This document describes a study investigating the potential of anti-adhesion therapies for treating bacterial skin infections. Synthetic peptides of the tetraspanin CD9 were tested for their ability to reduce adhesion of Staphylococcus aureus and Pseudomonas aeruginosa, two common causes of skin infections, to human keratinocyte cells. Both peptides showed a reduction in bacterial adherence individually and when the bacteria were present together, demonstrating the peptides' potential as an alternative treatment to combat antimicrobial resistance by disrupting bacterial adhesion.

1. 1
Investigating the potential for anti-adhesion therapies ina
bacterial infection model
Supervisor: Dr. Rahaf Issa
Constantinos Demetriou
Registration No.:140158799
University of Sheffield

2. 2
1.0 Abstract
Antimicrobial resistance is a public health issue that leads to an increased mortality in humans and
healthcare costs. Resistant strains of pathogenic bacteria arise from the over-use of antibiotic drugs
and are difficult to eradicate due to their fast acquisition of genetic mutations. The search for new
therapiesthatdonotencourage antimicrobial resistanceisrequired,oneof whichisthe disruptionof
adherence of pathogenic bacteria to host cell receptors. Staphylococcusaureus and Pseudomonas
aeruginosa are the leadingcausesof skinandsofttissueinfections(SSTIs)andoftenco-existinwound
and respiratory infections. Bacterial adherence is considered a critical step for colonisation and
infection and pathogenic bacteria must first pass through a damaged skin and adhere to host cell
receptors,one of which are the tetraspanins. The tetraspanins are a familyof eukaryoticmembrane
receptors and are used as an anti-adhesion therapy to prevent the adhesion of S. aureus and P.
aeruginosa to human skin cells. Synthetic peptides of CD9 tetraspanins were used on human
keratinocyte cells to determine anti-adhesive properties against the bacteria and in co-infections,
representing polymicrobial infections. Both peptides shown a reduction in adherence of the two
bacteriaand were alsoeffective whenthe bacteriaco-existed,showingthatthey have a potential of
contributing to therapeutic treatments against skin infections.
2.0 Lay Abstract
The over-use of antibioticdrugscausescertainbacteriato mutate andeventuallybecomeresistantto
them,these resistantbacteriabecomedangerousastheyare difficulttokillandincrease themortality
in humans. Pathogenic bacteria invade the host by passing through a damaged skin. The skin is a
protective organ with many immune functions that prevent the entry of such microorganisms,
althoughsometimesitcanbecome compromisedthroughacut,burn,ora bite.The bacteriacanthen
bindto hostcellsand can thenmultiplyleadingtoinfection.Bindingtohost cell membrane proteins
is an essential step for infections and preventing this process can have the potential of becoming a
therapeutic treatment. Tetraspanins are proteins found on cells that are important for different
cellularfunctionsandcanbe exploitedbyinvasive bacteria. Staphylococcusaureus andPseudomonas
aeruginosa are used in this experiment as they are of clinical relevance and often infect together in
the skin. In thisproject, syntheticproteincomponentsof tetraspaninsare usedin humanskincells to
detect if they would reduce the binding of S. aureus and P. aeruginosa to the cells. These synthetic
proteinswere able toreduce the infectionof these bacteriatothe cellsandwere alsoeffective when
the bacteria were present together. Thus, this type of therapy has a potential of becoming an
alternative treatment for fighting resistant pathogenic bacteria.
3.0 Introduction
3.1 Rise in antimicrobial resistance
Inthe last decade there hasbeenadramaticincrease inthe numberof pathogenicbacteriathatshow
multidrug resistance to anti-bacterial agents and it is now considered a public health problem.The
forced exposure of antibiotic drugs to the bacteria provides selective pressure and leads to the
emergence of resistant pathogens through genetic mutations or acquisition of mobile genetic
elements that carry resistant genes (Vila, J., 2015).
It is an important issue as it is costly in both human and financial terms. Infection of resistant
microorganisms leads to an increase in health care costs, length of hospital stay, and mortality of

