Discovery of antimicrobial peptides active against antibiotic resistant bacterial pathogens

243 Pages·2015·6.76 MB·English

by  Arif Felek

Discovery of antimicrobial peptides active against
antibiotic resistant bacterial pathogens
A thesis submitted to the University of Manchester for the degree of
Doctor of Philosophy in the Faculty of Medical and Human Sciences
2015
Arif Felek
School of Medicine
List of contents
LIST OF CONTENTS
List of contents ....................................................................................................... 2
List of figures ......................................................................................................... 8
List of tables ......................................................................................................... 18
List of abbreviations ............................................................................................. 20
Declaration ........................................................................................................... 23
Copyright statement ............................................................................................. 24
Dedication ............................................................................................................ 25
Acknowledgements .............................................................................................. 26
Publications from this work ................................................................................. 27
1 Chapter 1 General Introduction .................................................................... 28
1.1 Downfall of novel antimicrobial discovery ........................................... 32
1.1.1 Movement of “big pharma” away from antimicrobial drug
discovery ....................................................................................................... 32
1.1.2 Overview of target based genetic approaches and their failure to
discover novel antibiotics ............................................................................. 34
1.2 Revitalising natural product screening as a source of novel antibiotics 36
1.3 Bacterially derived natural products of interest..................................... 39
1.4 Bacteriocins ........................................................................................... 41
1.5 Bacteriocins of Gram positive bacteria ................................................. 43
1.5.1 Class I - Lanthionine containing bacteriocins (Lantibiotics) ......... 43
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1.5.2 Class II non-lanthionine containing bacteriocins ........................... 48
1.5.3 Class III large proteins: .................................................................. 50
1.5.4 Class IV Cyclic peptides ................................................................ 51
1.6 Bacteriocins of Gram-negative bacteria ................................................ 52
1.6.1 Colicins .......................................................................................... 52
1.6.2 Microcins ....................................................................................... 54
1.7 Miscellaneous RiPP bacteriocins .......................................................... 58
1.7.1 Sactibiotics (Sulphur to α-carbon antibiotics) or sactipeptides ...... 58
1.7.2 Thiopeptides ................................................................................... 58
1.7.3 Bottromycins .................................................................................. 59
1.7.4 Glycocins ....................................................................................... 59
1.7.5 Linear azol(in)e-containing peptides (LAPs) and lasso peptides ... 60
1.8 Polyketides ............................................................................................ 61
1.9 Non-ribosomal proteins ......................................................................... 62
1.10 Lysins: ................................................................................................... 64
1.11 Advances in natural product screening.................................................. 65
1.11.1 Purification and identification of the active compound ................. 65
1.11.2 Identification of producer strains ................................................... 67
1.11.3 Genome mining and scanning for natural products ....................... 67
1.12 Aims and objectives .............................................................................. 72
2 Chapter 2 Methods ........................................................................................ 74
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2.1 In vitro antimicrobial peptide screening and characterisation ............... 75
2.1.1 Bacterial isolates ............................................................................ 75
2.1.2 Identification of the producer strains using 16S rRNA gene
sequencing .................................................................................................... 76
2.1.3 Agarose gel electrophoresis ........................................................... 78
2.1.4 Assays used for the assessment of antimicrobial activity .............. 78
2.1.5 Optimisation of antimicrobial peptide production ......................... 83
2.2 Proteomic analysis of the identified antimicrobial peptides.................. 84
2.2.1 Purification of antimicrobial peptides from the culture media ...... 84
2.2.2 SDS-PAGE analysis and gel diffusion agar overlay assay ............ 88
2.2.3 MALDI-ToF mass determination and ESI-MS scan of the RP-
HPLC Purified Active Fractions ................................................................... 89
2.2.4 De novo peptide sequencing (MS/MS) .......................................... 90
2.2.5 Genome sequencing and annotation of the draft genome of
producer strains ............................................................................................. 93
