purification and characterization of an antimicrobial peptide … · 2017-10-11 · purification...
TRANSCRIPT
Purification and characterization of an antimicrobial peptide
produced by Bacillus sp. strain P7
A dissertation submitted to the University of Manchester for the degree of Master of
Science in Medical Microbiology in the Faculty of Medical and Human Science
2014
Paulina Fernández Soto
School of Medicine
2
Table of Contents
List of Figures .............................................................................................................. 3
List of Tables ............................................................................................................... 3
Abstract ........................................................................................................................ 4
Declaration .................................................................................................................. 5
Intellectual Property Statement ................................................................................ 6
Acknowledgments ....................................................................................................... 8
Preface .......................................................................................................................... 9
Dedication .................................................................................................................... 9
1. Introduction ....................................................................................................... 10
2. Materials and Methods ..................................................................................... 15
2.1. Bacterial strains ............................................................................................ 15
2.2. Indicator strains ............................................................................................ 15
2.3. Screening for antimicrobial peptide production ........................................... 15
2.4. Broth optimization for antimicrobial peptide production ............................ 16
2.5. Assays to test antimicrobial peptide activity ................................................ 16
2.6. Purification of the antimicrobial peptide ...................................................... 17
2.7. Characterization of the antimicrobial peptide .............................................. 19
2.8. Strain identification by sequence analysis of the 16S rRNA gene ............... 21
3. Results ................................................................................................................. 24
3.1. Screening of bacteriocin production using simultaneous antagonism ......... 24
3.2. Broth Optimization ....................................................................................... 25
3.3. Antimicrobial peptide production and purification ...................................... 26
3.4. Characterization of the peptide .................................................................... 28
3.5. 16S rRNA sequence analysis ....................................................................... 30
4. Discussion ........................................................................................................... 31
5. References .......................................................................................................... 36
Word Count ( 6,181 )
3
List of Figures
Figure 1: Well diffusion results of NB optimization ..................................................... 25
Figure 2: Well diffusion assay: AMP-P7 produced by P7 from NB with 5% yeast
extract. ............................................................................................................................ 26
Figure 3: Range of activity of purified peptide from P7 ............................................... 28
List of Tables
Table 1: Antimicrobial peptide activity of the environmental bacteria isolates. ........... 24
Table 2: Nutrient Broth optimization for antimicrobial peptide production. ................ 25
Table 3: Oasis fractions and zones of inhibition ........................................................... 27
4
Abstract
Background: Antimicrobial peptides are considered to be an alternative to combat
antibiotic-resistant bacteria due to their broad-spectrum activity and lack of interaction
with specific protein binding sites. This project aimed to screen a library of
environmental samples for antimicrobial-peptide-producing bacteria, and attempted to
purify and characterize potential novel agents produced by these bacteria.
Methods: A library of environmental strains were screened for antimicrobial peptide
production against several indicator strains, using the simultaneous antagonism method.
The best producer strain was designated as P7 and its antimicrobial peptide as AMP-P7.
Antimicrobial peptide production from P7 was achieved in Nutrient Broth with 5%
yeast extract, and AMP-P7 inhibitory activity was monitored by either the well
diffusion or spot-on-lawn methods. Partial purification of AMP-P7 was achieved by
using Oasis HLB column chromatography.
Results: A potential novel antimicrobial peptide (AMP-P7) targeting S. aureus NCTC
7447 was identified from a library of environmental strains. A BLAST search revealed
that the 16S rRNA gene sequence of P7 strain was 99.9% similar to Bacillus tequilensis.
Conclusion: This study reports a potential novel antimicrobial peptide with narrow
spectrum activity against S. aureus NCTC 7447, produced by Bacillus tequilensis.
Further studies to improve AMP-P7 purification and characterization are required to
establish its potential used in medicine and industry.
5
Declaration
I, Paulina Fernandez Soto, hereby declare that no portion of the work referred to in the
dissertation has been submitted in support of an application for another degree or
qualification of this or any other university or other institute of learning.
6
Intellectual Property Statement
i. The author of this dissertation (including any appendices and/or schedules to this
dissertation) owns certain copyright or related rights in it (the “Copyright”) and s/he has
given The University of Manchester certain rights to use such Copyright, including for
administrative purposes.
ii. Copies of this dissertation, either in full or in extracts and whether in hard or
electronic copy, may be made only in accordance with the Copyright, Designs and
Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in
accordance with licensing agreements which th University has entered into. This page
must form part of any such copies made.
iii. The ownership of certain Copyright, patents, designs, trade marks and other
intellectual property (the “Intellectual Property”) and any reproductions of copyright
works in the dissertation, for example graphs and tables (“Reproductions”), which may
be described in this dissertation, may not be owned by the author and may be owned by
third parties. Such Intellectual Property and Reproductions cannot and must not be
made available for use without the prior written permission of the owner(s) of the
relevant Intellectual Property and/or Reproductions.
iv. Further information on the conditions under which disclosure, publication and
commercialisation of this dissertation, the Copyright and any Intellectual Property
and/or Reproductions described in it may take place is available in the University IP
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Policy (see http://documents.manchester.ac.uk/display.aspx?DocID=487), in any
relevant Dissertation restriction declarations deposited in the University Library, The
University Library’s regulations
(see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s
Guidance for the Presentation of Dissertations.