3. 3
carriers compared to infections with non-resistant microorganisms (Shlaes, D.M., 1997). Around
25,000 people dieacrossEurope everyyearduetohospitalinfectionscausedbythefollowingresistant
bacteria;Escherichia coli,Klebsiella pneumoniae,Enterococcusfaecium,Pseudomonasaeruginosa and
Methicillin-ResistantStaphylococcusaureus (MRSA) (PublicHealthEngland,2015).Itisestimatedthat
the failure of tacklinganti-microbialresistancewouldleadtoaworldpopulationdecrease of11million
to444 millionpeoplebythe year2050(Taylor,J.,2014a). The reduction of the populationandincrease
inmorbiditywouldalsoreducethe worldGrossDomesticProduct(GDP),by2050 the worldeconomy
will be smaller by 0.06%-3.1% (Taylor, J., 2014b).
There are ways of preventing the emergence of resistance, these include and active system of
surveillance for resistance, an infection control program to prevent the spread of further resistant
microorganisms and an effective program of antimicrobial use stewardship. Although, due to the
complexityof drugresistance inmany microorganisms,itishighlylikelythatnotall control measures
will be successful which leads to the search of new ways of fighting bacterial infectionsinevitable
(Ofek, I., Hasty, D.L., Sharon, N., 2003).
3.2 The skin, structure and function
3.2.1 The skin as a barrier
The skin plays a fundamental role in the immune system, it acts as a semi-permeable barrier and
preventsthe entryof pathogenicmicroorganisms.It hasarange of immune functionssuchassecreting
anti-microbial peptides, cytokines and chemokines in response to wound formation or invasion of
pathogens, it also maintains surface pH (Ong, P.Y., 2002).
3.2.2 Skin Structure
The structure of the skinmakesit a safeguardingorganpreventingmicro-organismsfromentering,it
iscomposedof three layers;the epidermis, dermisandthe subcutaneouslayer. The uppermostlayer
is the epidermis, it comprises mainly keratinocytes along with pigmented melanocytes, nerve
receptors and Langerhans cells. The keratinocyte cells begin as undifferentiated cells at the base of
the epidermis and differentiate as they move up. Eventually these cells lose their nuclei and die
formingthe StratumCorneumwhichisthe uppermostlayerof theepidermis.Due tothe accumulation
of lipids and the close cell junctions, the Stratum Corneum becomes impermeable to pathogens
(Haake, A., Scott, G., Holbrook, K., 2001a).

4. 4
Figure 1 Shows the 3 main layers of the skin from most exterior to interior; Epidermis, Dermis and
Hypodermis, taken by (Metclafe, A.D., Ferguson, M.W.J., 2007)
Deeptothe epidermisis the dermis andformsthe bulkof the skin(Menon,G.K., 2002). The dermisis
mainlycomposedof connectivetissueand protectsthe bodyfrommechanical injuries,aidsinwound
healing and the immune system. It is made up of two layers, the papillary and the reticular dermis.
The papillarydermishasahighdensityof fibroblasts thatsecrete extracellularmatrixproteinssuchas
elastinandcollagento protectit from mechanical stress. The reticulardermis ismainlycomposedof
collagenandelasticfibresthatprovide strongmechanical properties.Macrophagescanalsobe found
in the dermis along with adipocytes which are involved in lipid secretion. Below the dermis is the
subcutaneoustissue whichiscomposedof fatthat insulatesthe body andacts as an energyreservoir
(Haake, A., Scott, G., Holbrook, K., 2001b).
3.2.3 Skin defencemechanisms:
The skin has many defence mechanisms that prevent pathogens from damaging the body. For
example, keratinocytes secrete lysozymes, RNase 7, dermcidin as well as anti-microbial peptides
(AMPs) whichcause the degradationof many microorganisms(Gläser,R., 2005). Cytokinesactas the
first immune response upon wounding and promote inflammationto the woundedarea to increase
the blood flow and bring immune cells to the area. Langerhans cells found in the epidermis are
dendritic cells that phagocytose pathogenic microorganisms and also secrete cytokines and
chemokines to further boost the immune system (Pasparakis, M., Haase, I., Nestle, F.O., 2014).
Wound healingisanotherprocessby which the skinprotects the body from infections.Thisinvolves
the immediate clottingof plateletsto the site of trauma, followed by granulation and production of
new extra cellular matrix proteins by fibroblasts. The last step is the remodelling stage where
keratinocytes form new epidermis (Barrientos, S., 2008).

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3.3 Skin Pathogens
3.3.1 Staphylococcus aureus
StaphylococcusaureusisaGram positive cocci thatcanbe a humancommensal ora potentiallylethal
pathogen.Itis one of the mostcommon causesof bacteraemiaandcarriesa higherrate of mortality
than any other; current 20-40% mortality at 30 days without appropriate treatment. It frequently
invades the skin but it can also cause deep-seated infections (Chambers,H.F., 2009). The greatest
cause of illnessby S.aureus isviaskinandsofttissue infections(SSTIs),whichincludeboils,impetigo,
cellulitis and skin abscess.
Figure 2 Skin and soft tissue infections caused by Staphylococcus aureus; abscess and cellulitis
(Gandara, M.P., 2015).
Infections continue to occur due to the rise of antibiotic-resistant strains of S. aureus that have
reached global epidemic proportions.Methicillin resistant S. aureus (MRSA) strains are increasing in
both healthcare and community settings (Brown A.F., 2014), and appear to be virulent leading to
overwhelminginfectionssuchasnecrotizingpneumoniaandfasciitis.Theseresistant S.aureusstrains
are the consequence of the selective pressure caused by antibiotics (Vayalumkal, J.V., 2007).
S. aureus invade skinepithelial cellsviaspecificadhesionproteinscalledfibronectin-bindingproteins
(FnBPs). The FnBPs bind to fibronectin receptors found on host target cells leading to bacterial
colonisation. It has been shown that the FnBPs are sufficient to allow successful invasion to human
skin cells and do not require any specific co-receptors on the target skin cells (Sinha, B., 2000).
3.3.2 Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative bacterial pathogen which causes a broad range of
chronic and acute infections such as urinary tract, skin (folliculitis, burn infections) and bloodstream
infections. It is a leading cause of nosocomial infections and is responsible for 10% of all infections
acquired in hospitals (Aloush, V., 2006).
Furthermore, P.aeruginosa isknownto complicate patientswithCysticFibrosis(CF),a disorderthat
involvesamutationinthe cysticfibrosisconductance regulator(CFTR) gene.Infectionbythisbacteria
in CF patients leads to a decrease in pulmonary function and a rise in morbidity and mortality.

