protease. For this purpose, we produced β-peptide mutants, in which some amino acids were replaced by Ala.

The results indicated that LicP functionality is not impaired by single amino acid replacements. It was also shown that the Bliβ maturation process should, indeed, include the proteolysis of the leader sequence before the activity of LicP since an introduced mutation in the LicT cleavage site most probably inhibited the Bliβ full maturation.

i

TABLE OF CONTENTS

1. Introduction ... 1 1.1. Lantibiotics ... 2 1.1.1. Classification of Lantibiotics ... 5 1.1.1.1. Class I lantibiotics ... 5 1.1.1.1.1. Nisin group ... 6 1.1.1.1.2. Epidermin group ... 7 1.1.1.1.3. Pep 5 group ... 7 1.1.1.2. Class II lantibiotics ... 8 1.1.1.2.1. Lacticin 481 group ... 8 1.1.1.2.2. Mersacidin group ... 9 1.1.1.2.3. Cynnamicin group... 10 1.1.1.2.4. Two­component lantibiotics ... 10

1.1.1.3. Class III lantibiotics... 12

1.1.2. Gene organization ... 12

1.1.3. Biosynthesis of Lantibiotics ... 15

1.1.3.1. Precursor peptides... 15

1.1.3.2. Modification enzymes... 16

1.1.3.3. Proteases and Transporters ... 17

1.1.3.4. Regulation ... 19

1.1.4. Self­immunity of the producing strains ... 22

1.1.5. Mode of action ... 22

1.1.6. Applications of lantibiotics ... 25

1.1.7. Lantibiotics bioengineering ... 27

1.1.8. Lichenicidin... 28

1.1.8.1. Lichenicidin gene cluster and biosynthesis ... 28

1.1.8.2. Lichenicidin structure ... 30

1.2. Objectives ... 30

2. Experimental procedures ... 33

2.1. Strains and vectors ... 35

2.1.1. General strains ... 35

2.1.2. Plasmids and fosmids... 36

2.2. Culture media... 36

ii

2.3. Preparation of E.coli DH5α, BLic5�A2 and BW25113�tolC:kan competent cells . 38

2.4. Transformation of E.coli DH5α, BLic5�A2 and BW25113�tolC:kan competent

cells... 38

2.5. Extraction of plasmid DNA from E. coli ... 38

2.6. Production of Bliβ mutants by site­directed mutagenesis ... 39

2.7. Amplification of genes cloned in pET­vectors ... 41

2.8. Production of BW�tolC_ p15licP strain ... 41

2.8.1. Extraction of plasmid pET­15b ... 41

2.8.2. Amplification of licP gene ... 41

2.8.3. Restriction enzyme digestion of licP gene and pET­15b vector ... 42

2.8.4. Insertion of licP gene in the pET­15b vector and transformation ... 42

2.8.5. Transformation of BW�tolC:kan cells with the p15licP plasmid ... 43

2.8.6. Evaluation of LicP extracellular activity ... 43

2.9. Evaluation of Bliβ mutants antibacterial activity by colony bioassay ... 43

2.10. Detection of Bliα and Bliβ peptides ... 43

2.10.1. Preparation of extracts for mass spectrometry analysis... 43

3. Results and discussion ... 45

3.1. Analysis of the Bliβ’ peptide homology with other lantibiotics that undergo a two­step proteolysis ... 47

3.2. Analysis of LicP homology with other proteases ... 47

3.3. In silico prediction of LicP structure and function ... 49

3.4. Impact of the hexapeptide amino acid substitutions in the production of Bliβ ... 50

3.5. Evaluation of the proteolytic dependence of LicT and LicP ... 53

3.6. In silico prediction of LicP cellular location ... 54

3.7. Influence of the outer membrane protein TolC in the export of LicP ... 55

4. Conclusions and future perspectives ... 59

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LIST OF FIGURES

Fig. 1 A) Schematic representation of a lantibiotic prepropeptide. B) Formation of lanthionine (Lan) or methillanthionine (MeLan) in lantibiotic prepropeptides (adapted from Willey et al., 2007)………..……….…………. ₃

Fig. 2 Lantibiotic posttranslational modifications reported to date (adapted

from Willey et al., 2007)...……….…….……… 5

Fig. 3 Representative examples of Class I lantibiotics (adapted from Willey et al., 2007)..……….…...………...…….………….………. 6 Fig. 4 Structures of lacticin 481, cypemycin and sublancin (adapted from Willey

et al., 2007 and Chatterjee et al., 2005)..….……….………. 9 Fig. 5 Structures of mersacidin (adapted from Willey et al., 2007) and ala(0)­

actagardine (adapted from Chatterjee et al., 2005)...….……… 10 Fig. 6 Structure of cinnamycin (adapted from Willey et al., 2007) and

duramycin (http://www.freepatentsonline.com/7199230.html)………….…. 10

Fig. 7 Structure of α­ and β­ peptides of lacticin 3147 and haloduracin (adapted

from Willey et al., 2007)…….………….……..……….……… 11

Fig. 8 Representative examples of Class III lantibiotics (adapted from Willey et al., 2007).………..……….……… 12 Fig. 9 Representative biosynthetic gene clusters of lantibiotics (adapted from

Willey et al., 2007).………….……….…..……….……….. 13

Fig. 10 Schematic representation of the process of subtilin biosynthesis (Willey

et al., 2007).…...…………...……….……… 20 Fig. 11 Schematic representation of the process of cytolysin biosynthesis (Willey

et al., 2007).………..……….………. 21 Fig. 12 Schematic representation of bacterial cell wall biosynthesis

iv

Fig. 13 Mode of action of nisin (Breukink et al., 2004).……….. ₂₄

Fig. 14 Organization of lichenicidin gene cluster according to the genome annotation for B. licheniformis ATCC 14760 (Caetano et al., 2011)..………… 29 Fig. 15 Proposed structures of lichenicidin α­ and β­ peptides (Caetano et al.,

2011)……….. 30

Fig. 16 Proposed structures of lichenicidin H2N­NDVNPE­ Bliβ peptide (Caetano

T)……… 31

Fig. 17 Sequence alignment of lantibiotic peptides that suffer a second proteolysis for leader sequence removal. The residues that are removed in this proteolysis are shown inside the black box. Bliβ’ (lichenicidin β­ peptide); Mrs’ (mersacidin); Halβ’ (haloduracin β­peptide); Plwβ' (plantaricin W β­peptide); CylLL’ and CylLS‘ (cytolysin L and S peptides)... 47 Fig. 18 Amino acid sequence of LicP protease. The predicted catalytic residues