2.3 Characterization of the purified antimicrobial peptides ........................ 94
UV absorbing material leakage assay ........................................................... 95
2.3.1 Physicochemical analysis of purified antimicrobial peptides (heat
and enzyme stability tests) ............................................................................ 95
2.3.2 Determination of minimum inhibitory concentration .................... 96
2.3.3 Haemolysis assay ........................................................................... 96
2.3.4 Eukaryotic cell toxicity studies ...................................................... 97
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2.3.5 UV Absorbing material leakage assay ........................................... 99
2.4 In silico Genome mining and identification of putative bacteriocin leads
………………………………………………………………………...99
2.5 Cloning and expression methods ......................................................... 102
2.5.1 Insert construction and primer design for cell free protein
expression ................................................................................................... 102
2.5.2 In vitro cell-free expression ......................................................... 106
2.5.3 Proteomic analysis of peptide products generated following
expression studies ....................................................................................... 107
3 Chapter 3 Pumicin NI04, a novel antimicrobial peptide from Bacillus
pumilus, is homologous to the EsxA virulence determinant .............................. 108
3.1 Introduction ......................................................................................... 109
3.2 Results ................................................................................................. 113
3.2.1 Two antimicrobial agents are produced by B. pumilus J1 ........... 113
3.2.2 Bacteriocin production can be optimised by manipulation of
growth conditions ....................................................................................... 115
3.2.3 De novo peptide sequence determination of peptide NI04 using
mass spectrometry analysis and genome interrogation facilitates
identification of the pumicin NI04 locus .................................................... 117
3.2.4 Pumicin NI04 is stable to heat treatments and protease degradation
confirms a proteinaceous nature ................................................................. 125
3.2.5 Pumicin NI04 has potent activity against resistant Gram positive
pathogens .................................................................................................... 126
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3.2.6 UV absorbing material leakage assay .......................................... 127
3.2.7 Pumicin NI04 has low levels of in vitro toxicity ......................... 130
3.3 Discussion ........................................................................................... 132
4 Chapter 4 Discovery and Analysis Of Peptide NI05 produced by Klebsiella
pneumoniae strain A7 ........................................................................................ 141
4.1 Introduction ......................................................................................... 142
4.2 Results: ................................................................................................ 147
4.2.1 Simultaneous antagonism assays for preliminary analysis of the
antimicrobial activity spectrum of peptide NI05 ........................................ 147
4.2.2 Optimisation of antimicrobial peptide production ....................... 148
4.2.3 Purification ................................................................................... 149
4.2.4 Matrix Assisted Laser Desorption Ionisation – Time of Flight
(MALDI-ToF) mass determination ............................................................ 150
4.2.5 Genome sequence and annotation ................................................ 151
4.2.6 In silico analysis of the draft genome and discovery of the
MccE492 gene cluster:................................................................................ 152
4.3 Discussion ........................................................................................... 158
5 Chapter 5 Genome mining of anaerobic bacteria, known producer bacteria
and cell free expression of pumicin NI04 .......................................................... 166
5.1 Introduction ......................................................................................... 167
5.2 Results ................................................................................................. 170
5.3 Genome mining results ........................................................................ 170
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5.3.1 Lead 3 ........................................................................................... 176
5.3.2 Lead 12 ......................................................................................... 177
5.3.3 Lead 14 ......................................................................................... 178
5.3.4 Unique putative bacteriocin candidates ....................................... 178
5.4 Cloning Results ................................................................................... 181
5.4.1 Insert confirmation and validation following transformation ...... 182
5.4.2 In vitro expression of pumicin NI04 ............................................ 186
5.5 Discussion ........................................................................................... 188
Chapter 6 Concluding Remarks ......................................................................... 199
References .......................................................................................................... 205

