ABSTRACT
Staphylococcus aureus is one of
the most important and studies gram positive bacterial strains, which have a
great potential to infect human being as well as other mammals. Staphylococcus
aureus is a major cause of infections responsible for much life threatening
diseases like toxic-shock syndrome, staphylococcal scarlet fever, meningitis,
osteomyelitis etc. infection due to these strains are difficult to treat. This
research work focused on the identification of drug target enzymes in
staphylococcus aureus. These drug target enzymes development of the new
antibiotic which can kill or suppress the growth of pathogen with no effect in
the host. The E-value threshold cut-off was set to 0.001.
TABLE OF CONTENTS
Title
page i
Certification
ii
Dedication
iii
Acknowledgement
iv
Abstract
v
Table
of content vi
CHAPTER ONE
Introduction
CHAPTER TWO
Material
and methods
CHAPTER THREE
Result
CHAPTER FOUR
Discussion
CHAPTER FIVE
Conclusion
References
CHAPTER ONE
INTRODUCTION
Staphylococcus
aureus a member of staphylococcaceae is considered as an opportunistic pathogen
for the different mammals including livestock as well as humans (Lowy1998,
projan and novick, 1995). It has been reported that staphylococcus aureus is
resistance against varies antibiotics present in the market (Lowy, 1998; walsh
and bowe, 2002). this bacterial strain word widely known for causing many of
the severe and deadly disease like osteomyelitis bacteremia , endocarditic,
meningitis, scalded skin syndrome, toxic shock syndrome, food poisoning, etc(
Lowy, 1998; drekema et al ., 2001 ). It is the primary cause of lower
respiratory tract infections surgical site infection (Richard, et al., 1993)
and the second leading of nosocomial
bacteremia (wisplinghoff et al ., 2004) pneumonia, and cardiovascular
infections (Richard et al., 1993). Beside these disease, it is also found on
the skin of the human beings and causing major problems like pimples, sour
throat, hair follicle infection, acne, and sties (a sty is an inflammation of a
gland in the eyelid). It is also causes boils, which are deeper pus-filled
abscesses of the skin and underlying tissue (freeman-cook and freeman-cook,
2006 Carleton et al., 2004; king et al., 2006). In common with other
facultative aerobes, staphylococcus aureus can grow in the absence of oxygen
either by fermentation or by using an alternative terminal electron acceptor,
such as nitrate.
Various studies suggest that oxygen play a
role in the pathogenesis of staphylococcus aureus, in both is capacity to
produce virulence factors and its ability to persist and grow in different and
often hostile environment niches (Chan and foster, in 1998; clement and foster,
ohisen et al., 1997; Ross and onderdonk, 2000). The bacteria contain or can
produce a variety virulence factor like adhesion, colonization, exoenzyme and exotoxins,
capsule etc.These virulence factors help the bacteria to attack to the host
cell, it can bind to protein such as fibronectin, laminin, vitronectin, and
collagen, which form the extra cellular matrix of epithelia and endothelia
surfaces (Gillaspy et al., 1990; freeman-cook, 2006). The resistance to antibiotics
emerged and spread rapidly among strains of staphylococcus aureus, about 90% of
staphylococcus aureus strains are currently resistant to penicillin and
derivatives. To combat this problem, new derivatives of penicillin were introduced
(Lowry, 2003; freeman-cook, 2006).
Today, around 50% of all staphylococcus
infections are multidrug resistant (resistant to penicillin, methicillin,
tetracycline, and erythromycin). One antibiotic stood for years as a drug that
did not cause resistant bacteria to emerge. It often thought of as “last resort
“, the name implies exactly how it has been used. Thus, the battle between
human and bacteria continues (Freeman- cook et al., 2006). The computational
approach has been use to investigate novel drug targets in other pathogenic
organisms such as pseudomonas aeruginosa (sakharkar et al., 2004; perumal et
al., 2007) and in helicobacter pylori (outla et al., 2006). As most currently
known, antibacterial are essentially inhibitors of certain bacterial enzymes.