(Asp­His­Ser) were highlighted in red and the conserved sequences in

blue………. ₄₈

Fig. 19 LicP predicted 3D­structure, according to the I­Tasser web server……… 49 Fig. 20 Schematic representation of Bliβ’ mutants produced in this study by

substitution of each one of the amino acids of the NDVNPE

sequence………. 50

Fig. 21 Bioactivity of Bliβ’ mutants. Agar diffusion assay to evaluate the activity of the mutated lichenicidin β’­peptide using M. luteus as the indicator

strain. (A) Bliβ (control); (B) Bliβ'E­1A; (C) Bliβ'P­2A; (D) Bliβ'N­3A; (E) Bliβ'V­4A; (F) Bliβ'D­5A; (G) Bliβ'N­6A... 51

v Fig. 22 Schematic representation of substrate binding to a subtilisin­like serine

protease. The enzyme numbering is that of subtilisin BPN’ protease. The P4­P2’ represent the substrate amino acid residues with the respective side chains and S4­S2’ represent the enzyme binding sites (Siezen et al.,

1997)……….. ₅₂

Fig. 23 Schematic representation of Bliβ mutants produced in this study by deletion of the NDVNPE sequence and substitution of the “GG” motif by

“AA”……… ₅₃

Fig. 24 Bioactivity of Bliβ mutants. Agar diffusion assay to evaluate the activity of the mutated lichenicidin β’­peptide using M. luteus as the indicator

strain. (A) Bliβ (control); (B) Bliβ'hexa; (C) BliβGG_AA.………. ₅₄ Fig. 25 Sequence of LicP protease, where the predicted signal peptide sequence

is marked in blue and the cleavage site is indicated with an

arrow………...………...………...……….………… ₅₅

vii

LIST OF TABLES

Table 1 List of general strains used in this study. ATCC (American Type Culture Collection); MUL (University of Lisbon Microorganism Collection); LBM

(Molecular Biotechnology Laboratory)………. 37

Table 2 List of plasmids and fosmids used in this study. Clo (Chloramphenicol); Kan

(Kanamycin); Amp (Ampicillin)……… 37

Table 3 Summary of the selective agents used in this study. NR (not required).

*Protected from light with foil paper……… 39

Table 4 Table 4. List of primers designed for the construction of Bliβ mutants. The place corresponding to the introduced mutation is highlighted with bold

ix

ABBREVIATIONS

ABC ATP­binding cassette

allo­Ile allo­Isoleucine

Amp Ampicillin

Asp Aspartic acid

ATCC American Type Culture Collection

ATP Adenosine triphosphate

ATPase Enzyme that catalyses the decomposition of ATP into adenosine

diphosphate (ADP) and a free phosphate ion

AviCys S­�(Z)­2­aminovinyl�­D­cysteine

AviMeCys S­�(Z)­2­aminovinyl�­(3S)­3­methil­D­cysteine

Clo Chloramphenicol

Cys Cysteine

d’NTP Deoxyribonucleotide triphosphate

Dha 2,3­Dehydroalanine

Dhb (Z)­2,3­Dehydrobutyrine

DNA Deoxyribonucleic acid

Glu Glutamic acid

His Histidine

Kan Kanamycin

LA Luria­Bertani Agar

Lan Lanthionine

lan Generic designation for lantibiotic gene

LanA Generic designation for lantibiotic unmodified prepropeptide

LanB Generic designation for lantibiotic dehydratase

LanC Generic designation for lantibiotic cyclase

LanE Generic designation for component of ABC­transport protein involved

in lantibiotic self­ immunity

LanF Generic designation for component of ABC­transport protein involved

x

LanG Generic designation for component of ABC­transport protein involved

in lantibiotic self­ immunity

LanH Generic designation for protein that acts as helper peptide to the lantibiotic transporter

LanI Generic designation for lantibiotic immunity protein

LanK Generic designation for lantibiotic receptor­histidine kinase

LanM Generic designation of bifunctional enzymes catalyzing both

dehydration and cyclization reactions

LanP Generic designation of protease that removes the lantibiotic leader sequence

LanR Generic designation for lantibiotic response regulator protein

LanT Generic designation of lantibiotic ABC transporters

Lanα and Lanβ Generic designation for two­component lantibiotics mature peptides

LB Luria­Bertani broth

LBM Molecular Biotechnology Laboratory

Lys Lisine

MeLan Methillanthionine

MRSA Methicillin­resistant Staphylococcus aureus

MUL University of Lisbon

NCBI National Centre for Biotechnology Information

NMR Nuclear magnetic resonance

OBu 2­Oxobutyryl

OD Optical density

OM Outer membrane

OPr 2­Oxopropionyl

PCR Polymerase chain reaction

pDNA Plasmid DNA

SDM Site­directed mutagenesis

sec Secretory pathway

xi

Thr Threonine

TSA Tryptic soy agar

TSB Tryptic soy broth

Introduction

3

1.1.Lantibiotics

The term “lantibiotics” was introduced as an abbreviation for lanthionine­containing antibiotic peptides (Schnell et al., 1988). Presently, lantibiotics are defined as small (19­38

amino acids) ribosomally synthesized peptides produced by Gram­positive bacteria that have antibiotic or morphogenetic activity and are structurally defined by the presence of unusual amino acids introduced by post­translational modification (Willey et al., 2007).

These modifications occur at the C­terminal region of the prepropeptide (Fig. 1A) and include the dehydration of Ser and Thr residues to yield 2,3­didehydroalanine (Dha) and (Z)­2,3­Didehydrobutyrine (Dhb), respectively. This is followed by Dha and Dhb cross­ linking with the neighboring Cys residues resulting in the formation of the amino acids lanthionine (Lan) and methillanthionine (MeLan) (Fig. 1B), in that order.

Figure 1. A) Schematic representation of a lantibiotic prepropeptide. B) Formation of lanthionine (Lan) or methillanthionine (MeLan) in lantibiotic prepropeptides (adapted from Willey et al., 2007).

Introduction

4

The thioether­cross­linked amino acids such as Lan and MeLan were first isolated from the treatment of wool with sodium carbonate, from where the name “lanthionine” (Latin,

lana = wool) has its origin (Horn et al., 1941).

The formation of thioether rings converts the linear prepropeptide chain into a polycyclic form, which confers structure and function to the final lantibiotic, contributing for its enhanced proteolytic resistance and also contributing to increase its tolerance to oxidation (Field et al., 2010).

In general, some Dha and/or Dhb residues remain in their dehydrated form, but, ocasionaly, they may undergo alternate modifications involving, for example, spontaneous hydrolysis to yield 2­oxopropionyl (OPr) and 2­oxobutyryl (OBu) groups (Willey and van der Donk, 2007). Beyond Lan, MeLan, Dha and Dhb, the most commonly modified residues found in lantibiotics, more than 15 different posttranslational modifications have been documented (Fig. 2). The function of theses residues is less well defined, although it has been suggested that they may contribute to antimicrobial activity by interacting with free sulfhydryl groups on the cell envelope of the target organisms (Liu and Hansen, 1990).

After modification, the peptide is exported and the leader sequence (23­59 amino acids), located at the unmodified N­terminal region, is proteolytically removed, although not always in that order, to yield the active lantibiotic (Willey and van der Donk, 2007).

