research article participation of integrin 5 1 in the fibronectin

Research ArticleParticipation of Integrin 12057251205731 inthe Fibronectin-Mediated Adherence of EnteroaggregativeEscherichia coli to Intestinal Cells

Mariana Izquierdo1 Alejandra Alvestegui1 James P Nataro2

Fernando Ruiz-Perez2 and Mauricio J Farfan1

1 Centro de Estudios Moleculares Departamento de Pediatrıa Hospital Dr Luis Calvo Mackenna Facultad de MedicinaUniversidad de Chile Antonio Varas 360 Providencia 7500539 Santiago Chile

2 Department of Pediatrics University of Virginia School of Medicine Charlottesville VA 22908 USA

Correspondence should be addressed to Mauricio J Farfan mfarfanmeduchilecl

Received 9 May 2014 Accepted 11 July 2014 Published 7 August 2014

Academic Editor Fernando Navarro-Garcia

Copyright copy 2014 Mariana Izquierdo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection The aggregativeadherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells We and others havedemonstrated that fibronectin is a receptor for AAFII fimbriae Considering that the major cellular receptor of fibronectin isintegrin 12057251205731 in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain042 to intestinal cells We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin 12057251205731 direct bindingwas not mediated by AAFII fimbriae and indirect binding was mediated by AAFII and fibronectin Coimmunoprecipitationassays confirmed the formation of the complex AafAfibronectinintegrin 12057251205731 To evaluate EAEC adherence to intestinal cellsT84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin 12057251205731 to fibronectin theRGD site Under these conditions we found the number of adherent bacteria to epithelial cells significantly reduced Additionallyfibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin 1205725 shRNA Altogetherour data support the involvement of integrin 12057251205731 in the fibronectin-mediated EAEC binding to intestinal cells

1 Introduction

Enteroaggregative Escherichia coli (EAEC) is an importantcause of endemic and epidemic diarrheal disease worldwideAdherence to the intestinal epithelia is a key feature of EAECpathogenesis since it allows bacterial colonization of theintestinal mucosa the secretion of enterotoxins and cytotox-ins and the induction of proinflammatory cytokines frominfected epithelial cells EAEC adherence to intestinal cells ismediated by fimbrial adhesins designated aggregative adher-ence fimbriae (AAFs) Adherence of the prototype EAECstrain 042 to cells and abiotic surfaces requires the AAF pilusvariant called AAFII [1] Although the importance of theadherence of EAEC to intestinal cells has been established

the cell receptors involved in AAF fimbriae recognition havenot been fully characterized We and others have previouslyshown recognition of AAFII fimbriae by extracellularmatrix(ECM) protein fibronectin which enhanced bacterial adher-ence to the surface of polarized intestinal monolayers [2 3]

Fibronectin was the first ECM protein to be shown toact as a receptor for bacterial adherence to eukaryotic cells[4] This glycoprotein is composed of two nearly identical220 kDa disulfide-bound protein subunits and comprisesseveral structurally distinct domains which can bind tocellular surfaces fibrin collagens DNA gelatin integrinsheparin and heparan sulphate [5] To date over 100 bacterialfibronectin binding proteins have been identified and severalstudies have shown that adhesion to fibronectin would

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 781246 8 pageshttpdxdoiorg1011552014781246

2 BioMed Research International

contribute to the successful colonization of bacteria at theepithelial surface acting as a ldquomolecular bridgerdquo connectingthe bacteria with the host cell surface [6]

Integrins are heterodimeric glycoproteins that are criticalfor a variety of cell-cell and cell-matrix binding events Oncea particular ligand binds to integrin it triggers a specificsignaling pathway depending on the type of integrin thatis modulated [7] Fibronectin is a ligand for at least adozen members of the 1205731 integrin family where 12057251205731 is themajor integrin involved in indirect recognition of bacterialpathogens with the ability to bind to fibronectin [6 8]For example Staphylococcus aureus employs fibronectin as apathogenic mechanism to adhere modulate the intracellularsignaling pathway and invade host cells [7 9] Fibronectin-associated bacteria can be recognized by integrin 12057251205731through the RGD region and this engagement induces theinternalization of bacteria by host cells [10 11]

Considering the above in this study we sought to charac-terize the participation of integrin 12057251205731 in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cellsOur results support the participation of fibronectin as abridging molecule allowing the contact of EAEC strain 042and integrin 12057251205731 at the surface of intestinal cells