6. 6
Due to the increase in antibioticresistance,few classesof antibioticscanbe usedfor the treatment
of P. aeruginosa infections. An example is carbapenems, however, there are carbapenem-resistant
strains gradually increasing over time (Martinez, J.M.R., 2009).
As for P. aeruginosa and other pathogenic microorganisms, the ability to adhere to host tissues is
critical for colonisation and infection. P. aeruginosa adhesioninvolves binding of several adhesins,
includinglectinstospecifichostcellsurface glycoconjugates.Large quantitiesof bothlectins,LecA and
LecB are found on the outer membrane of the bacteria, which suggests that lectins play a role in
adhesion. LecA and LecB are also involved in virulence factors (Chemani, C., 2009).
3.3.3 Entry pathways for Infection
In order for pathogenic bacteria to successfully colonise and infect a host organism they must first
pass through the skin that is compromised by trauma or stress. The skin often becomes damaged
through a cut, burn or a bite.
Pathogenic microorganisms can then adhere to underlying skin cells that are normally hidden or
extracellular matrix components, including collagen, vitronectin, fibrinogen and mostly fibronectin.
Bacterial adhesins recognize specific host cell membrane elements such as integrins, selectins and
cadherins. After binding to cells through specific receptor adhesion, bacteria can form structured
bacterial coloniesknownasbiofilms extracellularlyorinternalizethe cellsthroughdifferentmethods
(Murillo, N.A., 2014a).
Bacterial internalization is a strategy that helps bacteria survive in the host cells and avoid the host
immune response.One example of aninternalizationmechanismisthe zipper-likeprocessused byS.
aureus whichdependsonremodellingof the membrane dynamicsandactincytoskeleton.Itinvolves
the bindingof an a5β1 integrinto fibronectin(Fn) whichisboundto the FnBP of S. aureus, thisthen
triggers the accumulation of a focal adhesion-like protein complex; actinin, paxillin, zyxin, focal
adhesion kinase (FAK), tensin and Src kinase. This then leads to the reorganization of the actin
cytoskeleton and thus the internalization of the bacteria. The detailed mechanism for S. aureus
internalization is shown below in figure 3 (Murillo, N.A., 2014b).
Figure 3 Zipper-like mechanism of Staphylococcus aureus internalization from (Murillo, N.A., 2014c)