8
Acknowledgments
I would like to thank my supervisor Dr. Guoqing for his corrections and comments
about this project. I would also want to express my sincere gratitude and recognition to
John Moat for all the time he spent with us in the laboratory, for his advice and
comments during this project. Likewise, my sincere recognition to Dr. Issam for his
help in the development of this project. Finally, my sincere gratitude to all the staff of
the Medical Microbiology Department.
My gratefulness also to my sponsor the Ecuadorian Government who granted me with a
scholarship to study at The University of Manchester with the program "Universidades
de Excelencia, 2013".
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Preface
I obtained my undergraduate degree in Clinical Biochemistry in 2011, and worked for
almost 2 years as microbiologist in charge of the diagnostic of zoonotic diseases such as
Tuberculosis and Brucellosis at the International Center of Zoonosis in Ecuador. After
that, to improve my knowledge and research abilities, I decided to come to England and
study at this University. My aim is to become a researcher in the antibiotic resistance
area.
Dedication
I dedicate all this year of study and MSc project to my parents and siblings who have
supported me during this time. Besides, to all my friends, and church family who have
helped me to face difficult times in England. Finally, all my effort is dedicated to God
who is the provider of all wisdom and intelligence.
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1. Introduction
Microbial resistance to antibiotics is a natural phenomenon that is accelerated by
selective pressure due to overuse and misuse of antibiotics in medical and agricultural
areas (WHO 2014). More than 50% of all the antibiotics prescribed for people are
neither needed nor effective (CDC 2013). Likewise, every year an estimated 10,000-
20,0000 tonnes of antibiotics are manufactured and most of them are used for
agricultural purposes (Laxminarayan et al. 2013). The reduction of antibiotic efficacy to
treat serious infections is a worldwide problem, at least 23,000 people die each year as a
result of these infections in the USA (CDC 2013).
Core actions to combat this overwhelming health problem include: appropriate
use of antibiotics, a global surveillance system of antibiotic use and resistance,
prevention of infections and spread of resistance, improvement of medical diagnostics
to assure correct antibiotic therapy, and development of new antibiotics (CDC 2013;
Laxminarayan et al. 2013; Metz & Shlaes 2014). Numerous investigations are being
conducted into membrane-permeabilizing antimicrobial peptides (AMPs) as an
alternative antimicrobial strategy (Wimley & Hristova 2011; Cotter et al. 2013). AMPs
present broad-spectrum activity against drug-resistant bacteria and fungi along with
showing no specific affinity for a particular protein binding site. Both properties
decrease the risk of induced bacterial resistance to antimicrobial peptides (Wimley &
Hristova 2011).
AMPs can be classified into either ribosomal or non-ribosomal peptides
according to their biosynthetic pathways (Hancock 1997). Ribosomally synthesized
11
AMPs are known as bacteriocins (Abriouel et al. 2011), and they are the most studied
subgroup of AMPs (Cotter et al. 2013). Bacteriocin activity was first demonstrated by
Gratia (1925) with Colicin produced by Escherichia coli, followed by the description
of food-grade lactic acid bacteria (LAB) in 1928 (Cotter et al. 2005; Yang et al. 2014).
LABs are used as food preservatives or antimicrobial peptides (Nishie et al. 2012).
Nonribosomal peptides (NRPs) are peptides produced by bacteria, fungi, and
streptomycetes synthesized on multienzyme complexes instead of being synthesized on
ribosomes (Hancock 1997). NRPs present broad spectrum activity and
immunomodulator, or antitumor activities (Caboche et al. 2010). NRPs are
distinguished from ribosomally synthesized peptides by two structural characteristics.
NRPs present often a cyclic primary structure rather than lineal, and they are made of a
vast biodiversity of monomers apart from the known 20 amino acids residues (Caboche
et al. 2010).
Bacteriocins characteristics make them ideal as alternatives to antibiotics.
Bacteriocins can inhibit the growth of bacteria of the same species (narrow spectrum) or
other genera (broad spectrum) and their spectrum of activity often depends on the
mechanisms of action of each bacteriocin (Cotter et al. 2005; Snyder & Worobo 2014).
They are small, heat-stable peptides made of short chains of around 20-60 amino acid
residues, however longer chains can also be found (Snyder & Worobo 2014). Most
bacteriocins are products of Gram-positive bacteria, as reported in BACTIBASE
dataset. A few bacteriocins from Gram-negative bacteria have been described and even
fewer from Archaea domain (Hammami et al. 2013).