2 Materials and Methods

21 Bacterial Strains Prototype EAEC strain 042 (O44H18)was originally isolated from a child with diarrhea in LimaPeru The EAEC 042aafA strain was constructed using thelambda red linear recombination method [12] as previouslydescribed [13] Prototype enterohemorrhagic E coli (EHEC)strain 86-24 and enterotoxigenicE coli (ETEC) strainH10407were used to evaluate binding to integrin 12057251205731 Bacteriawere grown overnight in Luria-Bertani (LB) broth with theaddition of kanamycin (50120583gmL) when appropriate

22 Cell Lines T84 and HEp-2 cells were routinely main-tained in Dulbeccorsquos modified Eaglersquos medium (DMEM) F12and DMEM high glucose (DMEMHG) (HyClone) respec-tively supplemented with 10 fetal bovine serum (FBS)penicillin (10UmL) and streptomycin (10 120583gmL) at 37∘Cunder 5 CO

2

23 Solid-Phase Binding Assay Microtiter plates (ThermoLabsystems FranklinMA)were coated overnight at 4∘Cwitha solution of 2 120583gmL of integrin 12057251205731 (RampD system) orbovine serum albumin (BSA Sigma) in integrin binding (IB)buffer containing Tris 25mM CaCl

22mM MgCl

21mM

MnCl21mM and NaCl 150mM pH 75 Unbound protein

was removed by washing three times with phosphate bufferedsaline (PBS HyClone) and subsequently blocked with 1skim milk in PBS for 1 h at 37∘C The blocking buffer wasremoved and the plates were washed and incubated with asolution of 1 120583gmL of fibronectin (Sigma) in IB buffer by 1 hat 37∘C Binding of fibronectin to integrin 12057251205731 was detectedusing an anti-fibronectin antibody (Sigma) which was incu-bated for 1 h at room temperature Anti-rabbit horseradishperoxidase conjugate (HRP KPL Gaithersburg MD) was

added following another PBS wash step Peroxidase activityassociated with each well was detected by the addition of 3 310158405 51015840-tetramethylbenzidine (TMB) substrate solution (KPL)Optical densities were read at 450 nm with a 96-well platereader (Infinite Tecan) To determine the adhesion of EAECor AafA-dsc protein to integrin 12057251205731 and fibronectinintegrin12057251205731 complex we performed the same protocol describedabove with some modification After the incubation withfibronectin plates were washed three times with PBS andincubated with bacteria in a 200120583L final volume or witha solution of 1 120583gmL of AafA-dsc protein Incubation withbacteria was performed for 2 h at 37∘C while incubationwith protein was carried out for 2 h at room temperaturePlateswerewashed and rabbit anti-O44 serum (Denka SeikenCo LTD Tokyo Japan) diluted 1 200 or rabbit anti-AafA1 2000 respectively was added to the wells and incubatedfor 1 h at room temperature Anti-rabbit HRP conjugatewas added following another PBS wash step and peroxidaseactivity associatedwith eachwell was detected by the additionof TMB substrate solution Optical densities were read at450 nm

24 Coimmunoprecipitation Assays We constructed colum-ns with anti-AafA antibodies and control columns (withoutantibodies) using pierce coimmunoprecipitation (Co-IP) kitfollowing manufacturer instructions To evaluate the forma-tion of the AafAfibronectin complex we prepared a solutionof 5 120583gmL of AafA-dsc protein and 10 120583gmL of purifiedfibronectin This mixture was incubated with gentle shakingfor 1 h at room temperature and then added to each columnfollowing manufacturer instructions Unbound protein wasremoved by washing three times with PBS and immunopre-cipitated proteins were eluted To evaluate the formation ofthe complex AafAfibronectinintegrin 12057251205731 we performedthe same procedure described above We incubated 5120583gmLof AafA-dsc 10 120583gmL of purified fibronectin and 5120583gmLof purified integrin 12057251205731 protein for 1 h at room temperatureFractions recovered from control column and column withanti-AafA antibodies were analyzed by Dot blot

25 Dot Blot Nitrocellulose membrane was washed withPBS and then coated with 10 120583L of fractions recoveredfrom control column and column with anti-AafA antibodiesThe membrane was blocked with BSA 1 for 1 h at roomtemperature and the presence of AafA-dsc fibronectin orintegrin 12057251205731 proteins was determined using anti-AafA anti-fibronectin (Sigma) or anti-integrin 1205725 (Santa Cruz Biotech-nology) Membranes were washed three times with PBS-Tween 005 and anti-rabbit HRP was added Peroxidaseactivity associated was detected by the addition of TMBsubstrate solution directly on membrane The resulting dotswere scanned and signal intensity was quantified using UN-SCAN-IT 61 software