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3.4 Anti-adhesion therapies
Thisnewapproachisbecomingincreasinglypopulardue tothe rise inantimicrobialresistance.Unlike
antibiotics, anti-adhesion therapies do not exert significant selective pressure on bacteria and
therefore there is less chance of a resistance to develop. The therapy involves preventing the
adherence of pathogenic microorganisms to host cell receptors, thus preventing infection. The
mechanisms of these therapeutic agents involve the inhibition of adhesinsand their host receptors,
using probiotics and dietary supplements to interfere with the adhesin-receptor interactions,
vaccination with adhesins or analogs or manipulating hydrophobic interactions (Cozens, D., 2014).
There is a number of anti-adhesion therapies currently being developed to treat bacterial SSTIs. An
example isbyinhibitingthe hostreceptorbiogenesis.Manybacterial adhesinsandtoxinsdependon
host glycosphingolipids(GSLs) forhost cell bindingandreducingGSLs from hostcell membraneshas
beenproposedasa strategyto preventinfectionsfromoccurring.Administeringspecificinhibitorsto
enzymesof the GSLpathwayhasbeensuccessful indiminishingbacterial colonization(Krachler,A.M.,
Orth, K., 2013a). GSL reduction has also been accomplished by an alternative therapy, enzyme
replacementtherapywithhumanglucosyl ceramide glucosidase fortreatingapatient sufferingfrom
Gauncher disease (Krachler, A.M., Orth, K., 2013b).
3.5 Tetraspanin function and structure
The tetraspaninsare a familyof eukaryoticmembrane proteins comprisingof 33 members.Theyact
as molecular facilitators in the cell membrane, as they dimerize with each other and interact with
other receptors including adhesion target molecules, signalling molecules and G-protein coupled
receptors. These broad interactions lead to the formation of tetraspanin enriched microdomains
(TEMs). Tetraspanins are involved in a range of cell functions such as cell adherence and fusion,
endocytosis,membrane traffickingandleukocyteadherence. Buttetraspaninscanbe usedindirectly
as gatewaysfor infectionbybacteriaand viruses throughreceptorsthat are embeddedinthe TEMs.
Therefore, by disrupting TEM function this could potentially affect binding of multiple species of
bacteria and have anti-infective results. Recentlyit has been shownthat pre-treatmentof cells with
tetraspaninantibodiesandrecombinantproteins of tetraspaninsleadtothe reductionin adherence
of Staphylococcus aureus, Neisseria lactamica, Escherichia coli and Streptococcus pneumoniae to
epithelial cells (Green, L.R., Monk, P.N., 2011a).
3.5.1 Tetraspanin structure
All 33 members of the tetraspanins share similar structural characteristics; they have 4
transmembrane domains that contain charged residues, an intracellular loop and two extracellular
loops; a small one (EC1) and a large one (EC2). EC2 is known to make specific protein-protein
interactions (Green, L.R., Monk, P.N., 2011b).

8. 8
Figure 4 - A representation of a typical tetraspanin receptor (Hemler, M.E., 2014).
3.5.2 CD9
One of the mostcommontetraspaninsexpressedincellsisCD9.CD9isa goodmodel forstudying anti-
adhesive properties in skin cells due to its high expression levels in keratinocytes.In a recent study,
syntheticpeptidesof tetraspaninCD9receptorswere showntoinhibitbacterial adhesiontocultured
keratinocytes and were effective in a tissue replica model of human skin infections without causing
any adverse effects on cell metabolism and function (Ventress, J.K., Monk, P.N., 2016).
However,notmuchwork hasbeendone on anti-adhesiontherapies againstpolymicrobial infections.
In polymicrobial infections,differentpathogenicpopulationsdisplaysynergisticinteractionsthatcan
enhance their colonisation, persistence and virulence in hosts. The most dominant types of
polymicrobial infectionsoccurinchronic wounds,andthe most commoncauses are S. aureus and P.
aeruginosa (DeLeon, S., 2014). Therefore, this project is aimed to use a more realistic approach, by
investigating a reduction in adherence by using synthetic peptides of CD9 tetraspanin receptors
against co-infections of both S. aureus and P. aeruginosa.
3.6 Outline of project
This research project involves an alternative way of fighting pathogenic bacteria through anti-
adhesiontherapy.Itisan attractive approach since the adhesionof bacteriato the hosts’tissue isan
essential step for colonisation and infection.
In orderfor pathogenicbacteriatoinfectahost theymustfirstpass throughthe stratum Corneumof
the skinand attach tounderlyingcells, Staphylococcusaureus andPseudomonasaeruginosa are used
in this project as they are the primary agents of skinand wound infections and oftenco-invade. The
aimis to use syntheticpeptidesof CD9tetraspaninreceptorstodetectanyreductioninadherence of
both S. aureus and P. aeruginosa in co-infection experiments.