All the elements involved in the lantibiotic biosynthesis have been, generally, designated by Lan, with a more specific term for each lantibiotic member (e.g. Nis for nisin, Epi for epidermin).

Introduction

5 Figure 2. Lantibiotic posttranslational modifications reported to date (adapted from Willey et al., 2007.

1.1.1. Classification of Lantibiotics

Lantibiotics can be classified based on their maturation pathway and antimicrobial activity. This classification scheme, proposed by Willey and Donk (2007), divides lantibiotics in three classes considering differences in the leader peptide sequence, biosynthetic operon structure, peptide function and, to a lesser extent, the structure of the mature lantibiotic.

1.1.1.1. Class I Lantibiotics

In Class I, the prepropeptides are modified by two distinct enzymes: i) the LanB, which dehydrates Thr and Ser residues, and ii) the LanC, which mediates cyclization reaction. The peptides are exported by LanT, an ATP­binding cassette transporter, and their leader

Introduction

6

sequences are removed by LanP, a subtilisin­like protease. These lantibiotics are more linear than those belonging to Class II.

Within this class, lantibiotics are divided in three major groups according to their similarity: nisin, epidermin and Pep5 (Fig. 3).

Figure 3. Representative examples of Class I lantibiotics (adapted from Willey et al., 2007).

1.1.1.1.1. Nisin group

Nisin, produced by Lactococcus lactis, is the prototype for Class I lantibiotics. The

structure of nisin A was firstly reported in 1971 (Gross and Morell, 1971). It is a flexible molecule with three dehydrated amino acids and five thioether rings, three of them located on its N­terminal (A, B and C), and two on its C­terminal (D and E). Nisin has three natural forms, two more common, nisin A and Z, which differ by a single amino acid at

Introduction

7 position 27, and a third, nisin Q, that differs at four positions from nisin A (Chatterjee et al., 2005).

Presently, three other lantibiotics belong to nisin group: subtilin, ericin S and A, and streptin. Subtilin and ericin S, produced by Bacillus subtilis ATCC6633 and B. subtilis A1/3,

respectively, have MeLan and Lan bridging patterns identical to that of nisin. The precursor peptide of ericin A, also produced by B. subtilis A1/3, has 75% homology with

the subtilin precursor. The structure of streptin, isolated from Streptococcus pyogenes,

was proposed exclusively based on its similarity to nisin.

1.1.1.1.2.Epidermin group

The structure of epidermin (Fig. 3), produced by Streptococcus epidermidis, was

elucidated in 1985 by Jung and co­workers. Epidermin contains one Dhb, three thioether rings (one MeLan and two Lan) and the unusual S­[(Z)­2­aminovinyl]­D­cysteine (AviCys) residue. This lantibiotic has a natural variant, gallidermin, produced by Staphylococcus gallinarium, which differs from epidermin only by a single amino acid (Ile 6 �Leu) (Chatterjee et al., 2005).

The lantibiotics that show similarity to epidermin are mutacin 1140, mutacin B­Ny266, mutacin I and mutacin III, isolated from various strains of Streptococcus mutans

(Chatterjee et al., 2005).

All the members of this group have the characteristic Lan ring between positions 3 and 7. However, only the mutacins have a Dha residue at position 5, which is also present in the nisin group and has been associated with their biological activity (Chatterjee et al.,

2005).

1.1.1.1.3.Pep5 group

The lantibiotic Pep5 (Fig. 3), isolated from Staphylococcus epidermidis 5, contains two

Dhb residues, two Lan and one MeLan rings, whose stereochemistry is identical to that of nisin and subtilin (Chatterjee et al., 2005).

The other members of this group are epilancin K7 and epicidin 280, also produced by S. epidermidis strains. Epilancin K7 is three residues shorter than Pep5 and contains two Dha

Introduction

8

residues that are absent in Pep5. Epicidin 280 contains one Dhb and three thioether rings (one Lan and two MeLan) presenting 75% identity at the amino acid level with the Pep5 lantibiotic.

1.1.1.2. Class II Lantibiotics

Class II lantibiotics are structurally diverse and are modified by LanM enzymes. These are large (900­1000 amino acids) proteins, exhibiting both dehydratase and cyclase activities. Despite such functionality, LanM enzymes bear no homology with LanB proteins and exhibit low sequence identity with the LanC enzymes (Siezen et al., 1996),

confined to the three zinc binding ligands (Okeley et al., 2003).

Class II lantibiotics secretion and leader processing is also performed by multifunctional proteins that, despite the functional differences, share the designation with the Class I exporters, Lan T. The LanT proteins involved in the biosynthesis of Class II lantibiotics possess an ATP­binding cassette transporter domain and a conserved N­ terminal cysteine protease domain (Pag and Sahl, 2002).

Within this class, the lantibiotics are further divided in four groups that include the two­component lantibiotics (Fig. 7).

1.1.1.2.1.Lacticin 481 group

Lacticin 481 (Fig. 4), produced by L. lactis, is one of about 20 related lantibiotics, all of

them being hydrophobic, with no or little net charge. These peptides are characterized by a linear N­terminus and the presence of three overlapping thioether bridges rendering a globular C­terminus (Willey and van der Donk, 2007).

All the other members of this group, except sublancin 168 and cypemycin, share high homology and a similar pattern of ring formation with lacticin 481 (Chatterjee et al.,

2005).

Sublancin 168 (Fig. 4), isolated from B. subtilis 168, differs significantly from the other

lantibiotics due to the presence of two disulfide bonds. The pattern of rings is also different from that of lacticin 481. The inclusion of sublancin in this group results from the similarity of its leader peptide with that of the other members (Chatterjee et al., 2005).

Introduction

9 Cypemycin (Fig. 4) is a special lantibiotic containing four Dhb residues, one allo­Ile, one

Avi­Cys and an N­terminal N,N­dimethylalanine residue. This molecule is a good example of the range of posttranslational modifications that may take place during lantibiotic maturation (Chatterjee et al., 2005).

Figure 4. Structures of lacticin 481, cypemycin and sublancin (adapted from Willey et al., 2007 and Chatterjee et al.,

2005).

1.1.1.2.2.Mersacidin group

Mersacidin (Fig. 5) is one of the smallest lantibiotics known, formed only by 20 residues and containing three MeLan rings and the residue AviMeCys. The C­ring of mersacidin is also conserved among the lacticin 481 group, the two­component lantibiotics and in plantaricin C (Willey and van der Donk, 2007).

Considering its bioactivity, mersacidin is active against methicillin­resistant

Staphylococcus aureus (MRSA) in a murine model (Kruszewska et al., 2004).

The other members of this group are actagardine, a tetracyclic lantibiotic, and its natural variant, Ala(0)­actagardine (Fig. 5) (Chatterjee et al., 2005).

Introduction

10

Figure 5. Structures of mersacidin (adapted from Willey et al., 2007) and ala(0)­actagardine (adapted from Chatterjee et al., 2005).