26 Adherence Assays Epithelial cells grown in 96-wellplates were incubated for 30minwith 100120583Lwell ofDMEM-F12 only or supplemented with fibronectin (1 120583gwell) anti-bodies anti-integrin 12057251205731 that block the interaction withRGD site in fibronectin (Abcam) or anti-integrin 1205725 (Santa

BioMed Research International 3

EAEC EHEC ETEC00

05

10

15

BSA

lowast

lowast

lowast

OD450

nm

Integrin 12057251205731

(a)

BSA00

02

04

06

08

042

lowast

lowast

OD450

nm

042aafA

Integrin 12057251205731

(b)

Figure 1 Diarrheagenic E coli bind to integrin 12057251205731 (a) EAEC EHEC and ETEC were added to integrin 12057251205731- and BSA-coated wells andbacterial binding was detected by ELISA using anti-O44 anti-O157 and anti-O78 serum respectively (b) EAEC strain 042 and 042aafAwere added to integrin 12057251205731- and BSA-coated wells EAEC binding was detected by ELISA using anti-O44 serum lowastSignificantly differentbetween treatments (119875 lt 005)

Cruz Biotechnology) Then medium was aspirated andbacteria were added to the monolayer (in triplicate) witha multiplicity of infection (MOI) of 10 The plates wereincubated at 37∘C in 5 CO

2for 3 h and then washed

three times with PBS Cells were lysed with a solutioncontaining 01 (vv) Triton X-100PBS and serial dilutionsof the lysates were plated on LB agarThe number of adherentbacteria was determined by counting colonies forming units(CFU)

27 shRNA Subconfluent cultures (sim40ndash50) of HEp-2 cellsgrown in 6-well plates were transfected with small hairpinRNA (shRNA) for integrin 1205725 (sc-29372-SH Santa CruzBiotechnology) following the manufacturerrsquos instructions(Santa Cruz Biotechnology) A solution of DMEMHGwithout antibiotics or serum containing shRNA transfectionreagent (Santa Cruz Biotechnology) and 1 120583g of integrin 1205725shRNA or scrambled shRNA was added to HEp-2 cellswhich were then incubated for 7 h at 37∘C in 5 CO

2 Later

mediumwas replaced byDMEMsupplementedwith FBS andantibiotics and after 48ndash72 h transfected cells were selectedby puromycin resistance To verify a reduction in integrin1205725 expression total RNA of HEp-2 cells transfected wasobtained using total RNA I (Omega Biotech) and treatedwith RNase-free DNase I (Omega Biotech) Two microgramsof RNA was reverse-transcribed by using kit Affinity ScriptPCR cDNA (Stratagene) The synthesized cDNA was used toquantify the expression of integrin 1205725 using the followingprimers forward 51015840 TGCAGTGTGAGGCTGTGTACA 31015840and the reverse 51015840-GTGGCCACCTGACGCTC-31015840 Levels ofmRNA expression were normalized to those of the humanhousekeeping gene glyceraldehyde 3-phosphate dehydroge-nase (GAPDH) Changes in cycle threshold (ΔCT) values for

each gene were obtained by subtracting the mean thresholdcycle (CT) of the reference GAPDH gene (data not shown)

28 Statistical Analysis Statistical significance between theindividual groups was analyzed using the unpaired Studentrsquos119905-test with a threshold of 119875 lt 005 Values are expressedas the means plusmn the standard errors of the mean of threeexperiments

3 Results

31 Diarrheagenic E coli Binding to Integrin 12057251205731 Firstwe evaluated binding to integrin 12057251205731 of three strains ofdiarrheagenic E coli EAEC EHEC and ETEC We obtainedthat all strains studied bind significantly to purified integrin12057251205731-coated wells compared to BSA-coated wells used asnegative control (Figure 1(a)) To characterize themechanisminvolved in the EAEC strain 042 binding to integrin 12057251205731we quantified binding of EAEC strain 042 and 042aafAmutant to recombinant purified human integrin 12057251205731 byELISA Binding of EAEC strain 042 and 042aafA to integrin12057251205731-coated wells was significantly higher than to BSA-coated wells but no difference in the binding to integrin12057251205731 between EAEC strain 042 and 042aafA was observed(Figure 1(b)) suggesting that AAFII fimbriae have no directrole in the binding of EAEC to integrin 12057251205731