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4.0 Materials and Methods
4.1 HaCaT cell culture
The maintenance of HaCaT, a human keratinocyte cell line required DMEM (Dulbecco's Modified
Eagle'smedium,Lonza,CatNO
:BE12-74IF);including10% (vol/vol) foetalbovineserum(FBS).The cells
were growninT-75culture flasksat37o
Cincubators with5% CO2.Forpassaging,thecellswerewashed
twice with 10ml of Hank’s solution (Lonza,Cat NO
: BE10-5L7F) and treated with 5ml of trypsin-EDTA
followedby8-10 minutesof incubation. The cellswerepassageduntil theyreached>90% confluency
with the splitting ratios and 15ml of the DMEM. The DMEM contained 10% FBS to neutralize the
trypsin.
SplitRatio Numberof days to reach 80% confluency
1 in 2 (500μl) ~2
1 in 3 (300μl) ~3
1 in 5 (200μl) ~4
1 in 10 (100μl) ~7
Table 1. Split ratios for HaCaT cell culturing, and number of days to reach 80% confluency
Sub-culturing was performed three times for the preparation of the experiment.
4.2 Bacterial strains
Twobacterial strainswere usedSH1000 andPAO1.SH1000 strainof StaphylococcusAureuscontained
a chloramphenicol resistantplasmidexpressinga greenfluorescentprotein(GFP),therefore SH1000
culturing prior to infections required 10μg/ml of chloramphenicol. PAO1 strain of Pseudomonas
aeruginosa also contained a chloramphenicol resistant plasmid, expressing a Discosoma sp. red
fluorescent protein (DsRed). PAO1 required 100μg/ml of chloramphenicol before culturing.
4.3 Expression of human membrane protein, tetraspanin CD9 on HaCaTs
Cellswere resuspendedinculture mediumuntil theyreached7x104
/ml andthen0.5ml aliquotswere
dispensedintotheLabTechchamberwells(eachslidehas8chambers),culturedovernight.Slideswere
examinedfor cell density and washed by adding and removing 0.5ml BSS (Balanced salt solution);
including 0.1% sodium azide and 0.2% bovine serum albumin (BSA). Cells were then fixed and
permeabilised using 0.5ml of acetone and incubated for 5 minutes. Using a Pasteur pipette the
acetone was removedandwashedby placingslide ina tank containingfreshPBS withstirring for 10
minutes. After the removal of remaining PBS, 100μl of appropriate dilution of primary, unlabelled
antibody (Mouse anti-human CD9 monoclonal antibody IgG2b) was added. Mouse IgG2b was also
added in one of the chambers as a negative control. This was followedby 30 minutes incubation at
room temperature,underhumidconditionsinthe dark.The slide wasthenwashedagainina tank of
fresh PBS and stirring for 15 minutes. PBS was removed from the slide and 100μl of appropriate
dilution of Fluorescein Isothiocyanate (FITC) labelled, secondary antibody (Anti-mouse IgG) was
added,followedbyincubationfor30minutesatroomtemperature inthe dark.The slide waswashed
and dried again in the same way with PBS. The cover slips were transferred from each chamber to
slidescontainingasmall dropof mountant (Vectashield/DAPI)tostainthe cellswithDAPI.Nailvarnish
wasaddedtoseal the coverslipsandstoredat4o
C indarkuntil examinedbyfluorescencemicroscopy.
DAPI and FITC channels were used at 60x magnification with oil.

10. 10
4.4 Exposure of HaCaT cellsto PAO1-DsRed andSH1000-GFPat a range of multiplicityof
infections; 5, 10, 50, 100, 150, 300
SH1000-GFP and PAO1-DsRed were cultured as 15ml liquid broths with shaking at 120rpm in a
humidified 37o
C incubator. SH1000 and PAO1 were then washed and centrifuged (4500xg for 10
minutes) twice with 15ml of PBS. Absorbance at OD600NM was recorded in order to find the quantity
needed for cell infection and diluted to OD600 of 1.0. The cells were washed twice with 1ml Hanks
solution.1ml of bacteriawas addedto the desiredMOI and thenincubated for 1 hour at 37o
C. After
incubation, the infected cells were washed with PBS and were treated withTriton 1% for lysing the
HaCaT cells, followed by incubation of 15 minutes. After this the bacteria were diluted for plating.
4.5 Co-culture infection of HaCaT cells with PAO1-DsRed and SH1000-GFPto determine
the differences in bacterial adhesion
Bacterial preparationforcellinfectionwasthe same,SH1000and PAO1were washedandcentrifuged
twice withPBS.Bacterialcultureswithappropriateamountof chloramphenicolwerediluteduntil they
reached absorbance of OD600 of 1.0. For co-infections, each desired MOI contained2ml of PAO1 and
2ml SH1000, vortexedbeforeinfection.HaCaTcellswerewashedwithHankssolutionandthen1ml of
bacteriawasaddedtothe desiredMOIandthenincubatedfor1hourat37o
C. AfterwashingwithPBS,
cellsthatwere co-infected,weretreatedwithTriton1%,followedbyincubationof 20 minutes.After
thisthe bacteriawere dilutedforplating.The S.aureus (SH1000-GFP) andP.aeruginosa (PAO1-DsRed)
colonieswere separatedbyplatingthe dilutionsinseparate agarplates;mannitol agar (figure 5) and
P. aeruginosa isolation agar (figure 6).
Figures 5&6 Shows the mannitol agar for restricting growth of S. aureus (SH1000-GFP) and the P.
aeruginosa isolation agar for P. aeruginosa (PAO1-DsRed) colonial growth. In order to determine
adhesion ability of each bacterium from the co-infection assays
4.6 Gentamicin protection assay to determine invasion of PAO1-DsRed and SH1000-GFP
to HaCaT cells
Bacterial preparation for cell infection was the same, for internalization infected cells were treated
with1ml of Gentamicin (200μg/ml) followedbyincubationfor1hour at 37 o
C.This wasperformedin
order to kill off the extracellular bacteria that have not internalized the HaCaT cells. The cells were
then treated with Triton 1% followedby dilutions and plating. Incubated overnight and internalized
PAO1-DsRed and SH1000-GFP were counted the next day.