12
Bacteriocins produced by Gram-positive bacteria are grouped in four classes
based on their biochemical, structural and genetic properties (Klaenhammer 1993;
Cintas et al. 2001; Hammami et al. 2013). Class I bacteriocins or lantibiotics are small
(<5kDa), thermostable peptides containing atypical amino acids (lanthionine and
methyllanthionine) that undergo post-translational modification. Class I is subdivided
into type A and type B. Class II non-lantibiotic bacteriocins are small (<10kDa),
thermostable, unmodified peptides which differ in their structure and give rise to four
subclasses: IIa (pediocin-like bacteriocins), IIb (dipeptide bacteriocins), IIc (non-
pediocin-like single-chain) and IId (differ from IIa-IIc e.g. enterocines). Class III are
large (˃30kDa) thermolabile bacteriocins and class IV are complex peptides containing
lipid or carbohydrate groups. In addition, there are new high molecular weight
bacteriocins which are phage-tail-like molecules. Bacteriocins produced by Gram-
negative bacteria are narrow spectrum antimicrobial peptides and present two groups
named colicins and microcins (Chen & Hoover 2003; Nishie et al. 2012; Hammami et
al. 2013; Karpiński & Szkaradkiewicz 2013).
Bacteriocins can present several modes of action. In general, bacteriocins act
either at the cell envelope or within the cell (Cotter et al. 2013). Bacterial cell death may
arise by different means such as leakage of cell contents, cell lysis (by peptidoglycan
inhibition), protein synthesis inhibition (by cleavage of 16S rRNA), removal of critical
ion gradients and DNA degradation (Vriezen et al. 2009; Hammami et al. 2013). Thus,
mode of action between bacteriocins and antibiotics differs significantly, and makes
bacteriocins a suitable alternative to antibiotics (Hammami et al. 2013).
13
The benefits of bacteriocins are seen not only in the food industry to improve
quality and safety, but also many of the bacteriocin properties suggest their potential
value in a clinical setting, including treatment for malignant cancers (Lancaster et al.
2007; Nishie et al. 2012). In food industry, bacteriocins are used as natural food
preservatives to replace chemical preservatives. In addition, bacteriocins present
sensitivity to proteases of the gastrointestinal tract, making them safe food additives that
can be easily digested in the gastrointestinal tract (Cleveland et al. 2001;Yang et al.
2014). Moreover, studies showed growth inhibition of important drug-resistant bacteria
such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci
(VRE) when lantibiotics such as nisin, mersacidin, mutacin 1140 and lacticin 3147 were
used (Nishie et al. 2012). Bacteriocin-producing Bacillus strains present antifungal
activity and may also be used as probiotics since they posses inhibitory activity against
C. perfringens and C. difficile (Abriouel et al. 2011). Bacteriocins are considered as
potential treatment against tumor cells. For example, nisin was used to treat head and
neck squamous cell carcinoma reducing cell proliferation (Joo et al. 2012). The fact that
bacteriocins are ribosomally synthesized allows them to be manipulated by genetic
engineering which may increase their antibacterial activity or structural stability (Nishie
et al. 2012).
The simplest way of screening bacteriocin activity is by direct simultaneous
antagonism in agar media contained in petri dishes (Chen & Hoover 2003). The
principle is based on Antonie van Leeuwenhoek´s studies in which the product from
one microorganism inhibit the growth of another (Chen & Hoover 2003). Although
known as a laborious method with some limitations, screening for inhibitory activity of
14
organisms has led to the discovery of novel antimicrobials such as penicillin (Snyder &
Worobo 2014).
This project aimed to screen a library of environmental samples for
antimicrobial-peptide-producing bacteria, and attempted to purify and characterize
potential novel agents produced by these bacteria.
15
2. Materials and Methods
2.1. Bacterial strains
A library of environmental strains was provided by the Department of Medical
Microbiology at the University of Manchester to be tested for bacteriocin production.
The strains were cultivated on Columbia Blood Agar media (CBA)(Oxoid Ltd.,
England) and incubated at 37 ºC aerobically and with CO2 for 24 hours.
2.2. Indicator strains
The environmental bacteria were assessed for activity against Staphylococcus
aureus NCTC 7447, Methicillin-Resistant S. aureus (MRSA) University strain 393,
Streptococcus pyogenes NCTC 8330, Enterobacter cloacae NCTC 5920, Klebsiella
pneumoniae 169, Bacillus subtilis NCTC 831, and Escherichia coli DH5 isolates
provided by the Department of Medical Microbiology.
2.3. Screening for antimicrobial peptide production
Screening for antimicrobial peptide production was achieved using the
simultaneous antagonism method (Burton et al. 2013). Indicator strains were suspended
in 3 ml of distilled water to gain a turbidity equivalent to a 0.5 McFarland Standard,
then diluted according to BSAC methods (British Society for Antimicrobial
Chemotherapy, May 2013) and plated onto Columbia Agar (CA) (Oxoid Ltd., England)
to produce a lawn of growth. Pure-producer strains were picked from the plates and
stabbed into the previous plated CA and incubated at 37 ºC aerobically and with CO2
(according to bacteria requirements), for 24 hours. The plates were then checked for
16
antimicrobial activity by observing inhibition zone around the stabbed bacterial
colonies. The zone of inhibition against indicator strains were assigned with arbitrary
score ranging from +1 as minimum inhibition to +4 as maximum inhibition.