32 Participation of Fibronectin in the Binding of EAECStrain 042 to Integrin 12057251205731 First we assessed the bindingof increasing concentrations of fibronectin to integrin 12057251205731and BSA-coated wells used as controls We found thatfibronectin binds significantly to integrin 12057251205731-coated wellsat all concentrations tested in a dose-dependent manner


Fibronectin (ngwell)0 50 100

00

05

10

15

BSA

lowast

lowast

lowast

lowast

OD450

nm

Integrin 12057251205731

(a)

Fn incubation (min)0 20 40 60

00

01

02

03

04

05

BSAIntegrin 12057251205731

lowast

lowast

OD450

nm

(b)

00

02

04

06lowast

lowast lowast

OD450

nm

BSA

Inte

grin12057251205731

Inte

grin12057251205731

Fn

BSA

Fn

(c)

00

02

04

06

08

10 lowast

OD450

nm

Integrin 12057251205731FnBSAFn

042042aafA

(d)

Figure 2 Fibronectinintegrin 12057251205731 complex increases the adhesion of EAEC strain 042 (a) Integrin 12057251205731- and BSA-coated wells wereincubated with increasing concentrations of fibronectin (25 50 and 100 ng) or (b) with 100 ng of fibronectin (Fn) for 15 30 or 60minBound fibronectin was detected by ELISA using anti-fibronectin antibodies (c) EAEC 042 was added to integrin 12057251205731- and BSA-coatedwells incubated or not with fibronectin (d) EAEC strain 042 and 042aafAwere added to integrin 12057251205731- and BSA-coated wells incubated withfibronectin EAEC binding was detected by ELISA using anti-O44 serumThe bars represent the mean for three experiments with the errorbars indicating standard deviation lowastSignificantly different between treatments (119875 lt 005)

(Figure 2(a)) Later we evaluated the time required forbinding of fibronectin to integrin 12057251205731 and we found thatfibronectin binds significantly to integrin 12057251205731-coated wellsafter 30min of incubation (Figure 2(b)) From these assayswe decided to use 100 ng of fibronectin for 60min to evaluatethe binding of EAEC strain 042 to fibronectinintegrin 12057251205731complex by ELISA EAEC strain 042 binding to the complexfibronectinintegrin 12057251205731 was significantly higher than thebinding to integrin 12057251205731 alone (Figure 2(c)) Consideringthat EAEC strain 042 binding to BSA or BSA incubatedwith fibronectin was similar we dismissed the possibilitythat the increase observed in the presence of the complex

fibronectinintegrin 12057251205731 was due to fibronectin incuba-tion Later we evaluated the participation of the AAFIIfimbriae in the binding of EAEC strain 042 to the com-plex fibronectinintegrin 12057251205731 and we found that bindingof EAEC 042aafA mutant strain was significantly lowercompared to the binding of wild-type EAEC strain 042(Figure 2(d))

33 Formation of Complex AafAFibronectinIntegrin 12057251205731EAEC strain 042 binding to fibronectin is mediated by themajor subunit of the AAFII fimbriae the AafA protein [2]


We evaluated the binding of purifiedAafA-dsc protein to inte-grin 12057251205731 and fibronectinintegrin 12057251205731 complex No directbinding of AafA-dsc protein to integrin 12057251205731 was foundhowever a significant binding to fibronectinintegrin 12057251205731complex was observed (Figure 3(a))These observations wereconfirmed by coimmunoprecipitation assays of a mixturecontaining the purified AafA-dsc and fibronectin Dot blotanalysis showed the presence of AafA and fibronectin in thefraction obtained from a column with anti-AafA antibodiesand not in the control column (Figure 3(b)) To evaluatethe formation of the complex AafA-dscfibronectinintegrin12057251205731 these proteins were incubated and immunoprecipitatedusing anti-AafA antibodies In the immunoprecipitated frac-tion with anti-AafA antibodies the three proteins were foundin higher amount compared to the proteins present in thefraction of control column (Figure 3(c))

34 Role of Integrin 12057251205731 in the Adherence of EAEC Strain042 to Cells To determine the participation of integrin 12057251205731in the fibronectin-mediated adherence of EAEC strain 042to intestinal epithelial cells prior to infection we incubatedT84 cells with fibronectin in the presence of two differentantibodies a polyclonal antibody against several epitopes ofthe integrin1205725molecule and amonoclonal antibody that onlyblocks the fibronectin-integrin12057251205731 interaction site the RGDsite EAEC strain 042 adherence to cells preincubated withfibronectin was significantly higher than cells incubated withmedium only used as control confirming previous observa-tions [2] EAEC strain 042 adherence was significantly lowerwhen cells were incubated with fibronectin and anti-integrin12057251205731 (RGD) antibodies compared to cells incubated withfibronectin alone The incubation with fibronectin and anti-integrin 1205725 antibodies did not affect the adherence to cellscompared to cells incubated only with fibronectin (Figure 4)