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4.7 Plating and bacterial counting
Tenfolddilutionswere made bytaking 20μl to 180μl of PBS from each well,making4 dilutions(10-1
,
10-2
, 10-3
and 10-4
). Agar plateswere platedbythe dilutionstoestimate the quantityof the growthof
viable bacteria.The agarplatesweresplitin4sectionsonwhich310μl aliquotsof eachdilutionswere
added. The mean number of bacteria was calculated by finding the average of the 3 spots of the 4
dilutions followed by calculations of the colony forming units per 1 ml (CFU/ml). The equation for
calculating CFU/ml is shown below.
CFU/ml = Average of three drops x
1
𝑉𝑜𝑙𝑢𝑚𝑒 𝑝𝑙𝑎𝑡𝑒𝑑 𝑖𝑛 𝑀𝐿
x Dilution Factor
4.8 Peptides
The peptidesthatwereusedare regions of the EC2of the CD9tetraspanin.The twopeptidesare 8005
and 800-cap. As can be seenin figures7and 8, peptide 8005 includesthe entirehelix of the EC2.800-
cap has a smaller peptide sequence than 8005 and it is capped with Asp (D). The peptides are also
shown in figure 9 generated by PyMOL. Scrambled peptides of 8005 and 800-cap were used as
controls that carry randomly generated CD9 peptide sequences (table 2).
Figure 7 8005 peptide of CD9 tetraspanin, includes the entire helix of EC2

12. 12
Figure 8 800-Cap peptide of CD9 tetraspanin, has shorter peptide sequence than 8005 and it is
capped with Asp (D)
Figure 9 800-Cap peptide is coloured blue and the longer peptide 8005 is coloured red in the EC2
domains of CD9 tetraspanin, generated by PyMOL

13. 13
Peptide Description Peptide sequence
8005
Extended peptide 8001 to include
entire helix
1 11 21 31 40
SHKDEVIKEVQEFYKDTYNKLKTKDEPQRETLKAIHYALN
8005
SCR
Scrambled control for extended
peptide 8001
1 11 21 31 40
THDAEKKNPINDLKKEVLERVKQKTYESTHFADLYQIEYK
800-Cap
Peptide 800 capped with Asp (D) 25 40
DEPQRETLKAIHYALN
800-Cap
SCR
Scrambled control for peptide 800
capped with Asp (D)
25 40
QEALKYNRAETPLDIH
Table 2. Peptide names, description and sequences
4.9 Effects of different tetraspanin CD9-derived peptides on HaCaTs infected with both
PAO1-DsRed and SH1000-GFP
MOI 100-S. aureus (SH1000-GFP) and MOI 35-P. aeruginosa (PAO1-DsRed) were usedforthe peptide
treatments. Asinthe bacterial multiplicityof infections,the bacterial liquidbroths were washedwith
PBS and centrifuged, followedby dilutions until they reached absorbance of OD600 of 1.0. The multi-
well plate containingthe HaCaTcellswasremovedfromincubationandthe mediawasremovedfrom
each well using a Pasteur pipette. The cells were washed twice with Hank’s solution, 1ml each time.
Then 200μl of peptides 8005, 8005 SCR, 800-cap, 800-cap SCR (200nM concentration) or media
(control) were added to the appropriate wells and incubated for 30 minutes. The solution was then
removedfromeachwell using1ml pipette.250μl of SH1000-GFP and PAO1-DsRedwere addedtothe
appropriate wellsandincubatedfor1hour.Afterincubation,the bacteriawereremovedandthe wells
were washedwith1ml of PBS three times. 200μl of 2% paraformaldehyde was lateraddedto fix the
cells/bacteria,followedbyincubation for1 hour.Then, the wellswere washedthree timeswithPBS
and transferredtoLU102 glassslides.One dropof Vectashield HardsetcontainingDAPIwasaddedto
stainthe cells,the coverslipsweretransferredtothe slidesandlefttodry for30 minutesat4o
C inthe
dark. The slides were then used for fluorescence microscopy to detect adherence of bacteria.
4.9.1 Quantitative Microscopy
AnOlympusBX61 microscope wasusedat100x magnificationforcountingthe DAPI-stainedcellsand
the number of infected cells. The following channels were used; DAPI-for the HaCaT cells, FITC- for
SH1000-GFP andTex red- forPAO1-DsRed. Eachcoverslipwasnumberedbyrandomof 100 cells.Cells
undergoing mitosis were not scored as they are considered abnormal.
5.0 Results
5.1 CD9 tetraspanins are highly expressed on HaCaT cells
Immunofluorescence (figures10) was usedto determine if CD9 isexpressedinHaCaT cells.CD9 FITC
expression was normalizedagainst a non-specific isotype control antibody IgG2b. The figuresbelow
show that CD9 is richly expressed in the HaCaT cells.