2.4. Broth optimization for antimicrobial peptide production
Among the four broths tested for antimicrobial peptide production: Nutrient
Broth (NB), Tryptic Soy Broth (TSB), de Man, Rogosa and Sharpe (MRS) Broth, and
Brain Heart Infusion (BHI); Nutrient Broth presented the greatest yield of peptide after
the well diffusion assay was performed. Further optimisation of this broth was carried
out by addition of sugars such as lactose, sucrose, glucose and yeast extract. All media
were purchased from Oxoid Ltd., England. The sugars and yeast extract were added
individually at a concentration of 5% to each liquid media, then 1 ml (5%) of a 0.5
McFarland suspension of the producer strain was added to a baffled Erlenmeyer flask
containing 20 ml of NB with the different additives. The samples were incubated on a
shaker at 37 ºC for 18 hours. After incubation, 1 ml of the samples were transferred to a
1.5 ml Eppendorf tube and centrifuged at high speed (13,300 RPM) for 5 minutes to
remove cellular debris, then the cell-free supernatants were assayed for antimicrobial
activity using the well diffusion method.
2.5. Assays to test antimicrobial peptide activity
2.5.1. Well diffusion assay
The antimicrobial peptide activity was monitored during the extraction and
purification procedures by well diffusion assay with the indicator strain Staphylococcus
17
aureus NCTC 7447 (Sharma et al. 2009). On the CBA plate, circular wells of 1 cm in
diameter were cut using a sterile cork borer and then partially sealed with low melting
temperature agarose (Sigma-Aldrich Company Ltd.). The wells were filled with 50 µl of
either cell-free supernatant or purified extracts. After complete diffusion of the
supernatant into the agar was observed, the plate was sterilized with chloroform vapour
for 20 minutes to kill any viable bacteria, and then removed and allowed to dry. The
plate was then inoculated with an indicator strain and incubated at 37 ºC for 24 hours.
Inhibitory activity of the peptide was indicated by the zone of inhibition produced after
incubation.
2.5.2. Spot-on-lawn method
Spot-on-lawn method was also used to check antimicrobial peptide activity. It
comprised in spotted 20 µl of sample into a CBA plate, then allowing it to be absorbed
by the agar. After that, the plate was sterilized with chloroform for 20 minutes, and then
proceeded in the same way as in the well diffusion method (Sandiford & Upton 2012).
2.6. Purification of the antimicrobial peptide
2.6.1. Concentration of the antimicrobial-peptide-containing supernatant
To extract and purify the antimicrobial peptide, a baffled Erlenmeyer flask
containing 150 ml of Nutrient Broth with 5% yeast extract was inoculated with 15 ml
(10%) of a 0.5 McFarland suspension containing the producer strain. The sample was
incubated on a shaking incubator at 37 ºC for 18 hours. After incubation, the sample
was divided in 50 ml Falcon tubes and centrifuged at high speed (3,000 RPM) for 20
18
minutes. The sample was pooled and assayed using well diffusion method to confirm
the presence and inhibitory activity of the antimicrobial peptide.
The extraction and purification of the antimicrobial peptide were performed by
Solid Phase Extraction methods (SPE) (Pingitore & Salvucci 2007). The SPE methods
used in this study were Strata-XL-C followed by Oasis HLB columns.
2.6.2. Strata-XL-C chromatography
A Strata-XL-C column was first conditioned using 40 ml of 90% methanol
(pH2) and 80 ml of 99.9% pure methanol (Fisher Scientific UK Ltd., UK). Then, 80 ml
of ultra-pure water was used to wash out the methanol. After that, 150 ml of the
supernatant adjusted to a pH 6.1 with 1 molar HCl (pH range from 5.8 to 6.2) was
loaded through the column using a sterile syringe. The obtained fractions were collected
in 50 ml Falcon tubes and labelled as "flow through". Next, the column was washed
with 40 ml of ultra-pure water, followed by a second wash with 40 ml of 50% methanol
(pH2). The active peptide was eluted with 60 ml of 90% methanol (pH2). All fractions
were collected and assessed by the well diffusion assay. The characteristics of the
Strata-XL-C column used was 100 µl Polymeric Strong Cation 5g/60ml Giga tube
(Phenomenex Ltd., UK).
2.6.3. Oasis HLB column chromatography
The previous active fractions (flows through) were pooled and centrifuged at
high speed (3,000 RPM) for 20 minutes. The supernatant was collected and acidified to
pH 2 with trifluoroacetic acid (TFA) (Sigma Aldrich, USA) before loading on the Oasis
19
HLB With LP Extraction Cartridge (Waters Corporation, USA) column. The column
was equilibrated first with 2 ml of pure acetonitrile (ACN) (Fisher Chemical, UK Ltd.)
followed by 2 ml of Buffer A (ultra-pure water with 0.1% TFA). After loading 20 ml of
the supernatant, the column was washed with 2 ml of Buffer A, and air was pushed
through the column to dry it. The antimicrobial peptide was eluted with 2 ml of
increasing concentrations (from 10% to 90%) of acetonitrile with 0.1% TFA. All the
fractions were collected at each concentration and assessed by spot-on-lawn assay.
