On the other hand, S. aureus possess a variety of enzymes
and regulatory proteins that play a role in detoxification of ROS and surviving
the oxidative stress conditions.
Superoxide dismutases (SOD) are metalloenzymes which catalyze dismutation of O2?
into oxygen and H2O2.
Therefore, they protect S. aureus from
O2? and also from the products of reactions that involve
O2? such as OONO?.
There are 2 SOD enzymes encoded by 2 monocistronic genes: sodA and sodM, with SodA
responsible for the main activity (Poyart et al., 1995).
Catalase protects S. aureus from H2O2 through
its degradation into oxygen and water. Catalase is encoded by monocistronic
gene katA. In addition to catalase, S. aureus possess alkyl hydroperoxide
reductase -encoded by ahpC- that
detoxifies alkyl hydroperoxides into their corresponding alcohols. Upon
treatment with H2O2, both catalase and AhpC are induced
in S. aureus with catalase
representing the major resistance against H2O2 (Poole 2005) (Cosgrove et al., 2007).
aureus possesses peroxide sensing regulatory
protein called PerR that controls the expression of many genes involved in
oxidative stress response. Upon complexation with iron or manganese, perR
functions as repressor of oxidative stress genes as katA and ahpC by binding
to a conserved motif called perR box. In oxidative stress, H2O2
reacts with iron in PerR generating HO• that
oxidizes iron-coordinating histidines leading to the loss of iron and
consequently loss of the DNA binding activity (Horsburgh et al., 2001) (Lee and Helmann 2006).
Iron is a transition metal
required for many metabolic processes where it acts as co-factor for the
activity of many enzymes and therefore, it is essential for viability of
bacteria (Griffiths et al., 1999).
However, high level of free intracellular iron is toxic to the cell and also
ferrous iron Fe2+ contributes to oxidative stress through generation
of ROS in Fenton reaction (Imlay et al., 1988).
Therefore, iron level in S. aureus is
under control of different proteins such as ferritin and Dps-homolog MrgA.
Ferritin is an iron storage protein that is encoded by ftnA. It has a ferroxidase center that oxidizes Fe2+
into Fe3+; hence it provides protection
from HO• generated from Fe2+
in Fenton reaction (Andrews and Simon 1998) (Andrews et al., 2003).
In addition to ferritin, S. aureus
possesses MrgA (Metallo regulated gene A) that is homolog to Dps of E. coli. MrgA has a bi-functional
activity in resistance against oxidative stress. First, it has iron chelating
function with ferroxidase center providing protection against Fenton reaction.
Second, it binds to DNA and induces nucleoid condensation which provides DNA protection
against any oxidative stressors that may target it (Ingmer
S. aureus also possesses ferric uptake regulator (Fur) which is a
transcriptional regulator which plays a role in maintaining iron homeostatsis.
When Fur is complexed with iron, it regulates transcription of genes involved
in iron acquisition and trasnport through binding to a conserved repeat
sequence called Fur box (Xiong et al., 2000)
Spx and its role in resistance to oxidative stress
Spx is a conserved global gene regulator
in Gram-positive bacteria where it directly interacts with ? subunit of RNA
polymerase (RNAP) and thus controls initiation of transcription, either positively
or negatively. Negative regulation is mediated through spx binding to residue
(Tyr263) in C-terminus of RNAP ? subunit, which is highly conserved in Gram-positive
bacteria leading to blocking the interaction of transcriptional activators with
RNAP (Zuber 2004) ( Nakano et al., 2003a).
Spx is also a positive regulator of genes involved oxidative stress where
activates the transcription of trxA and trxB, encoding thioredoxin and thioredoxin reductase,
respectively (Nakano et al. 2003b) (Pamp et al., 2006).
Spx has a
Cys-X-X-Cys (CXXC) motif at its N-terminus which seems to regulate its activity
through thiol/ disulfide switch. Spx was reported to activate trxA and trxB only during oxidative stress with the formation of
intramolecular disulfide bond (Nakano et al., 2005).
The thioredoxin system -comprised of thioredoxin and
thioredoxin reductase- maintains bacterial cytoplasm in a reduced state.
Thioredoxins contribute to oxidative stress resistance through facilitating H2O2
reduction and HO• radical scavenging. Thioredoxins
also donate reducing equivalents to peroxiredoxins (Arner et al., 2000).
Inactivation of Spx in S. aureus renders cells very sensitive
in different stress conditions such as oxidative stress and high and low
temperatures. In addition, Spx inactivation also leads to slow bacterial growth in non-stress conditions. Such
phenotype of slow bacterial growth and hypersensitivity to stress conditions
may be due the reduction of trxB expression when Spx is inactivated (Pamp et al., 2006).
1.7 Phenol Soluble
Modulins, Agr Quorum sensing and SarA regulatory system
One of S. aureus key strategies to evade host
immunity is to mediate cytolysis of immune cells through production of phenol
soluble modulins (PSMs). PSMs were first identified in S. epidermidis through isolation of a ‘pro-inflammatory complex’ by
phenol extraction, thus termed phenol soluble modulins (Mehlin et al., 1999). Later, PSMs were
identified in S. aureus with the
ability to lyse human neutrophils. PSMs are key impact of S. aureus pathogenesis and they are associated with the ability of
community acquired MRSA to cause infections. There are various PSMs that are
encoded from different locations where 4 PSM ? peptides are encoded from PSM ?
operon and 2 PSM ? peptides are encoded from PSM ? operon (Wang et al.,. 2007).