14. 14
A B
C D
E F
G
Figure 10 (A-C) Visualisation of protein distribution (1 in 50 dilution); (A), HaCaT cells only (B), CD9
only (C), HaCaT cells with CD9, figures (D-F) Protein distribution (1 in 100 dilution); (D), HaCaT cells
only (E), CD9 only (F), HaCaT cells with CD9, (G) Isotype control, Scale bar, 11.00 μm. DAPI shown in
blue, FITC shown in green.

15. 15
5.2 Adhesion assays of PAO1-DsRed and SH1000-GFP on HaCaT cells
Adhesionassaysof PAO1-DsRedandSH1000-GFP were performed onHaCaTcells toassessthe ability
of S. aureus and P. aeruginosa to adhere on HaCaT cells and if co-infection cultures will affect the
adhesion patterns of each bacterium. (Figure 11A) shows the adhesion for mono-infections and
(Figure 11B) for co-infectionassays. Figure 12 showsthe adhesionfor S. aureus and P. aeruginosa in
both mono- and co-infections.
A
B
Figure 11 (A&B) Showtheadhesion of S. aureus (SH1000-GFP) and P. aeruginosa (PAO1-DsRed) per
10,000 HaCaT cells in MOI=5-300 for mono- and co-infections.Thedata werecollected by plating and
CFU calculations.Valuesare means ± SD. *, significantdifference in the adhesion of P. aeruginosa to
HaCaT cells compared to S. aureus. **, P≤0.01, and ****, P≤0.001, by two-way analysis of variance
(ANOVA) with Sidak’s multiple-comparison test.

16. 16
Figure 12 showstheadhesion of S.aureus (SH1000-GFP) and P. aeruginosa (PAO1-DsRed) per 10,000
HaCaT cells in MOI=5-300 for both mono- and co-infections. Values are means ± SD. The data from
figure 11A and 11B are shown together to visualize any effects in adherence for each bacteria when
they co-infect.
A 2 way ANOVA,Tukey’smultiple comparisontestwasperformedtocompare if there is a significant
difference between the adherence of Mono-infection bacteria to Co-infection bacteria. Null
hypothesis-no significant difference. Test is shown below in table 3.
2 way ANOVA of Mono- and Co-infections using Tukey’s multiple comparison test
Mono-infectionvsCo-infection:S.aureus Not significant, p=0.5599
Mono-infectionvsCo-infection:P.aeruginosa Significant, p=0.0257
Table 3.
Infection images-FluorescentMicroscopy
A B
C D

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Figures13 Showstheimages of the HaCaTcells and the adhered/internalized S.aureus(SH1000-GFP)
and P. aeruginosa (PAO1-DsRed). (A) Control group, no infections. (B) S. aureus (SH1000-GFP)
infecting the HaCaTcells at FITC channel.(C) P.aeruginosa (PAO1-DsRed)infecting the HaCaT cells at
Tec Red channel and (D) shows the co-infection of HaCaT cells by both bacteria.
5.3 Invasion assays of PAO1-DsRed and SH1000-GFP on HaCaT cells
Invasionassayswere performedtoassessthe abilityof S.aureus andP. aeruginosa tointernalize the
HaCaT cells. (Figure 14) below shows the levels of internalizationof S. aureus and P. aeruginosa per
10,000 HaCaT cells.
Figure 14 Shows the invasion of S. aureus (SH1000-GFP) and P. aeruginosa (PAO1-GFP) per 10,000
HaCaTcells in MOI=5-100. Valuesare means ± SD. *, significant difference in the internalization of P.
aeruginosa to HaCaT cells compared to S. aureus. ****, P≤0.001, by two-way analysis of variance
(ANOVA) with Sidak’s multiple-comparison test
5.4 Peptide treatment of P8005 and P800-Cap
Treatmentof CD9-derivedpeptideswasperformedtodetermine if theycanreduce the adherence of
S. aureus and P. aeruginosa andif treatmentiseffective whenthe bacteriaco-exist. Figure 15below
show the effect of P8005 and P800-Cap on bacterial adherence.
A

18. 18
B
Figure 15 Level of infected HaCaT cells with peptide treatment. (A) Shows the P8005/SCR treatment
and its effect on HaCaTcell infection by S. aureus(SH1000-GFP),P. aeruginosa (PAO1-GFP) and in co-
infections.(B) Shows the P800-Cap/SCR treatment and its effect on HaCaT cell infection by S. aureus
(SH1000-GFP), P. aeruginosa (PAO1-GFP) and in co-infections. Data were collected by quantitative
microscopy.
A 1 way ANOVA, Tukey’s multiple comparisontest was performedfor both peptidesto determine if
there is a significant decrease in adherence of the bacteria to the HaCaT cells without peptide
treatment(Control)andwithpeptidetreatment(P8005andP800-Cap). Null hypothesis-nosignificant
difference between adherence of Control group and peptide treatment.
1 way ANOVA; Tukey’s multiple comparison test for adherence in control vs peptide
treatment
Control vs P8005: S. aureus Significant, p<0.01
Control vs P8005: P. aeruginosa Significant, p<0.01
Control vs P8005: Co-infection Not significant
Control vs P800-Cap: S. aureus Significant, p<0.05
Control vs P800-Cap: P. aeruginosa Not significant
Control vs P800-Cap: Co-infection Not significant
Table 4.