1.1.1.2.3.Cinnamycin group

Cinnamycin (Fig. 6), ancovenin and three duramycin (Fig. 6) variants are secondary products of Streptomycetes that are remarkable for the presence of unusual modifications. They are all 19 residues long and contain one Lan and two MeLan rings in conserved positions. Except for ancovenin, they all possess the unusual lysinoalanine ring that brings together Lys19 to Dha6.

Figure 6. Structure of cinnamycin (adapted from Willey et al., 2007) and duramycin (http://www.freepatentsonline.com/7199230.html).

1.1.1.2.4.Two­component lantibiotics

In this specific Class II group the antibacterial activity is a result of the assembly of two different peptides. Usually, the unmodified prepropeptides are designated by LanA1 and LanA2 and the mature peptides by Lanα and Lanβ, respectively. Each of these peptides is encoded by a different structural gene and modified by a separate LanM enzyme (LanM1 and LanM2) (McAuliffe et al., 2000; McClerren et al., 2006). However, a single LanT

Introduction

11 Generally, each of these peptides displays weak or no antimicrobial activity, but, the synergistic interaction of the α­ and the β­ peptides results in strong antibacterial activity (Willey and van der Donk, 2007).

The lantibiotics included in this group are plantaricin W, staphylococcin C55, cytolysin L, Smb, BHT­A, haloduracin, lacticin 3147 and lichenicidin.

In all but cytolysin, the α­peptide resembles mersacidin and the β­ peptide is relatively long and flexible (Fig. 7). At the present, only the structure of lacticin 3147 (Martin et al.,

2004) and lichenicidin (Shenkarev et al., 2010) has been fully elucidated by NMR spectroscopy, and the structure proposed for haloduracin was strongly supported by tandem mass spectrometry (McClerren et al., 2006). This structural information

combined with sequence homology suggests that the three C­terminally located rings in all α­peptides have the same topology. Regarding the β­peptides, the first MeLan ring identified in Halβ appears to be conserved in all of these peptides except lacticin 3147 and staphylococcin C55 (Willey and van der Donk, 2007). The B ring is also relatively conserved among currently known two­component lantibiotics, being absent only in BHT Smb. Finally the two most C­terminally located thioether rings are conserved in all the β­ peptides except cytolysin (Willey and van der Donk, 2007).

Introduction

12

1.1.1.3. Class III Lantibiotics

Class III lantibiotics are peptides that lack significant antibiotic activity. Instead, they have another function for the producing cell.

To date, three such peptides have been described: i) SapB, secreted by Streptomyces coelicolor (Kodani et al., 2004), ii) AmfS, produced by Streptomyces griseus (Ueda et al.,

2002) and iii) SapT, secreted by Streptomyces tendae (Kodani et al., 2005). All these

peptides are involved in aerial hyphae formation in the streptomycetes.

The structure of SapT has already been confirmed by NMR spectroscopy (Fig. 8) (Kodani et al., 2005) whereas the structure of SapB (Fig. 8) was predicted by molecular

modeling (Kodani et al., 2004). AmfS has been purified but its structure has not been

explored (Willey et al., 2006).

Figure 8. Representative examples of Class III lantibiotics (adapted from Willey et al., 2007).

The putative modification enzymes for SapB and AmfS bear homology to the C­ terminal cyclization domain of LanM enzymes, however, they lack the zinc ligands that are important for cyclization by LanM. Therefore, it is assumed that the mechanism of maturation of these lantibiotics could be distinct (Willey and van der Donk, 2007).

Since these lantibiotics do not present antibacterial activity, they will not be considered in the context of the present work.

1.1.2. Gene organization

The genes encoding the elements necessary for lantibiotic biosynthesis are normally found in clusters and have been designated generally by lan, with a more specific

Introduction

13 They may be found on conjugative transposable elements (e.g., nisin), on the chromosome of the host (e.g., subtilin), or on plasmids (e.g., epidermin, lacticin 481) (Chatterjee et al., 2005).

The sequencing studies of the lan gene clusters have demonstrated a high level of

similarity in the gene organization for production of the various compounds (Fig. 9).

Figure 9. Representative biosynthetic gene clusters of lantibiotics (adapted from Willey et al., 2007).

Although the gene order, complexity and transcriptional organization of the various clusters differ, three genes have been identified that are involved in the biosynthesis of all class II lantibiotics (lanAMT), and four genes are present in all class I lantibiotics gene

clusters (lanABCT). These essential genes include the structural gene encoding the

prepropeptide, which have been generally designated by lanA, except for subtilin whose

Introduction

14

The lanA genes encode prepropeptides that have an extension of 23­59 amino acids at

their N­terminus when compared to the mature lantibiotic product. This provided the first indication that an N­terminal leader peptide should play an important role in lantibiotic biosynthesis (Chatterjee et al., 2005). The nucleotide sequence of these genes also

indicated that Ser and Thr residues were the precursors to Dha and Dhb residues found in the mature lantibiotic, and that the reaction of Cys with these didehydrated amino acids should result in the formation of the characteristic Lan and MeLan structures (Chatterjee

et al., 2005).

The lanB and lanC genes, only found in gene clusters of class I lantibiotics code for

proteins that have no similarity with others found in databases, which are required for the dehydration of Ser and Thr residues (LanB), followed by the formation of Lan and MeLan rings (LanC) (Augustin et al., 1992; Hansen, 1993; Klein et al., 1992).

In the class II gene clusters, a single gene (lanM) encoding a protein with sequence

homology at its C­terminus with the LanC proteins is found. It was confirmed, both in vitro and in vivo, that the lanM products carry out both the dehydration and cyclization

reactions (McAuliffe et al., 2000; Xie et al., 2004). Therefore this protein exerts the same

function as LanB and LanC involved in the biosynthesis of class I lantibiotics. However, as no homology can be detected between the LanM and LanB proteins, the lanM genes did

not originate from a gene fusion event (Siezen et al., 1996).

An ATP­binding cassette (ABC) transport system (lanT) is encoded in all the

characterized lantibiotic gene clusters, except for epicidin 280 (Heidrich et al., 1998). The

LanT proteins are responsible for secretion of either the final mature product or the posttranslationally modified product still attached to its leader sequence. Gene disruption and heterologous expression studies have shown that these transport systems are absolutely required in some cases, as for instance for subtilin and nisin (Chung et al.,

1992; Qiao and Saris, 1996) but, for other lantibiotics, like Pep5 and epidermin, alternative transport systems are present (Augustin et al., 1992; Meyer et al., 1995;

Schnell et al., 1992).

Although all lantibiotics require proteolytic removal of the leader peptide, genes encoding the proteases are not always located in the biosynthetic gene clusters,

Introduction

15 suggesting that other cellular proteases can fulfill this role (e.g., subtilin and cinnamycin) (Corvey et al., 2003; Widdick et al., 2003).

The genes coding for proteases that are found in biosynthetic operons have been designated by lanP and code for subtilisin­type serine proteases. Some clusters lacking a lanP gene have lanT genes with an N­terminal protease domain fused to the ABC­type

transporter.