To confirm the participation of integrin 12057251205731 in theadherence of EAEC strain 042 to epithelial cells HEp-2cells were transfected with scrambled shRNA or integrin 1205725shRNA Prior to EAEC infection cells were preincubatedwith DMEM or medium supplemented with fibronectinThesignificant increase in EAEC adherence to nontransfectedHEp-2 cells orHEp-2 cells transfectedwith scrambled shRNAin presence of fibronectin compared to cell not incubatedwith this ECM protein was not observed in HEp-2 cellstransfected with integrin 1205725 shRNA (Figure 5)

Altogether these results support the role of integrin 12057251205731in the fibronectin-mediated EAEC adherence to intestinalcells

4 Discussion

Adherence to intestinal epithelia is a key process in EAECpathogenesis Although the cellular receptors involved inthe EAEC recognition are not fully characterized previousstudies have demonstrated that fibronectin is a receptor forthe AAFII fimbriae [2 3] one of the major adherencefactors of EAEC In the prototype EAEC strain 042 carryingthe AAFII fimbriae variant this binding is mediated byAafA protein Fibronectin can bind different receptors in the

eukaryotic cell being integrin 12057251205731 the most studied Thisinteraction has different roles in vivo such as fibronectin fibrilformation extracellular matrix assembly and transductionof biochemical signals that regulate cell adhesion migrationproliferation and apoptosis [14]

Fibronectin-integrin 12057251205731 interaction occurs through ashort polypeptide sequence Arg-Gly-Asp or RGD sequencepresent in the fibronectin molecule which interacts withthe interface between subunits 120572 and 120573 of integrin 12057251205731[15] In this study we evaluated the interaction betweenfibronectin and integrin 12057251205731 using purified proteins and wefound that this interaction is dependent on the fibronectinconcentration employed and the period of incubation Nextwe evaluated the participation of integrin 12057251205731 in theadherence of EAEC and we found that prototype EAECstrain 042 binds to purified integrin 12057251205731 but this bindingis independent of the AAFII fimbriae suggesting the con-tribution of other adhesins in this interaction (Figure 1) Theparticipation of integrins as pathogen receptors has beendescribed previously For example nonfimbrial adhesinsAFA(homologous to AAF fimbriae) present in pathogenic Ecoli strains have the ability to bind to 1205731 integrin [16]In uropathogenic E coli (UPEC) strains type 1 fimbriaebind to integrins 1205731 and 1205723 and the heterodimer formedbetween these proteins [17] In Gram-positive pathogensbinding to integrin 12057251205731 is mediated by fibronectin Thisprotein acts as a molecular bridge connecting bacteria withhost cell Fibronectin binding proteins (FnBP) located atthe surface of bacteria bind to fibronectin and the complexbacteriafibronectin is recognized by integrin 12057251205731 promot-ing the endocytosis of bacteria [10] Our data showed anincrease in the binding of EAEC strain 042 to the complexfibronectin-integrin 12057251205731 (Figure 2) suggesting that EAEChas the ability to bind to integrin 12057251205731 either directly ormediated by fibronectin Similarly we found that AafA-dsc protein binds to fibronectinintegrin 12057251205731 complex butnot to integrin 12057251205731 alone (Figure 3) Altogether theseresults suggest that EAEC can bind directly (not mediatedby AAFII fimbriae) and indirectly (mediated by AAFIIand fibronectin) to integrin 12057251205731 The latter observation issupported by coimmunoprecipitation assays using purifiedAafA-dsc protein that confirmed the formation of the com-plex AafAfibronectinintegrin 12057251205731 (Figure 3) To evaluatethe importance of this interaction in the context of EAECinfection to intestinal cell we assessed the adherence ofEAEC strain 042 to T84 cells by preincubating with anantibody that blocks the RGD binding site of integrin 12057251205731and by knocking down the integrin 1205725 expression withshRNA Under these conditions fibronectin-mediated EAECadherence was abolished (Figures 4 and 5)