Total words Count: 53,113

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List of figures
LIST OF FIGURES
Figure 1.1 Timeline of antibiotic discovery versus outbreak of resistance among
bacteria [from (Clatworthy et al., 2007)] ............................................................. 29
Figure 1.2 The amount of antibiotics being introduced into the clinic has been
drastically decreasing in recent years [From (Boucher et al., 2013)] .................. 30
Figure 1.3 Graphical representation and comparison of the number of antibiotic
candidates going through clinical trials between 2011 and 2013 [from (Pucci and
Bush, 2013)]. ........................................................................................................ 31
Figure 1.4 The process of target based antimicrobial drug discovery [adapted
from (Mills, 2006; Pucci, 2007)]. ........................................................................ 34
Figure 1.5 Advantages of natural products (NP) as antibiotic drug leads ........... 36
Figure 1.6 Natural products of interest in the current project. The figure above
lists the natural products produced by bacteria that have the potential to be used
as antibiotics. These products are shortly reviewed in this study. ....................... 40
Figure 1.7 The "Universal” bacteriocin classification scheme. This figure by
Heng and Tagg, (2006) illustrates the two major classification schemes that are
used and how they propose to combine the respective advantages to construct a
universal scheme. ................................................................................................. 42
Figure 1.8 Bacteriocin mode of action. The class II bacteriocins such as sakacin
can insert into the cell membrane by interacting with membrane proteins and
cause pore formation and depolarisation of the cell membrane. The class I
bacteriocins on the other hand can have a single or multiple modes of action such
as nisin. Large protein bacteriocins such as lysostaphin can also act on the cell
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wall by hydrolysing the interpeptide bridges of the peptidogycan layer [adapted
from (Cotter et al. 2005)]. .................................................................................... 44
Figure 1.9 Four classes of the lanthipeptide family can be separated by the
modification enzymes involved in generation of their lanthionine bridges. Solid
lines indicate the conserved regions thought to play roles in the catalysis
including zinc ligands that are missing in class III [from (Knerr and van der
Donk, 2012)]. ....................................................................................................... 46
Figure 1.10 Class II-A bacteriocin action is dependent on the presence of IID
and IIC proteins, which are membrane proteins of the Man-PTS mechanism.
Class II a bacteriocin first binds to the extracellular loop and then to the
transmembrane helices of these peptides causing conformational changes that
leads to efflux and cell death [from (Kjos et al., 2011)] ...................................... 49
Figure 1.11 Mechanisms of cellular entry used by colicins. ExbB, ExbD
components in the TonB pathway are used by group B colicins and the Tol
pathway (TolA, TolB, TolQ and TolR) is employed by group A colicins [from
(Lloubès et al., 2012)]. ......................................................................................... 53
Figure 1.12 Class I microcin action. A) Microcin (Mcc) J25 gains access to the
cell through the outer membrane (OM) receptor FhuA and then utilises the
TonB/ExbB/ExbD complex and SbmA protein to translocate into the cytoplasm
where it inhibits transcription by attacking the RNA polymerase enzyme
(Mathavan et al., 2014). B) Mcc B17 targets the DNA gyrase enzyme that is
responsible for supercoiling of the DNA, it gains access to the inner membrane
(IM) using the OmpF porin on the OM and is translocated into the cell by
exploiting the SbmA protein. C) Although the entry mechanism of Mcc C7/C51
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is unknown, it acts by inhibiting aspartyl-tRNA synthetase [from (Duquesne et
al., 2007a)]. .......................................................................................................... 55
Figure 1.13 Class IIb microcin action MccE492 (A), MccM (B), MccH47(C)
and MccI47 (D)* all exploit the catechol-siderophore receptors and are
translocated into the periplasm in a TonB pathway dependent manner. Once
inside, MccE492, using the ManY and ManZ membrane proteins of the mannose
permease complex as docking stations, inserts itself into the inner membrane
where it disrupts the IM potential by allowing protons to leak. On the other hand,
MccH47 inhibits ATP synthase by interfering with the F0 component once it
gains access to the periplasm. The exact mechanism of action for the remaining
microcins of this group is not known [modified from (Duquesne et al., 2007a)] .
*MccI47 peptide has not been isolated, but on the basis of predicted amino acid
sequence it contains the serine rich C-terminal that is associated with siderophore
interaction. ............................................................................................................ 57
Figure 1.14 A diagram of Microcin J25 structure, demonstrating the lasso fold
[from (Maksimov et al., 2012)] ........................................................................... 60
Figure 1.15 Examples of NRP structures. Tyrocidine A is a good example for
macrocyclic NRPs, while surfactine is a good a good example of branched
macrocyclic NRPs [from (Schwarzer et al., 2003)] ............................................. 63
Figure 1.16 A hybrid NRP/PK. NRPS derived structure is coloured in blue while
PKS derived structures are red [from (Walsh and Fischbach, 2010)].................. 64
Figure 2.1 Simultaneous antagonism test, the antimicrobial activity of potential
inhibitor producers against two indicator E. coli species (414 at the top and
DH5α at bottom of the plate) was observed as clear zones of inhibition............. 79
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