In addition to the transport system that excretes the lantibiotic, several gene clusters contain a second transport system comprised of three genes (lanEFG) that has been

associated with the self­immunity mechanism of the producing strains (Chatterjee et al.,

2005). Beside these, a protein encoded by lanI is also believed to be involved in self­

protection for some family members (Chatterjee et al., 2005).

Additionally, genes that are important for the regulation of lantibiotic production, comprising a two­component sensory system (lanKR) are often found in the lantibiotics

gene clusters (Chatterjee et al., 2005).

In certain clusters, other genes could be found, encoding proteins generally involved in additional, less frequently encountered posttranslational modifications. One such example is the hydroxylase that oxidizes the Asp residue of cinnamycin, resulting from the cinX gene (Lawton et al., 2007).

1.1.3. Biosynthesis of Lantibiotics 1.1.3.1. Precursor peptides

As already mentioned, all lantibiotics precursor peptides contain a C­terminal structural region that undergoes posttranslational modification (prepropeptide) and a relatively long N­terminal leader sequence which remains unaffected during biosynthesis (leader sequence) (Chatterjee et al., 2005).

Comparisons of the leader sequences of a large number of lantibiotics have revealed two different conserved motifs: i) the class I lantibiotics have a common “FNLD” motif between positions ­20 and ­15 and ii) the class II peptides contain a characteristic “GG” or “GA” cleavage site (the double Gly motif) (Nes and Tagg, 1996).

Introduction

16

One exception to this general rule are salivaricins, which contain the typical leader GG­ signature for the class II lantibiotics but are modified by SalB and SalC proteins (Upton et al., 2001). Other lantibiotics whose leader sequences do not follow this rule are

cinnamycin and lactocin S, which are modified by LanM enzymes but do not present the GG­motif (Skaugen et al., 1997; Widdick et al., 2003).

The molecular details of the function of the leader sequence remain unclear (Patton et al., 2008). In some cases, the leader sequence is required for secretion (Chen et al., 2001;

Kuipers, 2004; Qiao and Saris, 1996; Uguen et al., 2005; van der Meer et al., 1994) and

ensures complete maturation (Chen et al., 1998; Dufour et al., 2007; Li et al., 2006; Xie et al., 2004). For the lantibiotics that are proteolytically processed extracellularly, the leader

sequence may prevent activation of the lantibiotic until it is safely outside the cell (van der Meer et al., 1993), as lantibiotics with their leader sequence attached have no

antimicrobial activity (Kuipers et al., 1993; Li et al., 2006; van der Meer et al., 1993; Xie et al., 2004).

1.1.3.2. Modification enzymes

The LanB enzymes do not present homology with any other known proteins and among the LanB family, the overall sequence identity is only around 30% (Chatterjee et al., 2005). This low similarity might be due to the significantly different prepropeptide

substrates and the formation of products of vastly different three­dimensional structures. In cases where the products are structurally close, the dehydratase proteins also show higher homology, such as the subtilin (SpaB) and ericin S (EriB) dehydratases that share 83% identity (Stein et al., 2002a).

Although hydrophobicity plots indicate that LanB proteins are rather hydrophilic and contain no clear transmembrane domains, NisB and SpaB were found to co­sediment with membrane vesicles, suggesting a membrane­associated nature (Engelke et al., 1992).

Further, studies also suggested that LanB should form a membrane­associated multimeric complex with LanC and LanT, the transport protein (Kiesau et al., 1997; Siegers et al.,

Introduction

17 both cytoplasmic and membrane fractions, indicating a loose association with the cytoplasmic membrane (Chatterjee et al., 2005).

Regarding LanM proteins, their C­terminal domain presents about 20­27% sequence identity with LanC proteins including the conserved motifs with the possible metal ligands (Chatterjee et al., 2005). Direct evidence for the role of LanM proteins was provided

during the first reconstitution of an active lantibiotic synthetase (LctM) involved in the biosynthesis of lacticin 481 (Xie et al., 2004). In this report, mass spectrometry (MS)

studies showed that LctM converted the prepropeptide LctA into a 4­fold dehydrated species and that the correct cyclization reactions also occurred, confirming the dual­ functionality of these enzymes. LctM required ATP and Mg2+ to carry out the posttranslational modifications, although, at present, the exact role of the cofactor is unknown. It may activate the Ser and Thr for elimination by phosphorylation of their hydroxyl groups, or it may provide the energy for peptide translocation during the series of dehydration and cyclization reactions (Xie et al., 2004).

Both LanB and LanM enzymes demonstrate to have low substrate specificity. For instance, NisB (nisin dehydratase) is able to dehydrate non­lantibiotic peptides when fused to the NisA leader peptide and expressed in L. Lactis (Kluskens et al., 2005; Rink et al., 2005), and LctM can do the same in vitro with peptides containing both proteinogenic

and nonproteinogenic amino acids fused with the lacticin 481 leader sequence (Chatterjee et al., 2006). This was surprising, given the exquisite control of LctM

processing, where only 4 of the 14 Ser and Thr residues present in LctA are dehydrated (Chatterjee et al., 2005).

1.1.3.3. Proteases and Transporters

LanP proteases involved in the leader peptide processing of class I lantibiotics vary in size depending upon the presence or absence of an N­terminal sec­signal secretion

sequence and a C­terminal cell wall anchor sequence (Chatterjee et al., 2005). These

proteins share homology with the serine protease subtilisin, especially in the sequence near the residues involved in the catalytic triad (Chatterjee et al., 2005). It remains

Introduction

18

exported outside the cell. For instance, the nisin protease (NisP) contains a N­terminal

sec­dependent secretion signal and a C­terminal consensus sequence involved in

anchoring surface proteins in Gram­positive bacteria (Schneewind et al., 1995).

There are lantibiotics, like subtilin, whose gene cluster does not contain a dedicated protease. However, the observation that subtilin could be obtained by cleavage of the leader peptide upon incubation with culture supernatants from a nonproducing strain suggested the action of nonspecific proteases in this process (Stein and Entian, 2002).

In class I lantibiotics, the transport of the peptides to the extracellular medium is performed by proteins designated by LanT, which are ATP­binding cassette transporters. In some lantibiotic systems, it seems that a LanT protein is not absolutely necessary, since PepT is not required for the extracellular transport of Pep5 in S. epidermis (Meyer et al.,

1995), and the gene cluster of epicidin 280 does not have a lanT gene (Heidrich et al.,

1998). Conversely, in the case of nisin, NisT is an absolute requirement for extracellular transport (Qiao and Saris, 1996). However, NisT has low substrate specificity since it is able to transport peptide fragments from enkephalin and angiotensin when C­terminally fused with the nisin leader region (Kuipers, 2004).