In the intestinal epithelia fibronectin and integrins areprincipally expressed in the basolateral surface However ininflamed intestines upregulation in the expression of thesemolecules occurs promoting their presence in the apicalsurface of cells thus enabling interaction with bacteria [18ndash20] Previous studies have demonstrated that EAEC inducesthe expression and release of proinflammatory cytokines andloss of the epithelial barrier [21 22] In this condition the


00

02

04

06

BSA

lowast

Integrin 12057251205731 Integrin 12057251205731Fn

OD450

nm

(a)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

E1E2

E1 E2

120572-AafA

120572-Fn

(b)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

25

E3E4

E3 E4

Integrin 1205725

120572-AafA

120572-Fn

120572-1205725

(c)

Figure 3 AafA binding to fibronectinintegrin 12057251205731 complex (a) AafA-dsc protein was added to integrin 12057251205731- and BSA-coated wellsincubated or not with fibronectin (Fn) AafA-dsc binding was detected by ELISA using anti-AafA antibodies The bars represent the meanfor three experiments with the error bars indicating standard deviation lowastSignificantly different between treatments (119875 lt 005) (b) AafA-dsc and fibronectin or (c) AafA-dsc fibronectin and integrin 12057251205731 were mixed and the complex formed was added to control column or acolumn with anti-AafA antibodies for coimmunoprecipitation analysis The eluted fraction obtained from control column (E1 and E3) or acolumn with anti-AafA antibodies (E2 and E4) was analyzed by Dot blot using anti-AafA anti-integrin 1205725 and anti-fibronectin antibodiesThe resulting autoradiography was scanned and signal intensity was quantified by UN-SCAN-IT 61 software One representative experimentof three independent experiments with similar result is shown


10

15

20

25

30 lowastlowast

lowast

Con

trol

Fn

Fn120572

-12057251205731

(RG

D)

Fn120572

-12057251205731

(1205725

)

104

CFU

wel

l

Figure 4 Adhesion of EAEC strain 042 to T84 cells T84 cellspreincubated with DMEM only (control) or medium supplementedwith fibronectin (Fn) Fn and anti-integrin 12057251205731 (RGD) or anti-integrin 12057251205731 (1205725) were infected with EAEC strain 042The numberof adherent bacteria was determined by colony forming unit counts(CFU) lowastSignificantly different between treatments (119875 lt 005)

4

5

6

7

8

ControlFn

lowast

lowast

Scrambled shRNANontransfected Integrin 1205725 shRNA

106

CFU

wel

l

Figure 5 Integrin 1205725 expression knockdown reduces EAEC strain042 fibronectin-mediated binding to epithelial cells HEp-2 cellsnontransfected and transfected with scrambled or integrin 1205725shRNA were preincubated with DMEM only (control) or mediumsupplemented with fibronectin (Fn) and then infected with EAECstrain 042Numbers of adherent bacteriawere determined by colonyforming unit counts (CFU) lowastSignificantly different compared tocontrol treatment (119875 lt 005)

ability to bind to the fibronectinintegrin12057251205731 complexmightbe involved in the aggregative adherence phenotype displayedby EAEC on intestinal cells

In conclusion we described a novel potential receptor forEAEC strain 042 integrin12057251205731 whichmight be an important

factor in the fibronectin-mediated adherence of EAEC strain042 to intestinal cells

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byGrant no 1120809 fromFONDE-CYT to Mauricio J Farfan Work in the Nataro lab was sup-ported by Grant no AI-033096 from the National Institutesof Health to James P Nataro

References

[1] J R Czeczulin S Balepur S Hicks et al ldquoAggregative adher-ence fimbria II a second fimbrial antigenmediating aggregativeadherence in enteroaggregative Escherichia colirdquo Infection andImmunity vol 65 no 10 pp 4135ndash4145 1997

[2] M J Farfan K G Inman and J P Nataro ldquoThemajor pilin sub-unit of the AAFII fimbriae from enteroaggregative Escherichiacolimediates binding to extracellular matrix proteinsrdquo Infectionand Immunity vol 76 no 10 pp 4378ndash4384 2008

[3] M Konar O Sachin A Priya and S Ghosh ldquoIdentificationof key proteins of cultured human intestinal cells involvedin interaction with enteroaggregative Escherichia colirdquo FEMSImmunology and Medical Microbiology vol 66 no 2 pp 177ndash190 2012

[4] P Kuusela ldquoFibronectin binds to Staphylococcus aureusrdquoNature vol 276 no 5689 pp 718ndash720 1978

