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In Silico Drug Design of Biofilm Inhibitors of Staphylococcus epidermidis

by Al-mulla, Aymen Faraoun, MS

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epidermidis is the most common isolate and has been implicated in 34% of
cases in children and 51% in neonates (Hira et al., 2007; Rogers et al.,
2.2.3 Staphylococcus epidermidis virulence factors
S. epidermidis produces a very limited number of tissue damaging
exoenzymes and toxins when compared to S. aureus.
A cysteine protease and an extracellular metalloprotease of 32 kDa
molecular weight have been described in S. epidermidis (Sloot et al., 1992;
Teufel and Gotz, 1993).
Two very similar lipases are found in S. epidermidis, which may be
important for skin colonisation (Chamberlain and Brueggemann, 1997).
A delta-like toxin consisting of 25 amino acid residues has been
demonstrated in S. epidermidis (McKevitt et al., 1990). This was named
alpha toxin and it is encoded by the hld gene located in the regulatory agr
locus (Otto et al., 1998). This N-formylated alpha-helical peptide, alphatoxin,
causes the lysis of erythrocytes by forming pores in the cytoplasmic
membrane (McKevitt et al., 1990).
2.2.4 Biofilms
A biofilm is a structure composed of bacterial cells growing on a surface
and enclosed in an exopolysaccharide matrix (Dworniczek et al., 2003).
Bacterial biofilms are found in many aspects of life, including industry,
nature, and in human life. In industry, biofilms participate in biofouling
pipelines, material corrosion, and introduce problems in water remediation
(Coetser and Cloete, 2005). In the human domain, bacterial biofilms dwell
in the oral cavity as oral plaques as well as on the skin as part of protective
microflora against other more aggressive pathogens (Stewart et al., 2004).

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The biofilm is an extracellular polymeric substance (EPS) that allows
adhesion to various material surfaces and cohesion to each other. This
material includes polysaccharides, and nucleic acids (Flemming and
Wingender, 2010). EPS comprises nearly 90% of the biofilm biomass and
contributes significantly to the structural qualities that characterize
biofilms. This extracellular matrix provides the three-dimensional
structure of the biofilm; it is described as “cathedral” and resembles
mushroom-like formations (Stewart and Franklin, 2008).
Glycoproteins, are also found within this organic matrix (Humphrey et
al. 1979). EPS varies in chemical and physical properties, but in the case
of Gram-negative bacteria it is primarily composed of neutral or
polyanionic polysaccharides. Uronic acids (such as D-glucuronic, D-
galacturonic, and mannuronic acids) or ketal-linked pyruvates are known
to constitute part of the EPS matrix. These give anionic properties to the
biofilm allowing cross-linking of divalent cations such as calcium and
magnesium (Flemming et al., 2000; Sutherland, 2001).
EPS is highly hydrated, and can be both hydrophilic and hydrophobic
with varying degrees of solubility. The polysaccharide content of an EPS
has a marked effect on the biofilm as the composition and the structure will
determine their primary conformation (Sutherland, 2001). A bacterial EPS
contains backbone structures of 1,3- or 1,4-b-linked hexose residues, which
are rigid and generally poorly soluble or insoluble, whereas other EPS
molecules are more readily soluble in water. The fluid channels present
throughout the biofilm allow nutrient and waste transfer. In addition,
digestion can occur outside the cell in order to recycle raw cellular material.
Biofilms employ a form of chemical communication called quorum
sensing (QS). This characteristic allows this network of cells to work
collectively and coordinate various tasks such as cell growth, adhesion, and
death. Quorum sensing may act in response to external factors with the

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release of virulence factors to protect against foreign invaders, including
antimicrobials (Gasper, 2011). The signal molecules of QS systems are
small molecules called autoinducers (AIs). So far, three different types of
AIs have been described: N-acylhomoserine lactones (HSL, AI-1)
produced by Gram-negative bacteria, LuxS/autoinducer-2 (AI-2) is
common in Gram-positive as well as Gram-negative organisms, and
oligopeptides produced from pre-proteins found in many Gram positive
bacterial species (Mack et al., 2007). The accumulation of high
concentrations of AIs due to the increase in cell population density
activates QS systems which in turn regulate the expression of various
genes. In S. epidermidis two QS systems have been described to date: the
agr system (Kong et al., 2006) and luxS/AI-2 system (Xu et al., 2006; Li
et al., 2008). Both QS systems appear to activate the formation of biofilms.
In S. epidermidis, biofilm formation is regarded as a major
pathomechanism as it renders S. epidermidis highly resistant to
conventional antibiotics and host defenses. This can be caused by slow
diffusion of these compounds through the extracellular polymeric matrix
and slow growth of the bacteria (Schoenfelder et al., 2010; Mah and
O’Toole, 2001).
Possible mechanisms of biofilm resistance to antimicrobial agents
include: (i) Delayed penetration of the antimicrobial agent through the
biofilm matrix, (ii) Altered growth rate of biofilm organisms, and (iii)
Other physiological changes due to the biofilm mode of growth (Donlan
and Costerton, 2002). The biofilm production is mediated by icaADBCdependent
and independent pathways (Gotz, 2002; O'Gara, 2007). The ica
operon encodes enzymes that are involved in the production of
polysaccharide intercellular adhesion (PIA), which mediates the
intercellular adherence of bacteria and the accumulation of a multilayered
biofilm (Gotz, 2002).

