EXECUTIVE
SUMMARY

Staphylococcus
Aureus (S.
Aureus) is a major human pathogen
that can infect people. This bacterium has been known nowadays as it has become
resistant to certain antibiotics. Recent studies have demonstrated virulence of
S. Aureus is regulated by agr locus.
Agr locus is an important system for cell-to-cell communication within Staph
family due to its quorum sensing
(QS). This study is about to overcome
this problem by taking advantage of agr locus to develop biosensor to detect S. Aureus. This biosensor will be
comprised with red fluorescent protein under specific promoter; it will
fluoresce in red due to auto-inducing peptides (AIPs) from QS system. This assay
will allow rapid and efficient biosensor system to diagnose staphylococcal
infection without using any costly machines.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

OBJECTIVE

1.     
To
establish binding of AgrC and AgrA system to P3 promoter in AIP’s presence

2.     
To
construct P3 expression system to encode RFP gene in bacteria

3.     
To
develop reporter system using RFP to detect S.
Aureus

 

INTRODUCTION

Problem Statement

Pathogenic
S. Aureus can be detected by using
various biochemical assays in hospital. These biochemical assays are PCR,
catalase, coagulase and DNase tests. They are important as they can identify
the presence of S. Aureus. However,
there are limitations to use these assays to detect this bacterium.

 One of the biochemical assay to detect S. Aureus is polymerase chain reaction
(PCR). PCR can amplify virulence gene in S.
Aureus such as mecA but it takes time to detect this gene. It also needs a
good purity of sample to get better result; This is due to its extreme
sensitivity as it would have possibility of carryover contaminations. Primer
set must be designed to anneal at exact location to amplify virulence gene in S. Aureus.

Next,
catalase test can detect the presence of S.
Aureus. This bacterium respires using oxygen and it produces catalase
enzyme to protect it from toxic by-products of oxygen metabolism. This test is
performed with 3% hydrogen peroxide (H­2O2) which it will
change to water and oxygen when it mixes with culture of S. Aureus. The disadvantage of using this method is H2O­2
should undergo a control check daily as it is unstable. This test detects other
staphylocci and organisms such as Escherichia
coli and Klebsiella pneumoniae.
The Organisms also lose their catalase activity after 24 hours which might give
a result of false-positive.

There
are two types of coagulase tests which are slide coagulase and tube coagulase
tests. S. Aureus can cause rapid cell
agglutination due to bound coagulase on the surface of the cell wall; fibrin
will be formed when suspended in plasma that contains fibrinogen  (Jr
2006). Although this test can determine S. Aureus, there are limitations of using this method especially
using plasma as medium. One of the limitation is outdated human plasma may
contain anti-staphylococcal antibodies that might counteract with S. Aureus. Other factor is human plasma
might put in danger to researcher due to viral infections such as HIV, AIDS,
Hepatitis B and C (Kateete
et al. 2010). 

Some
S. Aureus strains produce negative
reaction when they are tested with slide coagulase test. They will go through
tube coagulase test to determine whether it is S. Aureus or not. The strains may produce free coagulase to form
clot in the presence of fibrinogen. The limitation of this test is S. Aureus will produce fibrinolysin that
can dissolve the clot when the incubation is after 4 hours in 35°C (Jr
2006). The cost of this test is also expensive.

Deoxyribonuclease
test (DNase) is a test that determines S.
Aureus as this bacterium produces DNase. Methyl green is an indicator that
is crucial for this method as it can bind to negatively charged DNA. S. Aureus can be determined when methyl
green does not attach to DNA as DNA has broken down. The limitation of this
test is other bacteria such as Streptococcus
pyogenes, Moraxella catarrhalis and others may produce DNase. Some
Methicilin Resistant S. Aureus
strains also do not give positive result of this test. This test also can be
determined by using HCl but 1N HCl that is used in DNase agar is bactericidal.
Thus, this test must be observed within 5 minutes.

 

Hypothesis

There
have been various biochemical assays that can detect the presence of S. Aureus.  However, these biochemical assays have their
own limitations that gives disadvantage when determining this bacterium. Hence,
this study is about biosensor that can determine the presence of S. Aureus. This biosensor will be
comprised with P3 promoter, RFP, AgrC and AgrA. This biosensor will show that
AgrC and AgrA can detect auto-inducing peptides (AIPs) from S. Aureus; this is due to quorum sensing
(QS) that is important to Staphylococcus species
for cell to cell communication. Then, these AgrC and AgrA will drive RNA
polymerase to bind to P3 promoter for expression of virulence gene but instead
of using virulence gene, we change it to red fluorescent protein (RFP) to
detect this bacterium. 

Background

Staphylococcus Aureus has long been recognized to cause
diseases in humans (Kluytmans
& Verbrugh 1997). It is common bacteria that is
found on the skin and it usually does not cause disease; but it can lead to
serious infections when the bacteria invade deeper in body. Commonly, these
infections can be treated by antibiotic treatment. However, S. Aureus has become huge problem due to
its evolution that against antibiotic resistant (Davies
& Davies 2010). Methicilin-resistant S. Aureus is difficult to treat than
most of S. Aureus strains due to its
ability to resist to certain antibiotics (Chambers
& Deleo 2010).