In class II lantibiotics, the proteins responsible for the transport of the peptides are also designed by LanT however, they are different from the class I LanT. These proteins possess a C­terminal ATP­binding cassette transporter domain, a transmembrane domain and a conserved N­terminal cysteine protease domain (Nishie et al., 2009). This dual

functionality allows these proteins to be also responsible for removing the class II prelantibiotics leader sequences. The N­ and C­ terminal domains of these enzymes are, presumably, cytoplasmic, which implies that the processing of the prelantibiotics takes place at the cytosolic side of the membrane (Havarstein et al., 1995).

Little is known about the substrate specificity of class II LanT enzymes. In the lacticin 481 system, the helical structure of the leader peptide is important for the recognition by the N­terminal domain of LctT (Furgerson Ihnken et al., 2008). In nukacin ISK­1, the

unusual amino acids present in the leader sequence are required for its processing by NukT (Nishie et al., 2009). In mutacin II, the interaction between the MutT protein and

Introduction

19 the prepeptide is presumably mediated by residues located at positions ­2 and ­1 from the cleavage site (Chen et al., 2001).

Some lantibiotics (e.g. Halβ, Plwβ, CylLL and CylLS) have leader sequences that undergo

stepwise proteolysis to be completely removed (Altena et al., 2000; Booth et al., 1996;

Caetano et al., 2011; Holo et al., 2001; McClerren et al., 2006). In this process, two

different proteases are involved.

1.1.3.4. Regulation

For nisin, subtilin, mersacidin, SA­FF22, salivaricin, and ruminococcin A the biosynthesis is controlled by a two­component regulation system that consists of a receptor­histidine kinase (LanK) and a transcriptional response regulator (LanR) (Stock et al., 2000). In bacteria, these systems are often involved in quorum sensing, the

intraspecies communication process that allows cells to sense other organisms in their surroundings in a cell­density­dependent­manner (Fuqua et al., 1996; Lyon and Muir,

2003; Podbielski and Kreikemeyer, 2004). The LanK present on the cellular surface detects an extracellular change, initiating a signal cascade by autophosphorylation of a histidine residue. This cascade originates the final adaptative response, which is mediated by the intracellularly located LanR (Fig. 10).

In all the cases mentioned above, except ruminococcin, the lantibiotic itself serves as quorum sensor that induces the transcription of biosynthetic and self­protection elements (Gomez et al., 2002; Kuipers et al., 1995; Schmitz et al., 2006; Stein et al.,

2002b; Upton et al., 2001; Wescombe et al., 2006).

Several lantibiotics have accessory gene regulators in their gene clusters: EpiQ (epidermin) (Peschel et al., 1993), LtnR (lacticin 3147) (McAuliffe et al., 2001), MutR

(mutacin II) (Chen et al., 1999) and MrsR1 (mersacidin) (Guder et al., 2002). For instance,

in mersacidin, the two­component regulatory system MrsR2/MrsK2 governs immunity, whereas MrsR1 is involved in the regulation of biosynthesis (Guder et al., 2002).

Lantibiotic biosynthesis can be regulated by the accumulation of active compound in the supernatant, by the growth state of the producing cells and by the culture conditions. For example, the production of mutacin I and the two peptide lantibiotic SmbAB by

Introduction

20

Streptococcus mutans is stimulated along with competence and biofilm formation

(Merritt et al., 2005; Petersen et al., 2006; Tsang et al., 2006); in Lactococcus lactis the

production of lacticin 481 is induced by the decrease of pH via RcfB (Petersen et al.,

2006); and in Ruminococcus gnavus ruminococcin biosynthesis is stimulated by the

presence of trypsin and a high cell density (Gomez et al., 2002).

Figure 10. Schematic representation of the process of subtilin biosynthesis (Willey et al., 2007). Presubtilin is encoded

by the spaS gene, which is modified by the membrane­associated subtilin synthetase complex SpaBTC (Kiesau et al.,

1997). Once in the extracellular environment, the subtilin leader sequence is cleaved by one of three exoproteases (subtilisin, WprA or Vpr) produced by B. subtilis (Corvey et al., 2003). The mature subtilin will serve as ligand for the

sensor kinase (SpaK) which, after being activated, phosphorylates SpaR. Subsequently, the SpaR will positively regulate

the expression of the spaBTC, spaS and spaIFEG operons. The spaRK operon itself is dependent on the alternative sigma

factor (σH) which is regulated at both the transcriptional and translational levels.

Cytolysin, belonging to the two­component lantibiotics, is produced by Enterococcus faecalis, and is the only lantibiotic that plays a role as virulence factor. Consequently, its

production is stimulated in the presence of potential target cells. In the absence of these cells, cytolysin production is repressed by CylR1, which dimerizes and binds specifically to the cytolysin operon promoter (Fig. 8a) (Rumpel et al., 2004). Basal transcription of the

Introduction

21 biosynthetic operon results in low­level production of CylLS and CylLL, which form an

insoluble complex that has neither cytolytic nor regulatory activity. When target cells are present, CylLL binds to phosphatidylcholine:cholesterol lipid bilayers and is no longer free

to bind CylLS whose accumulation results in high­level cytolysin expression (Coburn et al.,

2004). Although it remains unclear how CylLS causes derepression, a second, membrane

binding protein of unknown function, CylR2, is also involved ( Fig. 8b) (Cox et al., 2005).

Introduction

22

1.1.4. Self­protection of the producing strains

Any strain producing a lantibiotic must protect itself from its own product since it is active against strains that are closely related to the producer (Chatterjee et al., 2005).

Lantibiotic immunity is usually achieved through the LanI and/or Lan EFG elements. The LanI peptides vary in size and show no homology to each other, although they could function in a similar manner. The PepI is a short peptide (57­69 amino acids) that is attached to the outer side of the membrane and is thought to shield the membrane target from the lantibiotic pep5 (Hoffmann et al., 2004). On the other hand, immunity to

nisin is provided by both NisI and NisEFG. NisI is also coupled to the cell membrane (Qiao

et al., 1995), but its C­terminal domain is involved in the binding of nisin, probably

interacting with NisFEG via its N­terminal part (Koponen et al., 2004; Takala and Saris,

2006). LanFE(G) elements are specialized ABC transporter systems constituted by three subunits, where LanF corresponds to the ATPase domain that provides the energy for transport and LanE/G constitute integral membrane domains of the transporter (Bierbaum and Sahl, 2009).

Some systems have an additional LanH protein which acts as an helper peptide to the transporter, for example in epidermin (Peschel et al., 1997), and recognizes the

lantibiotic, in the case of nukacin (Okuda et al., 2008). Nukacin is bound by NukH and

subsequently removed from the membrane by NukFEG.

1.1.5. Mode of action

Lantibiotics are active mainly against Gram­positive bacteria, including MRSA, enterococci and Clostridium difficile (Castiglione et al., 2007; Kruszewska et al., 2004; Rea et al., 2007). So far, nisin and microbisporicin also have shown to be active against the

Gram­negative bacteria Moraxella catarrhalis, Neisseria gonorrhoeae and N. meningitidis

(Castiglione et al., 2008). The other Gram­negative bacteria are not susceptible mainly

because the outer membrane prevents the access of the lantibiotics to the cytoplasmic membrane (Kordel et al., 1988; Stevens et al., 1991).