2.7. Characterization of the antimicrobial peptide
The active fractions were pooled and rotary evaporated for 2 hours using a
Savant SpeedVac Concentrator (Thermo Scientific, Germany) to eliminate the ACN
and concentrate the antimicrobial peptide.
Methanol evaporation of active fractions obtained from Strata-XL-C column
was performed in a water bath at 70 ºC for 38 hours.
The samples were neutralized to pH 7 with 1 molar NaOH to perform tests such
as: range of activity, MALDI-TOF, Minimum Inhibitory Concentration (MIC) and
Haemolytic Assay. Due to the small quantity of the sample pH variation was measured
with strips (SIGMA-pH Test Strips 6.0-7.7).
2.7.1. Testing range of activity
The range of activity of the partially purified antimicrobial peptide was
performed by well diffusion assay on CBA plate with five indicator strains
Staphylococcus aureus NCTC 7447, MRSA University strain 393, Escherichia coli
20
DH5, Streptococcus pyogenes NCTC 8330 and Enterobacter cloacae NCTC 5920. The
indicators were streaked in a line (Figure 3).
2.7.2. MALDI-TOF Mass spectrometry
The molecular mass of the partially purified antimicrobial peptide was
determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)
mass spectrometry . MALDI-TOF was performed by the Core Protein Facility at the
Michael Smith Building of the University of Manchester.
2.7.3. Minimum Inhibitory Concentration
The MIC was determined according to the protocol recommended by the
Clinical Laboratory Standards Institute (formerly National Committee for Clinical
Laboratory Standards) (CLSI 2008). To test the MIC of the partially purified
antimicrobial peptide, suspensions of Staphylococcus aureus NCTC 7447, MRSA
University strain 393, and Escherichia coli DH5 were prepared based on CLSI
methods. The test was performed in a sterile 96-well U-shaped microplate (Thermo,
Scientific, England). First, using sterile pipettes, 100 µl of Müller-Hinton Broth (Oxoid
Ltd., England) was added in each row from well 1 to 12 for each strain to be tested.
Then, 100 µl of purify peptide was added to the first well, mixed and 100 µl were taken
from the first well to do the 2-fold dilutions of the peptide until well number 11, well 12
was used as a growth control. After that, 5 µl of each bacterial suspension previously
prepared was added to each well. The microplate was incubated at 37 ºC for 24 hours.
The lowest concentration that inhibited the growth of the organisms was defined as the
21
MIC. In this study, the MIC was measured by comparing the results obtained with the
positive controls. The purify peptide was inoculated in a CBA plate to test its sterility.
2.7.4. Haemolytic Assay
To determine the haemolytic activity of the partially purified antimicrobial
peptide a haemolytic assay was performed (Fernandez-Lopez et al. 2001). First, horse
blood (Thermo Scientific, Oxoid, England) was centrifuged for 10 minutes (3,000
RPM) and the supernatant was removed. Next, the erythrocytes were washed 3 times
with 0.9% saline solution and then re-suspended to a concentration of 5% in 0.9% saline
solution. Subsequently, 100 µl of saline solution was added until well number 10 of a
U-shape microplate. An amount of 100 µl of peptide was added to the first well and 2-
fold dilutions were performed until well number 10, well 11 containing a 100 µl saline
only (Negative control-0% haemolysis) and well 12 containing a 100 µl of 1% triton in
saline (Positive control-100% haemolysis). Finally, 100 µl of 5% horse blood was
added until well number 12 and the plate was incubated at 37 ºC for 30 minutes. The
haemolysis results were measured by comparing the positive and negative controls.
2.8. Strain identification by sequence analysis of the 16S rRNA gene
To identify the producer strain, the region coding for 16S rRNA was sequenced.
First, a Gram stain of the producer was performed to select the correct chromosomal
DNA extraction method, which in this case was focused on Aerobic Spore-bearers
(ASB). In an Eppendorf tube a loopful of cells from a fresh culture were suspended in 1
ml of lysis buffer pH 7.6 with 50 µl of lysozyme (20mg/ml) (SIGMA-ALDRICH,
England) and heated at 37 ºC for 30 minutes. Next, proteinase K from Tritirachium
22
(35mg/ml) (SIGMA-ALDRICH, England) was added to the mixture and heated at 57 ºC
for 30 minutes. As soon as the turbidity of the sample was reduced, it was taken out
from the heater. The DNA extraction was confirmed by running the sample on a E-gel
(Invitrogen E-gel 2% agarose, Life Technologies, Paisley, UK).
