(novel) antimicrobial substances

176 Pages·2017·10.72 MB·English

Identification, purification and
elucidation of the mode of action
of (novel) antimicrobial substances
Sabine Oppedijk
Identification, purification and elucidation of the mode of action
of (novel) antimicrobial compounds
PhD thesis, Utrecht University, 2017
In English, with a Dutch summary
Sabine Oppedijk
Cover design: Sabine Oppedijk
Printed by: ProefschriftMaken || www.proefschriftmaken.nl
ISBN: 978-90-393-6858-9
The majority of the research presented in this thesis was conducted at the Membrane
Biochemistry and Biophysics group, Bijvoet Centre for Biomolecular Research,
Faculty of Science, of Utrecht University, the Netherlands.
Identification, purification and elucidation of
the mode of action of (novel) antimicrobial
substances
Identificatie, zuivering en ontrafelen van het werkingsmechanisme
van (nieuwe) antimicrobiële substanties
(Met een samenvatting in het Nederlands)
Proefschrift
ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag
van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit
van het college voor promoties in het openbaar te verdedigen op woensdag
25 oktober 2017 des middags te 2.30 uur
door
Sabina Franka Oppedijk
geboren op 23 oktober 1987
te Utrecht
Promotoren: Prof. dr. J.A. Killian
Prof. dr. J. den Hertog
Copromotoren: Dr. E.J. Breukink
Dr. N.I. Martin
Table of Content
Table of Content
Chapter 1 Introduction: The peptidoglycan precursor lipid II as an 7
effient target for antibiotics against Gram-positive bacteria 1
Chapter 2 The search for novel lipid II targeting antibiotics from fungal 33
sources: Activity based isolation of antimicrobial active
compounds from Talaromyces atroroseus
Chapter 3 Mode of action studies of Laspartomycin C and analogues 59
of Daptomycn
Chapter 4 Characteristics of the mode of action of Pep5: a highly 75
active and specific lantibiotic
Chapter 5 Energy metabolism as a target for antibiotics: metabolism 99
studies on Staphylococcus simulans under the influence of
Pep5
Chapter 6 Genetic sequencing as a tool to study the mode of action 127
of Pep5
Chapter 7 General discussion and concluding remarks 157
Appendix 165
Nederlandse samenvatting 166
Acknowledgements 170
Curriculum Vitae 175

Chapter 1
Introduction
The peptidoglycan precursor Lipid II
as an efficient target for antibiotics
against Gram-positive bacteria
This chapter is based on:
Sabine F. Oppedijk(1), Nathaniel I. Martin(2) and Eefjan Breukink(1),
Hit ‘em where it hurts: The growing and structurally diverse family of
peptides that target Lipid-II, BBA Biomembranes, 2016, 1858(5): 947-
57
(1) Membrane Biochemistry and Biophysics, Utrecht University, Padualaan 8,
3584CH, Utrecht, The Netherlands
(2) Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for
Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht,
The Netherlands
Chapter 1: Introduction
Abstract:
1 The peptidoglycan precursor lipid II has been widely used as a target for antibiotics
and was dubbed by some as the “bacterial achilles heel”. This review provides a
comprehensive overview of different antimicrobial peptides that interact with lipid
II. Different classes of antimicrobial peptides and their interaction with lipid II are
discussed ranging from lantibiotics, defensins and the most recently discovered
teixobactin. We speculate that future antibacterial agents operating via lipid
II-mediated modes of action will play an important role in the battle against
pathogenic bacteria by effectively hitting ‘em where it hurts!
8
1.2. Peptidoglycan Biosynthesis Pathway
1.1. Introduction: The era of discovery of lipid II binding antibiotics