Presently, two modes of action are known for lantibiotics. For some peptides, the antibiotic activity involves only one mechanism but, for others, it relies in more than one

Introduction

23 that may include disturbance of the cell wall biosynthesis (Fig. 12) and pore formation induction in the cytoplasmic membrane (Chatterjee et al., 2005).

For instance, the nisin and epidermin group belonging to class I lantibiotics have the dual mode of action abovementioned (Wiedemann et al., 2001).

Introduction

24

More specifically, nisin forms complexes with lipid II and uses it as a docking molecule for pore formation: the two N­terminal rings of nisin form a binding pocket that envelops the undecaprenyl pyrophosphate moieties of the lipid intermediates of cell wall biosynthesis (Fig. 12 and Fig. 13). After binding to lipid II, the C­terminus of nisin inserts into the membrane, oligomerizes and forms a pore containing eight nisin and four lipid II molecules (Breukink et al., 2003; Hasper et al., 2004) (Fig. 12). This action leads to an efflux of molecules, like potassium and amino acids, and dissipation of the membrane potential, resulting in the interruption of cellular biosynthesis (Bierbaum and Sahl, 2009).

The inhibition of the bacterial spores outgrowth (e.g. Bacillus cereus (Morris et al., 1984), Clostridium sporogenes (Wijnker et al., 2011) and Bacillus anthracis (Gut et al., 2011)) by nisin might be equally dependent on pore formation in the cell membrane of the germinating spores (Rink et al., 2007).

Figure 13. Mode of action of nisin (Breukink et al., 2004). G and M represent N­acetylmuramic acid and N­acetyl glucosamine, respectively, which are the main constituents of lipid II. Nisin interacts with lipid II with its N­terminus (a), establishing a cage­like structure (b) and subsequently inserts into the membrane (c), forming a pore consisting of 4 lipid II and 8 nisin molecules (d).

The lantibiotics of the epidermin group gallidermin and mutacin 1140 also bind to lipid II however, pore formation could only be observed for some indicator strains (Bonelli et

Introduction

25 lipossomes showed that these peptides, much shorter than nisin, only have the capability of pore formation in membranes whose thickness would not exceed 40 Ȧ. Together, these findings can indicate why some lantibiotics are only active against a small spectrum of strains (Smith et al., 2008).

The class II lantibiotics mersacidin and actagardine inhibit cell wall biosynthesis also by complexing with lipid II but do not form pores (Brotz et al., 1998a; Brotz et al., 1995; Brotz

et al., 1997; Somma et al., 1977). It is believed that the mersacidin group peptides target

a region of lipid II that includes the N­acetylglucosamine moiety and, most probably, comprises the sugar and phosphate residues. Both mersacidin and actagardine possess a conserved motif that is believed to play an essential role for such binding (Szekat et al., 2003). Other class II lantibiotics interact with lipid monolayers (lacticin 481 and nukacin ISK­1) (Asaduzzaman et al., 2006; Demel et al., 1996) and complex with lipid I and lipid II (plantaricin C) (Wiedemann et al., 2006). Moreover, the latter one is also able to form pores in the membrane of some strains.

The two­component lantibiotic lacticin 3147 is able to form a complex with lipid II, inhibiting peptidoglycan biosynthesis and allowing pore formation. In this process, the α­ peptide binds to lipid II and the β­peptide recognizes and binds to this complex, followed by insertion into the membrane, inducing the pores (Bierbaum and Sahl, 2009). As already mentioned in a previous section, the α­peptides of two­component lantibiotics resemble mersacidin and they also possess the conserved motif that is presumably involved in binding lipid II. The binding of an antibiotic to a complex biosynthetic intermediate like lipid II has certain advantages over binding to a single enzyme involved in peptidoglycan assembly because it is easier to the microbe to change the structure of the enzyme active site than to change the structure of lipid II, thereby, the odds of bacterial resistance are diminished (Chatterjee et al., 2005).

1.1.6. Applications of lantibiotics

Presently, lantibiotics are used mainly in food technology.

Nisin, the first characterized lantibiotic, has been used as a biopreservative in over 50 countries worldwide to prevent microbial spoilage and inhibit the outgrowth of clostridial

Introduction

26

spores (DelvesBroughton et al., 1996). Lacticin 481, variacin, and lacticin 3147 have also been tested as potential preservatives (Dufour et al., 2007; O'Mahony et al., 2001; Ross et

al., 2002). However, lantibiotics are of particular interest since they can be several orders

of magnitude more potent than classical antibiotics and many target the essential cell wall precursor lipid II (Field et al., 2010).

So far, several lantibiotics have been presented as good candidates for biomedical application.

Nisin is active against MRSA, vancomycin­resistant enterococci (VRE) (Brumfitt et al., 2002), Streptococcus pneumoniae (Goldstein et al., 1998) and Clostridium difficile (Bartoloni et al., 2004). Therefore, it has also been proposed its use as a chemotherapeutic agent (Lubelski et al., 2008). This lantibiotic might be also potentially used, in the treatment of peptic ulcers due to its ability to eliminate the Gram­negative

Helicobacter pylori (Blackburn and Goldstein, 1995). Also, along with lacticin 3147, nisin

also showed to have spermicidal activity, which makes them both good candidates for being used as contraceptive agents that, at the same time, might help to prevent the spread of some sexually transmitted diseases (Aranha et al., 2004; Silkin et al., 2008).

Streptococcus salivarius K12 produces two lantibiotics, salivaricin B and salivaricin A2

(Wescombe et al., 2006), and has been employed in New Zealand as a probiotic ingredient in oral rinses due to its ability to control the growth of Streptococcus pyogenes and reduce the severity of halitosis (Burton et al., 2006a; Burton et al., 2006b; Dierksen et

al., 2007).

Epidermin has shown to be capable of preventing Staphylococcus epidermidis adhering to catheters currently used in hospitals. This lantibiotic, along with gallidermin, is also effective against Propionibacterium acnes and, therefore, both peptides might be used to treat acne topically (Lawton et al., 2007).

The two peptide lantibiotics are also of particular medical interest since they are active against a broad range of Gram­ positive microbes. Lacticin 3147 exhibits activity against MRSA, VRE, penicillin resistant Pneumococcus, Propionibacterium acnes and

Introduction

27 Experimental studies verified the effectiveness of lacticin 3147 in the treatment of mastitis in cattle caused by Streptococcus dysgalactiae (Ryan et al., 1999), thus suggesting that it could also be effective in the treatment of such infections in humans (e.g. acute pharyngitis in children). This lantibiotic also inhibits Streptococcus agalactiae (Twomey et

al., 2002), a pathogen that has been associated with perinatal morbidity and neonatal

mortality, as well as post partum endometritis. For all this aspects, lacticin 3147 may be a viable alternative to conventional antibiotics, with the advantage that little or no resistance to this lantibiotic has been observed.