PCR (model GeneAmp PCR system 9700 Applied Biosystems) reaction was
performed using 2X BioMix Red (Bioline Ltd., London, UK) ready to use reaction
mixture that includes an ultra-stable Taq DNA polymerase with 1μl of template DNA
extract. PCR amplification was performed with both forward and reverse universal PCR
primers at a concentration of 10 µmol/µl. These were 63f (5'-CAG GCC TAA CAC
ATG CAA GTC-3') and 1387r (5'-GGG CGG WGT GTA CAA GGC-3') (Eurofins,
Germany). The PCR cycle was run at 94 ºC for 2 minutes, followed by 35 cycles at 94
ºC for 1 minute, primer-annealing step at 60 ºC for 1 minute and an extension step at
72ºC for 1 minute. The reaction was completed with an extension step at 72 ºC for 5
minutes, and the product was kept at 4 ºC.
The PCR product was confirmed by running it into an E-gel. For sequencing, the
PCR product was cleaning up using the Illustra ExoProStar 1-step method by adding 2
µl of it to 5 µl of PCR product in a 0.2 ml PCR tubes. The sample was incubated at 37
ºC for 15 minutes to activate the enzymes followed by incubation at 80 ºC for 15
minutes to inactivate the enzymes. Sequencing was performed by staff at the University
of Manchester, department of DNA Sequencing facility at Stopford Building using
3730 48-capillary Applied Biosystems Genetic Analyzer.
23
Finally, the DNA sequence data was analyzed by BioEdit and searched using the
Basic Local Alignment Search Tool (BLAST) of NCBI (http://blast.ncbi.nlm.nih.gov),
and compared against the 16S ribosomal RNA sequences in the database.
24
3. Results
3.1. Screening of bacteriocin production using simultaneous antagonism
Among the 14 strains tested, strain P7 was chosen for peptide extraction and
purification since it presented the best inhibitory activity against S. aureus NCTC 7447,
MRSA University strain 393, Strep. pyogenes NCTC 8330 and E. coli DH5 as observed
in Table 1.
Table 1: Antimicrobial peptide activity of the environmental bacteria isolates.
Indicator strains
Producer Strain and
Zone of Inhibition
P7 P9 P14
S. aureus NCTC 7447 4+ 3+ 2+
MRSA University strain 393 4+ - -
Streptococcus pyogenes NCTC 8330 4+ - -
Enterobacter cloacae NCTC 5920 - - -
Klebsiella pneumoniae 169 - - -
Bacillus subtilis NCTC 831 - - -
Escherichia coli DH5 1+ - -
25
3.2. Broth Optimization
P7 strain grew and produced the potential antimicrobial peptide named AMP-P7
in Nutrient Broth (NB) with a zone of inhibition of 10 mm in diameter. Incubation in
other broths showed no antimicrobial peptide activity when supernatants were assessed
by the well diffusion method. Once additives were incorporated to NB the zones of
inhibition slightly increased (Table 2 & Figure 1).
Table 2: Nutrient Broth optimization for antimicrobial peptide production.
Producer
Strain
Nutrient Broth and Zone of inhibition
(Well diffusion method)
NB+
lactose 5%
NB+
glucose 5%
NB+
sucrose 5%
NB+
yeast extract 5%
P7 strain 14 mm 13 mm 11 mm 15 mm
Figure 1: Well diffusion results of NB optimization
Nutrient broth with: lactose (1), sucrose (2), yeast extract (3), glucose (4)
Indicator strain: S. aureus NCTC 7447
1
2 3
4
26
3.3. Antimicrobial peptide production and purification
NB with 5% yeast extract was selected for AMP-P7 production. After 18 hours
of incubation, the supernatant was tested by the well diffusion assay, and a zone of
inhibition of 20 mm in diameter was observed (Figure 2). This result was considered as
the positive control for AMP-P7 activity presence. Following this results, AMP-P7
purification was initiated with Strata-XL-C chromatography
Figure 2: Well diffusion assay: AMP-P7 produced by P7 from NB with 5% yeast
extract. Indicator strain: S. aureus NCTC 7447
3.3.1. Strata-XL-C chromatography
A bigger zone of inhibition was found in the "flow through" fractions than in the
eluted fractions after assessment of the supernatants by the well diffusion method. This
result suggests that AMP-P7 bound weakly to the column, as zone of inhibition was
also found when the sample was eluted with methanol 90%. Therefore, "flows through"
27
fractions were processed to be purified through Oasis column. Moreover, the fractions
eluted at methanol 90% containing AMP-P7 peptide were assessed by the well diffusion
method after methanol evaporation.
3.3.2. Oasis HLB column chromatography
After adjusting the previous "flows through" to pH 2, the supernatant was
assessed to verify AMP-P7 presence. A zone of inhibition was observed (10 mm)
showing its presence but in small amounts. No zone of inhibition was observed in the
loading and washing fractions which indicates that the AMP-P7 bound to the column.