[5] R Pankov and K M Yamada ldquoFibronectin at a glancerdquo Journalof Cell Science vol 115 no 20 pp 3861ndash3863 2002

[6] B Henderson S Nair J Pallas and M A WilliamsldquoFibronectin a multidomain host adhesin targeted by bacterialfibronectin-binding proteinsrdquo FEMSMicrobiology Reviews vol35 no 1 pp 147ndash200 2011

[7] C R Hauck and K Ohlsen ldquoSticky connections extracellularmatrix protein recognition and integrin-mediated cellular inva-sion by Staphylococcus aureusrdquo Current Opinion in Microbiol-ogy vol 9 no 1 pp 5ndash11 2006

[8] C R Hauck M Borisova and P Muenzner ldquoExploitation ofintegrin function by pathogenic microbesrdquo Current Opinion inCell Biology vol 24 no 5 pp 637ndash644 2012

[9] M Grundmeier M Hussain P Becker C Heilmann G Petersand B Sinha ldquoTruncation of fibronectin-binding proteinsin Staphylococcus aureus strain newman leads to deficientadherence and host cell invasion due to loss of the cell wallanchor functionrdquo Infection and Immunity vol 72 no 12 pp7155ndash7163 2004

[10] B Sinha P P Francois O Nuszlige et al ldquoFibronectin-bindingprotein acts as Staphylococcus aureus invasin via fibronectinbridging to integrin 120572 5120573 1rdquo Cellular Microbiology vol 1 no 2pp 101ndash117 1999

[11] C Hoffmann K Ohlsen and C R Hauck ldquoIntegrin-mediateduptake of fibronectin-binding bacteriardquo European Journal ofCell Biology vol 90 no 11 pp 891ndash896 2011

[12] K A Datsenko and B L Wanner ldquoOne-step inactivationof chromosomal genes in Escherichia coli K-12 using PCR


productsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 12 pp 6640ndash6645 2000

[13] M Izquierdo F Navarro-Garcia R Nava-Acosta J P NataroF Ruiz-Perez and M J Farfan ldquoIdentification of cell surface-exposed proteins involved in the fimbria-mediated adherenceof enteroaggregative Escherichia coli to intestinal cellsrdquo Infectionand Immunity vol 82 no 4 pp 1719ndash1724 2014

[14] L A Cary D C Han and J Guan ldquoIntegrin-mediated signaltransduction pathwaysrdquo Histology and Histopathology vol 14no 3 pp 1001ndash1009 1999

[15] J D Humphries A Byron and M J Humphries ldquoIntegrinligands at a glancerdquo Journal of Cell Science vol 119 no 19 pp3901ndash3903 2006

[16] L Plancon L du Merle S Le Friec et al ldquoRecognition ofthe cellular 1205731-chain integrin by the bacterial AfaD invasin isimplicated in the internalization of afa-expressing pathogenicEscherichia coli strainsrdquoCellularMicrobiology vol 5 no 10 pp681ndash693 2003

[17] D S Eto T A Jones J L Sundsbak and M A Mul-vey ldquoIntegrin-mediated host cell invasion by type 1-piliateduropathogenic Escherichia colirdquo PLoS Pathogens vol 3 no 7 pe100 2007

[18] B Walia F E Castaneda L Wang et al ldquoPolarized fibronectinsecretion induced by adenosine regulates bacterial-epithelialinteraction in human intestinal epithelial cellsrdquo BiochemicalJournal vol 382 part 2 pp 589ndash596 2004

[19] V L Kolachala R Bajaj L Wang et al ldquoEpithelial-derivedfibronectin expression signaling and function in intestinalinflammationrdquo Journal of Biological Chemistry vol 282 no 45pp 32965ndash32973 2007

[20] J F Beaulieu ldquoIntegrins and human intestinal cell functionsrdquoFrontiers in Bioscience vol 4 pp D310ndashD321 1999

[21] S M Harrington M C Strauman C M Abe and J P NataroldquoAggregative adherence fimbriae contribute to the inflamma-tory response of epithelial cells infected with enteroaggregativeEscherichia colirdquo Cellular Microbiology vol 7 no 11 pp 1565ndash1578 2005

[22] M C Strauman J M Harper S M Harrington E J Boll and JP Nataro ldquoEnteroaggregative Escherichia coli disrupts epithelialcell tight junctionsrdquo Infection and Immunity vol 78 no 11 pp4958ndash4964 2010

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Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


contribute to the successful colonization of bacteria at theepithelial surface acting as a ldquomolecular bridgerdquo connectingthe bacteria with the host cell surface [6]