In an era where microorganisms develop resistance faster than we can develop
new antibiotics, interest in antimicrobial substances produced by competing 1
microorganisms is growing. Microorganisms themselves are an important source
of novel antimicrobial substances as they commonly produce toxins to combat
other microbes. Screening for novel antimicrobials has led to the discovery of
numerous active compounds in the past decades. In addition to the search for novel
antibiotics, understanding their associated mode(s) of action is also of increasing
importance. Studying different types of antimicrobial peptides (AMPs) reveals that
AMPs have sustained their effectiveness throughout evolution.1,2 Many AMPs have
an amphipathic nature and are positively charged due to the high content of arginine
and lysine residues. This gives them the ability to specifically interact with negatively
charged membranes and subsequently disrupt the bilayer structure. Several general
mechanisms have been assigned for these various AMP modes of actions including
the carpet model3,4 and the ‘sand-in-the-gearbox’ model5 where amphipathic peptides
aggregate on the membrane followed by membrane disruption. However, in order for
an AMP to be broadly applicable as a possible therapeutic, it is important that it acts
on a specific target, so as to circumvent toxicity and general membrane distortion.
In this regard, there are a number of AMPs that are effective at low nanomolar
concentrations indicating that they work via a target-specific mechanism rather than
via general membrane distortion. One of those very essential target pathways in
Gram positive bacteria is the peptidoglycan synthesis pathway.

1.2. Peptidoglycan Biosynthesis Pathway
The bacterial cell wall is of vital importance for bacteria because it counters the
osmotic pressures that arise between the cell’s cytosol and its surroundings.
Peptidoglycan is synthesized from Lipid II, a building block unique to bacterial cells.
Lipid II synthesis starts at the cytosolic side of the membrane where the membrane
embedded enzyme MraY links UDP-N-Acetyl-Muramic acid pentapeptide (UDP-
MurNAc-pentapeptide) to the carrier lipid undecaprenyl phosphate forming lipid-I.
Next, the transferase MurG couples UDP-N-Acetyl Glucosamine (UDP-GlcNAc) to
the muramoyl moiety of lipid I to form lipid II. Lipid II is then flipped to the other side
of the bacterial membrane by FtsW after which penicillin binding proteins (PBPs)
incorporate lipid II into the growing peptidoglycan network. The residual undecaprenyl
pyrophosphate is then dephosphorylated to the monophosphate and flipped back to
9
Chapter 1: Introduction
1
Fig. 1: Schematic overview of the peptidoglycan biosynthesis pathway.
MurNAc-pentapeptide is coupled to undecaprenylphosphate by mraY. Then GlcNAc is
coupled to lipid I by MurG to form lipid II. The peptidoglycan precursor is then flipped to the
periplasmic space by ftsW and incorporated into the peptidoglycan by the penicillin binding
peptides (PBPs).
the cytosolic site to be reused in the lipid II synthesis cycle. (Figure 1, for reviews on
the whole process see 6-9)
The bacterial cell wall in Gram-positive bacteria is readily accessible and as such,
the bacterial cell wall biosynthesis machinery is a prominent target for different types
of antibiotics.6,7,10 The viability of lipid II as a drug target is clearly demonstrated by
vancomycin. Belonging to the glycopeptide class of antibiotics, vancomycin was first
isolated from the soil bacteria Streptomyces orientalis in 1953 by Eli Lily. The use
of vancomycin has significantly increased since the development of penicillin- and
methicillin- resistant isolates. Since its introduction into the clinic, vancomycin remains
the only antibiotic that targets lipid II and is a last resort treatment against many highly
resistant Gram-positive bacteria. During vancomycin’s early development phase,
researchers were unable to isolate vancomycin resistant bacteria.11,12 However, after
more that 50 years of clinical use vancomycin resistance slowly emerged. Nowadays
six different phenotypes of vancomycin resistant bacteria are known, designated
as the VanA- to VanG-gene clusters.12 Bacteria with VanA-, VanB- and VanD-type
resistance possess an altered form of lipid wherin the vancomycin-binding D-Ala-
D-Ala motif is replaced with D-Ala-D-Lac. Other vancomycin resistant organisms
instead employ a D-Ala-D-Ser although this results in less severe resistance.
Binding of vancomycin to the D-Ala-D-Ala moiety of lipid II is well studied and is
driven by the formation of five hydrogen bonds. Substituting the terminal D-alanine
10