Table 3: Oasis fractions and zones of inhibition
Fraction
(%)
Increasing concentrations of acetonitrile with 0.1% TFA
10 20 30 40 50 60 70 80 90 100
Zone of
Inhibition
(mm)
-
-
-
-
-
-
11
11
11
8
AMP-P7 (Table 3) was eluted at 70%, 80%, 90% and 100% acetonitrile. Similar
clear inhibition zones were obtained for 70%, 80% and 90% fractions; therefore, these
three fractions were combined and processed again with Oasis column to concentrate
the AMP-P7 peptide. After rotary evaporation of the acetonitrile from the fractions and
adjustment of pH (pH 7.7), fraction 90% was used for MALDI-TOF mass spectrometry
analysis, the 80% fraction was used for range of activity, MIC and haemolytic assays.
28
3.4. Characterization of the peptide
3.4.1. Testing range of activity
The inhibitory activity of AMP-P7 was tested against the indicator strains that
showed sensitivity in the initial simultaneous antagonism assay. However, inhibitory
activity was detected only against Staphylococcus aureus NCTC 7447 which since the
beginning showed to be greatly inhibited by AMP-P7 (Figure 3) suggesting that higher
concentration of AMP-P7 is required to confirmed its inhibitory activity.
Figure 3: Range of activity of purified peptide from P7
a) S. aureus NCTC 7447, b) Enterobacter cloacae NCTC 5920, c) MRSA University
strain 393, d) Strep. pyogenes NCTC 8330 and e) E. coli DH5
In addition, no zone of inhibition was observed when the methanol 90% fraction
was tested for presence of AMP-P7 after water-bath evaporation, suggesting that either
Zone of
inhibition a
e
a
d
a
b
c
a
29
the amount of AMP-P7 in the sample was low or that AMP-P7 is not heat stable at 70
ºC. Besides, all the active fractions containing the AMP-P7 also underwent pH
adjustment from 2 to 8 which could inactivate its antimicrobial activity.
3.4.2. Minimum Inhibitory Concentration
MICs were examined against S. aureus NCTC 7447, MRSA University strain
393, and E. coli DH5. MICs for all the indicator strains were ˃1/2 neat concentration
since no growth inhibition of the indicator strains was observed. These results suggest
that the amount of AMP-P7 was insufficient to determine the MIC necessary to inhibit
bacterial growth especially when serial dilution of AMP-P7 was required.
3.4.3. Haemolytic Assay
AMP-P7 showed haemolytic activity only at the first dilution (1:2 the Neat),
after this dilution no haemolysis was detected.
3.4.4. MALDI-TOF Mass spectrometry
The molecular weight of AMP-P7 eluted at 90% acetonitrile was measured by
MALDI-TOF mass spectrometry. The analysis gave 5 molecular masses: 565.598 Da,
615.943 Da, 889.426 Da, 928.159 Da. It was not possible to obtain a chromatogram of
the results due to technical problems with the mass spectrometry machine.
30
3.5. 16S rRNA sequence analysis
After a BLAST search was performed, it revealed that the 16S rRNA gene
sequence of P7 strain was 99% similar to Bacillus tequilensis strain 10b. No data of
any antimicrobial peptide produced by Bacillus tequilensis strain was found in
BACTIBASE dataset.
31
4. Discussion
A potential narrow spectrum antimicrobial peptide targeting S. aureus produced
by Bacillus tequilensis was obtained. Narrow spectrum bacteriocins are considered as
'designer drugs' and may be used to combat specific microorganism (Riley & Wertz
2002). As known S. aureus is part of the normal human microbiota, especially found in
the nose of 30% healthy people (Humphreys 2007). Infections cause by S. aureus
comprises from minor skin infections to severe pneumonia (Den Heijer et al. 2013). It is
speculated that resistance genes are acquired first by the normal commensal microbiota,
and this may increase the acquirement of resistance in pathogens (De Lastours et al.
2010).
DNA data result of P7 strain was similar to Bacillus tequilensis. In 16S rRNA
sequence analysis, it is likely to obtain sequences that share a great level of similarities
due to sequencing errors, amplification errors, and the possibility of microheterogeneity
(Fox et al. 1992). In addition, there is no a universal primer of sufficient length that is
100% match to all bacteria (Baker et al. 2003). Due to time constraint, the designing of
Bacillus specific primers to confirm the identification of the P7 strain as Bacillus
tequilensis could not be performed. Further identification of P7 strain should combine
confirmation of specie by specific primer design, and comparison of its phenotypic
characteristics described by Gatson et al. (2006).
Bacillus tequilensis was first describe in 2006, after isolation from an
approximately 2000-year-old shaft-tomb at a site called Huitzilapa, near the city of
Tequila in the Mexican state of Jalisco (Gatson et al. 2006). Up to now the only
32
reported clinical application was described by Pradhan et al. (2013), who found an
halophilic biosurfactant produce by B. tequilensis CH able to inhibit biofilm formation
of E. coli and S. mutans on both hydrophilic and hydrophobic surfaces. The rest of
applications found involve biotechnological or industrial focus. (Amulya et al. 2014;
Wang et al. 2014; Sondhi et al. 2014; Chiliveri & Linga 2014). No report of any
antimicrobial peptide produced by B. tequilensis has been documented; thus, the
described AMP-P7 peptide might be considered as a potential novel antimicrobial
peptide produced by this bacterium.