In this study, we have adopted a strategy for comparative pathway analysis to
find out some potential target against staphylococcus aureus. Only those
enzymes which show unique properties than the host were selected as the target.
Staphylococcus aureus is a vanguard
for both nosocomial and community- acquired infection. It is the primary cause
of surgical infection and lower respiratory tract infections, (Richard et al.,
1993) and the second leading cause of cardiovascular infections, and pneumonia
(Richards et al., 1999). Because of evolved resistance to antimicrobial drugs
such as penicillin newer narrow-spectrum β-lactamase-resistant penicillin
antimicrobial drugs (e.g. methicillin, oxacillin) infection with staphylococcus
aureus are especially difficult to treat and this resistance appeared soon
after they were introduced into clinical practice respectively in 1940s and
1960s (Lowy et al., 2003 ). Initially resistance to penicillin was restrained
to small number of hospitalized patients, but as use of penicillin increased,
resistance spread first to other hospitals and then into the community
(chambers at el., 2001). Greater than 80% of community and hospital- acquired
staphylococcus aureus isolated were resistant to penicillin by the late 1960s
(Lowy et al., 2003). Current report suggests that the spread and evolution of
methicillin-resistant staphylococcus aureus (MRSA) seems be following identical
wavelike emergence pattern (Chamber et al., 2001). In many US hospitals, MRSA is
now endemic and epidemic along term care facilities and communities (Straus
Baugh et al., 1960). National nosomial infections surveillance system's data
suggest that the proportion of staphylococcus aureus isolate that are resistant
to methicillin has increased to 59.5%-64.4%, in intensive care unit (klevens et
al ., 2006). Accurate national estimate of incidence are needed for
understanding the magnitude of the problem. Nevertheless, national studies
examine the effect of staphylococcus aureus or MRSA on the healthcare system
are greater than 5years old (Kuehnert et al., 2005). Noskin et al., estimated
that there were 290,000 staphylococcus aureus related hospitalization for
1999-2000 and reported that125, 969 (43.2%) were likely resistant to
methicillin (Kuehnert et al., 2005). The infections caused by this pathogen
range from mild infections such as skin infections, poisoning to life
threatening infections such as pneumonia, sepsis, osteomyelitis and infection endocarditic.
Methicillin- resistant staphylococcus aureus is considered as super bug which
was first reported in 1961 and now-a-days cause mortality rate of 39% while MRSA
cause 24% death [laupland et al .., 2008].
The currently known targets of staphylococcus
aureus include PBP (penicillin binding protein) of peptidoglycan biosynthesis
pathway. Previously, beta-lactam antibiotics were knows to be effective against
them, but due to production of altered form of PBP protein as well as
beta-lactamase enzyme synthesis those drugs are not effective now, hence, our
current study involves identifying targets apart from PBP(Hao et al., 2012).
Another antibiotics prescribed is fluroquinolone. Fluroquinolone target DNA
Gyrase A enzyme-essential for the replication, supercoiling of DNA. But
according to Stephen et al., a highly signification associate between levofloxaxin
and ciprofloxacin treatment and subsequent isolation of MRSA is reported (Weber
et al., 2003). Linezolid, a new class of antibiotic called oxazolidinones is
used to treat MRSA, which involve the mechanism of binding to bacterial 23s
ribosomal RNA but of the 50s subunit and
thus inhibiting the formation of functional 70s initiation complex but between
April 13 and June 26 ,2008,12 patient were identified by LRSA (linezolid resistant
staphylococcus aureus). All the isolates were detected with a point mutation in
23s RNA. It was concluded that the clinical outbreak of LRSA medicated by the
(Fr gene was related with extensive usage of linezolid (Tsiodras et al.,
2001). In this study, we have tried to
search for some potential therapeutic targets other than the target discussed
above. We have implemented an approach considering two pathogen in the senses
that it must be associated with replication as well as viability of the
pathogen. Secondly, the target should not be homologous to human. The non
homologous property of these proteins helps to establish highly selective drug
against the pathogen preventing the possibility of the cross reaction.