Many S. aureus virulence determinants are
under control of the global virulence system Agr. This system triggers changes
in gene expression in S. aureus in
high cell density through a process called quorum sensing. The agr locus encodes for 2 primary
transcripts, RNAII and RNA III that are generated from P2 and P3 promoter,
respectively. P2 operon encodes four proteins responsible for agr sensing mechanism which are AgrB
transmembrane protein, AgrD precursor peptide, AgrC receptor and AgrA response
regulator. The AgrD is a precursor peptide that is processed by AgrB
transmembrane protein to generate auto-inducing peptide (AIP). Upon secretion
of AIPs into external environment, they can be detected by AgrC receptor on
cell surface of S. aureus leading to
phosphorylation of AgrA response regulator. Phosphorylated AgrA activates
transcription P2 and P3 operons in an auto-feedback loop. P3 operon encodes for
RNAIII which is the regulatory effector of Agr system where it activates
expression of different virulence factors (Novick 2003).
It was found that both PSM ?
operons are under positive control of virulence regulator Agr quorum-sensing system.
In contrast to most genes which are regulated by RNAIII effector molecule, PSMs
were found to be expressed after direct binding of AgrA response regulator to
PSM operons independent of RNAIII (Wang et al.,
(Queck et al., 2008). An overview of
agr system and its effect on psm expression is shown in figure (1.7).
Another regulatory system
controlling gene expression in S. aureus
is SarA protein family which is a collection of DNA binding proteins. SarA, the
prototypic member of this family, is a 124
residue protein that affects expression of many genes involved in many processes such as virulence genes, amino-acid
and nucleotide metabolism genes (Cheung et al.,
SarA regulates gene
expression either by direct DNA binding or
indirectly through activation of agr quorum
sensing system (Cheung et al., 1997).
SarA is encoded by sarA locus which comprises
3 promoters P1, P3 and P2 encoding for three overlapping transcripts sarA, sarC, sarB respectively as shown
in figure (1.8).
The three transcripts are under regulation of bacterial growth cycle where sarA and sarB levels are high at early log phase while sarC level is highest at stationary phase (Bayer et al., 1996).
analysis of sarA locus have shown that SigB, which is a stress-induced transcription factor, activates
P3 promoter of sarA (Deora et al., 1997).
Illustration of sarA locus in S. aureus chromosome. It is composed
of 3 promoters P1, P3 and P2 that encode for three
overlapping transcripts sarA, sarC,
sarB respectively (Bischoff et al. 2001).
Most bacteria adapt to stress conditions of nutrient limitation
through stringent response. Stringent response is characterized by slow
bacterial growth, inhibition of DNA replication and activation/repression of
some genes. At molecular level, stringent response is characterized by the
production of GTP derivatives which are Guanosine pentaphosphate (pppGpp) and
Guanosine tetraphosphate (ppGpp) and they are collectively known as alarmones.
Alarmones reorganize cellular transcriptional activities and gene metabolism
leading to adaptation to stress conditions. In S. aureus, the level of alarmones is
regulated by RelA/SpoT homologue (RSH) in addition to small alarmone synthases,
RelP and RelQ. RSH has an enzymatic N-terminal domain (NTD) and regulatory
C-terminal domain (CTD). NTD of RSH enzyme has synthetase and hydrolase domains
that are responsible for synthesis and degradation of alarmones, respectively
and its CTD has TGS, DC and ACT domains whose functions are not fully clear,
while RelP and RelQ consist only of a synthetase domain. Nutrient limitation,
particularly amino-acid starvation induce RSH enzyme that synthesizes
alarmones, mainly pppGpp. On the other hand, RelP and RelQ are induced in
response to cell wall stress and they preferentially synthesize ppGpp. The
difference between ppGpp and pppGpp in gene expression is still not
We addressed this question and RNA sequencing was performed on the
strain of HG001 (p)ppGppo transduced with plasmids encoding for RSH
(synthetase part) and RelQ that synthesize pppGpp and ppGpp, respectively upon
induction. Results show significant differences between pppGpp and ppGpp in expression of oxidative stress response
genes such as sodA, ftnA, mrgA and perR. In order
to confirm RNA sequencing results, we aimed to perform several northern
analysis using probes for oxidative stress response genes (sodA, ftnA mrgA, perR, katA and ahpC). In
addition, we tested probes for regulatory genes as agrA, sarA and virulence
gene PSM. Furthermore, we aim to
investigate how alarmones affect bacterial growth in conditions of oxidative
stress through performing growth curves in HG001 strain. We also aim to mutate spx gene- a positive regulator of
oxidative stress response genes- in HG001 strain in order to check how it
affects the expression of other oxidative stress genes in Northern analysis.
Another scope of our investigation was to test protein-protein
interactions among RSH since interactions seem to play an important role for
the correct function of RSH. It has been shown that in E. coli, interactions are crucial for regulating the activity of
RelA where the enzyme is active in monomeric state and inactive in oligomeric
state. We used ‘Bacterial two hybrid assay’ with the aim of testing interactions
among RSH enzymes and identifying which domains of RSH are responsible for the