Bacillus species produce both types of AMPs: ribosomal (e.g., subtilin) and
nonribosomal (e.g., daptomycin) peptides (Lee 2011). It is important to distinguish
whether or not an AMP is a true bacteriocin, due to the emergence of resistance to some
nonribosomal peptides by certain bacteria (Abriouel et al. 2011; ). Further studies, of
AMP-P7 peptide, will involve its classification in any those two types of AMPs. There
are around 1164 NRPs (Caradec et al. 2014), however the study of them are beyond the
scope of this project.
Abriouel et al. (2011), proposed a new classification for bacteriocins produced
by Bacillus species that includes three classes: Class I contains bacteriocins that
undergo post-translational modifications (e.g., subtilin, subtilosin A, mersacidin). Class
II bacteriocins are small (0.77-10kDa), heat and pH stable, non-modified and linear
peptides (e.g., coagulin, lichenin). Class III contains large bacteriocins (˃30kDa) (e.g.,
megacin) with phospholipase activity. Bacteriocins that have not being able to be
grouped are called bacteriocins-like inhibitory substances (BLIS). (Abriouel et al. 2011;
33
Mongkolthanaruk 2012). It was suggested that AMP-P7 may belong to the Class II
small bacteriocins as its molecular mass ranged between 0.56kDa to 0.92kDa.
Previous studies showed that the production of bacteriocin may be affected by:
amount of carbon, nitrogen and phosphate, cations, surfactants, inhibitors and pH
(Parente & Ricciardi 1999). In addition, bacteriocin operon is commonly regulated by
specific environmental conditions and stress; therefore, bacteria may not express the
same range of activity under laboratory conditions (Snyder & Worobo 2014). Although
some studies suggest MRS, BHI and TSB mediums for peptide production from
Bacillus species (Marrec 1998; Aunpad & Na-Bangchang 2007; Xie et al. 2009), the
greatest yield of peptide production compare with the other broths was obtained using
NB with 5% yeast extract.
Production of high amounts of bacteriocins by some producer strains is not
always possible (Pingitore & Salvucci 2007). In this study, although NB with 5% yeast
extract allowed the greatest peptide production, few amount of AMP-P7 was obtained
according with the zone of inhibition observed when the well diffusion method was
performed. Therefore, to concentrate antimicrobial peptides prior purification,
ammonium sulfate concentration, absortion-desortion, organic solvent extraction, or
lyophilization can be performed (Aunpad & Na-Bangchang 2007; Pingitore & Salvucci
2007).
The best purification method will depend on the previous knowledge of the
peptide (Parente & Ricciardi 1999; Pingitore & Salvucci 2007). Solid-Phase Extraction
(SPE) methods were used to purify the AMP-P7 peptide. Oasis HLB column allowed
34
partial purification of AMP-P7. However, although Oasis HLB column allows the
extraction of low-concentration analytes, generally the analyte after elution steps ends
up in a large solution volume making difficult its detection (Kitt & Harris 2014).
Reverse-phase high-performance liquid chromatography (RP-HPLC) has shown great
contribution to obtain high purification of bacteriocins, combination of SPE and RP-
HPLC methodology are performed in numerous studies (Chaney 2009; Aunpad & Na-
Bangchang 2007; Pingitore & Salvucci 2007; Sandiford & Upton 2012).
After successful purification is performed, further studies will involve
quantification of AMP-P7 peptide by gas chromatography, mass spectrometry or high-
performance liquid chromatography (HPLC) (Kitt & Harris 2014). In addition, to
elucidate primary structure of peptides, Tandem mass spectrometry techniques are
commonly used; however, because some AMPs undergo post-translational modification
it is difficult to identify a complete sequence of the peptide by using only mass
spectrometry. Hence, to identify uncommon acids that are part of AMPs, Nuclear
Magnetic Resonance spectroscopy (NMR) is recommended (Kim et al. 2010).
As stated previously, production of high amounts of bacteriocins by some
producer strains is not always possible although optimization of extraction and
purification methodology is achieved. The application of the methodology here
described may be beneficial to obtain previous characterization of numerous potential
novel antimicrobial peptides. However, once characterization and potential importance
of the antimicrobial peptide is found, studies may be focus on obtaining bacteriocins by
genetic engineering (Nishie et al. 2012) using either mutating bacteriocin-encoding
genes or by fusing genes from different bacterial species (Gillor et al. 2005).
35
In conclusion, the present study described a partially purified antimicrobial
peptide produced by Bacillus tequilensis. AMP-P7 initially exhibited an antimicrobial
activity against S. aureus NCTC 7447, MRSA University strain 393, Strep. pyogenes
NCTC 8330 and E. coli DH5. However, after extraction and purification processes,
AMP-P7 inhibitory activity was found only against S. aureus. Numerous reports about
B. tequilensis applications has been conducted but none of them describe antimicrobial
peptide production by B. tequilensis. Further studies should focus on optimising
extraction and purification methods used to obtain more reliable characterisation of the
potential antimicrobial peptide isolated during this project.
36
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