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6.0 DISCUSSION
Immunofluorescence showed that CD9 Tetraspanins are highly expressedin the cell membrane of
HaCaT cells, thiswasdeterminedbythe highfluorescentintensityof the FITC-labelledantibodyof CD9
(figure 10). This finding reflects to current knowledge on Tetraspanindistribution and function, CD9
tetraspanins are richlyfoundonthe cell surfaceof the HaCaTcellsandcan formTetraspaninEnriched
Microdomains (TEMs) for various cellular functions.
CFU calculationsshowedthatbothS. aureus and P. aeruginosa stronglyadhere toHaCaT cellsand it
is thoughtis due to tetraspanin-mediatedadherence.Basedonthese findings, P.aeruginosa ismore
virulentthan S.aureus asit showssignificantlyhigheradhesion/internalizationtoHaCaT cells (figures
11 and 14), this was more evident when the bacteria co-infected HaCaT cells. A possible reason for
this is that P. aeruginosa may have more adhesins that can adhere to HaCaT tetraspanins than S.
aureus, leading to a higher persistency and colonisation.
An increase in adherence was expected in co-infections for both bacteria because S. aureus and P.
aeruginosa wouldbenefitfromeachotherthroughsynergisticinteractions leadingtostrongerbinding
to host cell receptors. Table 3 shows that there was only a significant increase in adherence for P.
aeruginosa,thistellsusthatthe presence of S.aureusenhancedthe adhesionabilityof P.aeruginosa.
For S. aureus there wasevena decrease inadherence inco-infections(figure 12,MOI5), a reasonfor
thismightbe becauseP.aeruginosasuccessfullycompetedagainst S.aureus forreceptorbinding,thus
lessS.aureus adheredtoHaCaTcellsthanexpected. More accurate resultscouldhave beenpresented
if the co-infectionassayswererepeatedbutdue tolackof time we were unable to. Inaddition, Figure
13 shows through fluorescent microscopy that P. aeruginosa is more aggressive than S. aureus and
that the bacteria were clumped together in co-infection possibly showing synergistic interactions.
MOI 100 forS. aureus andMOI 35 forP. aeruginosa were usedforthe infectionexperiments because
there was a reasonable amount of bacteria per 100 HaCaT cells to determine the difference in
infectionswithpeptidetreatment.Bothsyntheticpeptideswereeffectiveindecreasingtheadherence
of the bacteria, we expected that P8005 would be more effective than P800-Cap as it covered the
entire helixof the EC2.Asshowninfigures15(A&B) andtable 4, bothP8005 andP800-Cap decreased
the infectionof the cellsof bothbacteria,whileP8005was slightlymore effective.Interestingly,there
was a decrease in adherence in the Scrambled peptide controls, this may be due to some functional
aminoacidsequencesformed inthe EC2.Bothsyntheticpeptideswereable toreduce the adherence
of the bacteria when they co-exist (co-infections) (figure 15A&B), this was more evident with the
P8005 than P800-Cap, although there was no significant decrease. A possible reason for this is that
the bacteria in co-infectionsadhered non-specifically to other HaCaT cell adhesins such as integrins
and selectins that were independent of the synthetic peptides. An improvement of the experiment
couldbe to use higherconcentrationsof the syntheticpeptidesof CD9 tetraspaninssothat lessnon-
specific binding could occur and the effect of the peptides would be more accurate.
The use of syntheticpeptides of CD9tetraspaninsshowsusthatitcandisruptthe EC2 protein-protein
interactionswithothercellsurfacereceptors,preventingtheadherenceof pathogenicbacteriatohost
cell receptors.Therefore,thistherapy hasa potential of becominga treatmentagainstpolymicrobial
infections. As S. aureus and P. aeruginosa benefit from each other in chronic wound infections,
reducing their adherence may be the future therapy that does not induce selective pressure.

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7.0 ACKNOWLEDGEMENTS
I would like to thank my supervisor, Dr. Rahaf Issa for her amazing support, guidance and valuable
feedback thatallowedme toworkthroughthepractical workaswellasthe writtenlabreport.Iwould
also like to thank the students and the staff in the lab for helping me with any difficulties I faced
throughout the project. Special thanks to Dr. Peter Monk for giving me valuable information on the
project results and literature after giving my presentation.
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