INSILICO IDENTIFICATION 0F PUTATIVE DRUG TARGETS IN STAPHYLOCOCCUS AUREUS

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 AND 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.