With
the human host this may help to minimize the side effect of the proposed drug
(skaharkar et al.,2001). By this approach, we have found some targets which are
not only essential and non-homolog but also bypass the resistant mechanism of
existing targets and some targets are involved in the metabolic pathway of
pathogen of which is not exploited as potential target area (Nathan et al .,
2004). We have also identified the membrane-bound essential, non-human homolog
proteins emphasizing the fact that 60%
of the drug target is membrane-bound (arinaminpathy et al., 2009).
Staphylococcus aureus is an
important pathogen causing a wide range of infection in the hospital and
community setting. In order have adequate information for treatment of
staphylococcus aureus infections, it is crucial to understand the trend in the
antibiotic-resistance patterns. In addition, the occurrence and changes in
types of staphylococcus aureus, colonial identities, and their geographic
spread is essential for the establishment of adequate infection control
programmes. In this study, 68 staphylococcus aureus isolates obtained from
clinical and non-clinical sources Nigeria between January and April 2009 were
characterized using phenotype and molecular method.
Staphylococcus areus can exemplify
better than any other human pathogen the adaptive evolution of bacteria in the
antibiotic era, as it has demonstrated a unique ability to quickly respond to
each new antibiotic with the development of a resistant mechanism, starting
with penicillin and methicillin, until the most recent linezolid and
daptomycin. Resistant mechanism included inactivation of the antibiotic
(penicillinase and aminogylcoside-modification enzymes) alteration of the
target with decrease affinity for the antibiotic notable example being
penicillin-binding protein 29 of methicillin-resistant Staphylococcus aureus
and D-ala-D-lac of peptidoglycan precursors of Vancomycin-resistant strains]and
afflux pumps [fluroquinolone and tetracycline].Complex genetic
arrays[Staphylococcus chromosomal cassette mechanism elements or the Van A
Operonl have been acquired by Staphylococcus aureus through horizontal gene
transfer, while resistance to other
antibiotics, including some of the most recent ones [e.g fluroquinolones linezolid
and daptomycin have developed through spontaneous mutations and positive
selection. Detection of the resistance mechanisms and their genetic basis is an
important support antibiotics susceptibility and surveillance in Staphylococcus aureus.
The need for new drugs comes from the
widespread resistance to those in current use. New drugs targets are required
to allow the discovery of chemically diverse effective drugs. The search for
such new targets and new drug chemo types will likely be helped by the advent
of functional genomics and structure-based drug design. After validation of the
putative targets as those capable of providing effective and safe drugs,
targets can be used as the basis for screening compounds in order to identify
new leads, which in turn, will qualify for lead optimization work. Many enzyme
in macromolecular and metabolite synthesis are promising potential targets,
some of which have been established in many micro organisms.
Any gene necessary for the viability of a
pathogenic organism may be a possible drug target, particularly when the
sequence of that gene has little or no similarity to those of the host and the
pathogen can be effectively used for designing a drug specifically targeting
the pathogen. Detection of bacterial genes that are non-homologous to human genes
and are essential for the survival of the pathogen represents a promising means
of identifying novel drug targets (sakharkar et al.,2004).
The computation approach has been used to
investigate novel drug targets in other pathogenic organisms such as pseudomonas aeruginosa (sakharkar et
al., 2004; perumal et al., 2007) and in Helicobacter
pylori (dutta et al., 2006). In their study, the search for potential
vaccine and drug targets against staphylococcus
aureus was carried out by in silico.
AIM AN OBJECTIVES
AIM
To
identify different drug target enzymes in staphylococcus aureus.
PRIMARY OBJECTIVES
1.
To extract protein from NCBI( national
center for biotechnology information) database in staphylococcus aureus.
2.
To identify non-human homolog proteins
to serve as drug target.
3.
To find and locate those essential
genes of staphylococcus aureus that play important roles in the normal
functioning of the bacterium with the host and to short list them in the view
of drug targeting.
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