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2.2.5 Gene regulation of biofilm
Multiple genes are known to be involved in biofilm formation in bacteria .
These include:
1. icaADBC :
The ica locus is composed of an operon, icaADBC, which encodes the
structural genes required for PIA synthesis (Figure 2.4). The ica operon is
composed of four open reading frames, icaA, icaD, icaB and icaC (Rohde
et al., 2006). A fifth gene, divergently transcribed, is the icaR gene,
responsible for the regulation of the transcription of icaADBC, which is
located upstream of the icaA start codon. Conlon et al. (2002) reported that
the icaR gene encodes a transcriptional repressor involved in the
environmental regulation of the ica operon expression and biofilm
formation in S. epidermidis .
The open frame icaD is located between icaA and icaB, overlapping with
both genes. IcaA is responsible for N-acetylglucosaminyltransferase
activity during PIA synthesis; the presence of IcaD is required for optimal
transferase activity (Gerke et al., 1998). IcaB, a deacetylase, is responsible
for the deacetylation of the poly-N-acetylglucosamine molecule (Vuong et
al., 2004a). The transmembrane protein IcaC plays a putative role in
externalisation, elongation and translocation of the growing polysaccharide
chain to the cell surface (Rohde et al., 2006) and is responsible for the
production of the full-length PIA molecule.

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Figure 2.4 Schematic overview of PIA synthesis (Otto, 2009)

2. atlE:
AtlE is a multifunctional, surface-associated protein having both
enzymic and adhesive functions (Heilmann et al., 2003). It has been
demonstrated that AtlE is important in S. epidermidis pathogenicity. The
S. epidermidis mutant strain deficient in production of autolysin has been
shown to be less virulent than the wild-type parental strain in an
intravascular catheter-associated infection model in rats (Rupp et al.,
3. sarA:
The staphylococcal accessory regulator locus sarA encodes a DNAbinding
protein that is involved in the regulation of the biofilm formation
in S. epidermidis by affecting ica operon transcription in an icaRindependent
manner or by interfering with PIA production at a later stage
(Tao et al., 2006; Tormo et al., 2005a).

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4. sigB:
Bacterial RNA polymerase enzymes require a specialized subunit called
sigma factor to recognize and contact their promoter. Three sigma factors
A, σB, σH) have been identified in Staphylococcus spp., among which σB
has been intensively studied (Kies et al., 2001).The influence of σB on

biofilm formation appears to occur via repression of icaR transcription,
which in turn represses transcription of icaADBC (Knobloch et al. 2004).
5. agr:
The accessory gene regulator (agr) is part of a quorum-sensing system
and its expression is cell density dependent. The agr locus is activated by
an autoinducing peptide pheromone (AIP). Generally, when cell density is
low, agr activity will be low. This allows the expression of surface proteins
which are important for colonisation by these bacteria. But when cells
density increases, agr activity will be stimulated releasing exoenzymes and
toxins (Vuong and Otto, 2002).
6. luxS :
The LuxS gene is responsible for the production of AI2 during the
logarithmic growth phase and its expression is reduced in the stationary
growth phase in S. epidermidis. It has been demonstrated that AI-2
influences biofilm formation in vitro and enhances virulence in a rat model
of biofilm-associated infection. However, in contrast to the agr system, the
details of the mechanism of the AI-2 signaling function and the sensors or
transporters for AI-2 in S. epidermidis are not clear yet (Li et al. 2008).

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2.2.6 Mechanism of Biofilm Formation
1. Attachment
Initial attachment can be based on direct binding to abiotic surfaces. This
process is mediated by hydrophobic interaction, van der Waals forces,
electrostatic interaction between bacteria and implant surface, and several
surface proteins, e.g. Ssp-1, Ssp-2, Bhp, and AtlE and teichoic acids
(Veenstra et al., 1996; Heilmann et al., 1997; Gross et al., 2001; Tormo et
al., 2005b). Alternatively, indirect binding to surfaces of implanted
medical devices coated with host plasma and matrix proteins (conditioned
material) such as fibrinogen, fibronectin, collagen, vitronectin and elastin
can be mediated via microbial surface components recognizing adhesive
matrix molecules (Ribeiro et al., 2012).
2. Accumulation
Following adherence to the surfaces of implants, biofilms develop
through intercellular aggregation that is mediated by intercellular adhesins.
These include surface macromolecules, such as exopolysaccharide, surface
proteins, teichoic acids and extracellular DNA originating from lysed cells
(Shahrooei, 2010).
3. Maturation and Detachment
Once a mature biofilm has been established, cells or cell aggregates may
be continuously released into the flowing extracellular fluid (mainly blood
stream) on a chronic basis, with consequent bacteraemia as well as seeding
distant sites. In contrast to primary attachment and accumulation,
detachment is poorly understood in Staphylococcus spp. in comparison to
others. However, several factors have been proposed to be involved in
biofilm detachment, including mechanical forces (flow effect), changes in
nutrient concentration, cessation of production of biofilm building

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material, and production of detachment factors which is mainly controlled
by quorum-sensing (QS) system accessory-gene regulator (agr) (Yao et al.,
2005; Otto, 2008; Otto, 2009). The detachment factors have been proposed
to function via two detachment mechanisms: enzymatic degradation of
biofilm exopolymers (Boles and Horswill, 2008) and disruption of noncovalent
interactions by detergent-like molecules (Vuong et al., 2004b).

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Chapter three
Materials & Methods

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3. Materials and Methods
3.1 Materials
A- Equipment
Equipment Company (Origin)
Autoclave Raypa (spain)
Auto ELISA Reader Beckman (Austria)
Distillator GFL (Germany)
Incubator Binder (America)
Light microscope ALTAY (Taiwan)
Micropipettes Volac (England)
pH meter JENWAY (England)
Refrigerator Arçelik (Tukey)
Sensitive balance Sartorius (Germany)
Shaker incubator J.P. Selecta (Spain)
Spectrophotometer EMC LAB (America)
Vortex mixer IKA-WERK (America)
Water bath TAISITE (China)

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