Accessory
Gene Regulator (Agr) locus regulates virulence factors in S. Aureus especially during invasion phase of staphylococcal
infection (Bronner
et al. 2004; Yarwood & Schlievert 2003). This is due to its function to
repress the expression of adhesins and activates gene coding of exotoxin
secretion during stationary phase of this bacterium (Rutherford
& Bassler 2012). Agr locus generates quorum-sensing
(QS) for cell-to-cell communication within Staphylococcus
species (Yarwood &
Schlievert 2003). QS bacterium releases chemical
signal molecule that is called autoinducing peptides (AIPs) (Rutherford
& Bassler 2012). The mature AIP contains seven to
nine residues that form peptide thiolactone by covalently linking to C-terminal
amino acid carboxylate to shape a cyclic thioester (Jensen
et al. 2008). Increasing AIPs is crucial for Staphylococcus species to increase cell
population density (Federle
& Bassler 2003; Manuscript & Architectures 2015).

The
agr locus contains agrB, agrD, agrC and agrA(Sakoulas
et al. 2002). agrD is the signalling peptide
while agrB is the secretory protein; agrD and agrB are a crucial machinery of
QS system to produce and secrete mature AIP from S.Aureus (Koenig et al.
2004). This study only focuses on AgrC and
agrA; AgrC is the cognate sensory histidine kinase and agrA is the response
regulator (Koenig
et al. 2004). AgrC is comprised of six or seven
N-terminal transmembrane segments and a C-terminal cytoplasmic kinase domain
which can detect AIPs(Bronner
et al. 2004). Then, it phosphorylates aspartate
residue of agrA by the presence of AIPs. AgrA undergoes conformational changes
to upregulate the gene expression(Koenig
et al. 2004). Transcription starts when agrA
binds to promoter elements for gene coding(Jensen
et al. 2008).

The
primary effector of virulence responses is RNAIII polymerase (Koenig
et al. 2004). Agr locus controls the expression
of RNAIII polymerase to autoregulate QS system(Gordon
et al. 2013). Agr system controls P3 promoter.
This promoter is located at the upstream of RNAIII transcript. This promoter is
crucial to express virulence factor in S.
Aureus. P3 promoter will be activated when AgrA binds into it. Then, it
will express virulence gene of S. Aureus.

Red
fluorescent protein (RFP) can be used for whole organ imaging. Red light is
much more efficient at penetrating biological tissues due to its longer
wavelength. RFP also can be seen visibly instead of using GFP that needs UV
viewer to see its color.

 

 

MATERIALS AND METHODS

Strains and culture conditions

E. coli will be used for cloning of plasmid. This strain will be
cultivated in Luria Bertani (LB) medium. This strain will be cultured at 37°C
with shaking at 225 rpm. Antibiotics will be added according to selection
marker in plasmids.

 

Construction of plasmid

Plasmid
will be comprised with AgrA, AgrC, P3 promoter and RFP. AgrC and AgrA will be
cloned into shuttle bacterial plasmid under promoter in plasmid. These
fragments will be utilizing polymerase chain reaction (PCR) or gBlock synthesis
to amplify them using genomic DNA of S.
Aureus. P3 promoter will be needed to express RFP.  P3 promoter and RFP will be cloned using PCR
and gBlock synthesis into shuttle bacterial plasmid. P3 promoter will be using
genomic DNA of S. Aureus while RFP will
be amplified using certain plasmid as DNA template for PCR. Then, AgrCA will be
co-transformed with P3 and RFP to develop biosensor.

 

Cell Supernatant Assays

E. coli that contains pT7-agrBD will be cultivated in LB broth with
approriate antibiotic. Then, this culture will be measured its OD600
until its value is betweeen 0.5 – 1.0. Next, IPTG will be added to induce T7
promoter to express AgrBD gene to produce AIPs. The supernatant  will be sterile-filtered using 0.2µm syringe
and stored at -20°C after an hour of induction. This supernatant will be
measured using microplates that contain of 117µL LB medium with approriate
antibiotic and 3µL of overnight culture of bacterial biosensor. Then, this
microplate will be quantified its excitation and emission wavelength.

Co-culture Assays

S. Aureus will be cultivated in LB medium with recombinant bacteria.
Fluorescence of RFP will be measured by its excitation and emission wavelenght.
Colony forming unit also will be determined for both strains by using selective
BBC­TM CHROMagarTM.

 

 

 

EXPECTED RESULTS

This study focuses on
developing bacterial biosensor to detect S.
Aureus. This manipulated bacterial biosensor will detect auto-inducing
peptide from S. Aureus by AgrC and
AgrA. AgrC and AgrA will drive RNAIII polymerase to bind to P3 promoter. Then,
this bacterial biosensor will fluoresce in red due to RFP that has been
expressed by P3 promoter. This method will allow detection of S. Aureus rapidly and efficiently
without using any costly machines.