1.1.7. Lantibiotics bioengineering

Lantibiotics possess a key advantage over classical peptide antibiotics (nonribossomally synthesized) in that they are gene­encoded and, thus, they are much more amenable to engineering strategies designed to, for example, enhance their antimicrobial activity (Field et al., 2010).

In the last two decades, several strategies have been employed that revealed the importance to activity of certain residues and permitted to investigate the specificity of the lantibiotics biosynthetic machinery (Chen et al., 1998; Field et al., 2007).

By site­directed mutagenesis and in vitro experiments, some lantibiotics variants with improved properties have been obtained although the mechanisms underlying the enhanced antimicrobial activity have yet to be elucidated (e.g. nisin, mersacidin, lacticin 481, nukacin ISK­1, lacticin 3147 and actagardine) (Kuipers et al., 1996; Rink et al., 2007). Therefore, studies of structure­activity relationship accompanied by the investigation of mode of action could provide information for the design of more potent peptides and to improve target selectivity. Also, it would be useful to expand the range of lantibiotics action in order to target a broader range of bacteria. On the other hand, studies regarding the engineering of modification enzymes could also be of most importance since, in principle, it might be possible to increase the substrate tolerance and introduce the lantibiotic­associated structures into non­lantibiotic peptides (Field et al., 2010).

Introduction

28

1.1.8. Lichenicidin

Lichenicidin is a two­component lantibiotic produced by Bacillus licheniformis, composed by the two peptides Bliα and Bliβ (Begley et al., 2009; Dischinger et al., 2009).

Its producer is a saprophytic bacterium commonly found in soil that has been used, for over a decade, in the fermentation industry for production of proteases, amylases, antibiotics, and special chemicals. According with the information of the U.S. Environmental Protection Agency, there are no known reports of adverse effects of B.

licheniformis to human health or the environment.

The lichenicidin production was assigned to a biosynthetic gene cluster encoded in B.

licheniformis ATCC 14760 and DSM13 chromossome (Fig.14) (Begley, et al., 2009;

Dischinger, et al., 2009). However, although not recognized at the time, lichenicidin Bliα peptide was already detected in the supernatant extracts of B. licheniformis I89 (Mendo

et al., 2004). And recently, Bliα and Bliβ were identified in both cell wall­associated and

supernatant fractions of B. licheniformis I89 cultures (Caetano et al., 2011). Thus, in this strain, lichenicidin is not exclusively cell wall­associated as it has been described for the other two strains of this specie (Begley, et al., 2009; Dischinger, et al., 2009).

The lichenicidin complex has shown to be active against several MRSA, Listeria

monocytogenes strains, Micrococcus luteus, Bacillus subtilis, Bacillus megaterium and Rhodococcus sp. (Begley et al., 2009; Dischinger et al., 2009; Shenkarev et al., 2010).

1.1.8.1. Lichenicidin gene cluster and biosynthesis

The lichenicidin gene cluster contains 14 genes, encoding the lichenicidin structural genes as well as the proteins presumably involved in their modification, processing, transport, immunity and regulation (Fig. 14) (Caetano et al., 2011; Shenkarev et al., 2010). The licA1 and licA2 genes encode the Bliα and Bliβ prepropeptides, respectively.

Until recently, only the expression of licM1 and licM2 genes was directly associated with lichenicidin production (Dischinger, et al., 2009). However, Caetano and coworkers (2011) provided important information about the possible function of the other genes that compose the lichenicidin gene cluster.

Introduction

29 Figure 14. Organization of lichenicidin gene cluster according to the genome annotation for B. licheniformis ATCC 14760

(Caetano et al., 2011).

In that study the authors reported the successful expression of the lichenicidin gene cluster in Escherichia coli using a fosmid containing the complete lic gene cluster of B. licheniformis I89. Using the � RED recombinase technology, all the lic genes, except those

presumably related to immunity, were inactivated, which permitted to infer about their role in lichenicidin production. In this context, it was found that all the genes, except licX, were critical for lichenicidin production.

Both Bliα and Bliβ prepropeptides possess the Gly­Gly­motif and the member of the ABC transporter family with an integrated protease domain, LicT, is encoded in the lic

gene cluster. After the deletion of the licT gene, the lichenicidin peptides were not

detected in the I89 supernatant. Therefore, this protein should be responsible for removal of the leader sequences of Bliα and Bliβ peptides during substrate translocation.

However, an additional proteolytic cleavage is needed for the complete maturation of the Bliβ peptide (Shenkarev et al., 2010). This imply the removal of the six N­terminally located amino acids of the inactive Bliβ’ peptide (NDVNPE­ Bliβ).

According to the results presented by Caetano and coworkers (2011), LicP is probably the protease responsible for such reaction. In fact, the deletion of licP resulted in the absence

of the Bliβ peptide in I89 supernatants. The LicP is a putative uncharacterized serine

protease homologous to CylA (38% identity) from Enterococcus faecalis (CylLL and CylLS

producer) and to BH1491 uncharacterized protease (30% identity) from B. halodurans C­

125 (Halα and Halβ producer). Nevertheless, it is still unclear if LicP activity it is or it is not dependent of the proteolytical removal of Bliβ leader peptide by LicT.

Finally, the deletion of licR and licY genes permitted to conclude that LicR might have

an essential regulatory function in the biosynthesis of the mature Bliα peptide, whereas LicY is involved exclusively in the biosynthesis of Bliβ peptide.

Introduction

30

1.1.8.2. Lichenicidin Structure

As described earlier, two­component lantibiotics are formed from two precursor peptides that, after post­translation modifications, create two distinct products, α­ and β­ peptides, that act in synergy to provide full bactericidal activity (Willey and van der Donk, 2007). In the lichenicidin α­ and β­ peptides the dehydration and cyclization reactions result in the formation of four thioether rings and several Dha and Dhb residues in each peptide (Fig. 15). However, some of the Ser/Thr residues (one in Bliα and three in Bliβ) escape the action of the LanM1 and LanM2 modification enzymes. The structure of Bliα resembles its counterpart haloduracin, as well as that of mersacidin, while Bliβ is more similar to Halβ (Fig. 7). The high­resolution mass spectrometry analysis of lichenicidin peptides showed that Bliα and Bliβ have a molecular mass of 3249.7 Da and 3019.6 Da, respectively (Caetano et al., 2011).

Figure 15. Proposed structures of lichenicidin α­ and β­ peptides (Caetano et al., 2011).

1.2. Objectives

The results obtained by Caetano and coworkers (2011) demonstrated that in the absence of LicP, lichenicidin activity should be restored by the supernatant of an E. coli

strain expressing the licP gene (in the absence of other lic genetic determinants). All

together, the data obtained in the study allowed the assumption that LicP is responsible for the removal of the N­terminal six residues of the H2N­NDVNPE­ Bliβ peptide, (named