Integrins are heterodimeric glycoproteins that are criticalfor a variety of cell-cell and cell-matrix binding events Oncea particular ligand binds to integrin it triggers a specificsignaling pathway depending on the type of integrin thatis modulated [7] Fibronectin is a ligand for at least adozen members of the 1205731 integrin family where 12057251205731 is themajor integrin involved in indirect recognition of bacterialpathogens with the ability to bind to fibronectin [6 8]For example Staphylococcus aureus employs fibronectin as apathogenic mechanism to adhere modulate the intracellularsignaling pathway and invade host cells [7 9] Fibronectin-associated bacteria can be recognized by integrin 12057251205731through the RGD region and this engagement induces theinternalization of bacteria by host cells [10 11]

Considering the above in this study we sought to charac-terize the participation of integrin 12057251205731 in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cellsOur results support the participation of fibronectin as abridging molecule allowing the contact of EAEC strain 042and integrin 12057251205731 at the surface of intestinal cells

2 Materials and Methods

21 Bacterial Strains Prototype EAEC strain 042 (O44H18)was originally isolated from a child with diarrhea in LimaPeru The EAEC 042aafA strain was constructed using thelambda red linear recombination method [12] as previouslydescribed [13] Prototype enterohemorrhagic E coli (EHEC)strain 86-24 and enterotoxigenicE coli (ETEC) strainH10407were used to evaluate binding to integrin 12057251205731 Bacteriawere grown overnight in Luria-Bertani (LB) broth with theaddition of kanamycin (50120583gmL) when appropriate

22 Cell Lines T84 and HEp-2 cells were routinely main-tained in Dulbeccorsquos modified Eaglersquos medium (DMEM) F12and DMEM high glucose (DMEMHG) (HyClone) respec-tively supplemented with 10 fetal bovine serum (FBS)penicillin (10UmL) and streptomycin (10 120583gmL) at 37∘Cunder 5 CO

2

23 Solid-Phase Binding Assay Microtiter plates (ThermoLabsystems FranklinMA)were coated overnight at 4∘Cwitha solution of 2 120583gmL of integrin 12057251205731 (RampD system) orbovine serum albumin (BSA Sigma) in integrin binding (IB)buffer containing Tris 25mM CaCl

22mM MgCl

21mM

MnCl21mM and NaCl 150mM pH 75 Unbound protein

was removed by washing three times with phosphate bufferedsaline (PBS HyClone) and subsequently blocked with 1skim milk in PBS for 1 h at 37∘C The blocking buffer wasremoved and the plates were washed and incubated with asolution of 1 120583gmL of fibronectin (Sigma) in IB buffer by 1 hat 37∘C Binding of fibronectin to integrin 12057251205731 was detectedusing an anti-fibronectin antibody (Sigma) which was incu-bated for 1 h at room temperature Anti-rabbit horseradishperoxidase conjugate (HRP KPL Gaithersburg MD) was

added following another PBS wash step Peroxidase activityassociated with each well was detected by the addition of 3 310158405 51015840-tetramethylbenzidine (TMB) substrate solution (KPL)Optical densities were read at 450 nm with a 96-well platereader (Infinite Tecan) To determine the adhesion of EAECor AafA-dsc protein to integrin 12057251205731 and fibronectinintegrin12057251205731 complex we performed the same protocol describedabove with some modification After the incubation withfibronectin plates were washed three times with PBS andincubated with bacteria in a 200120583L final volume or witha solution of 1 120583gmL of AafA-dsc protein Incubation withbacteria was performed for 2 h at 37∘C while incubationwith protein was carried out for 2 h at room temperaturePlateswerewashed and rabbit anti-O44 serum (Denka SeikenCo LTD Tokyo Japan) diluted 1 200 or rabbit anti-AafA1 2000 respectively was added to the wells and incubatedfor 1 h at room temperature Anti-rabbit HRP conjugatewas added following another PBS wash step and peroxidaseactivity associatedwith eachwell was detected by the additionof TMB substrate solution Optical densities were read at450 nm

24 Coimmunoprecipitation Assays We constructed colum-ns with anti-AafA antibodies and control columns (withoutantibodies) using pierce coimmunoprecipitation (Co-IP) kitfollowing manufacturer instructions To evaluate the forma-tion of the AafAfibronectin complex we prepared a solutionof 5 120583gmL of AafA-dsc protein and 10 120583gmL of purifiedfibronectin This mixture was incubated with gentle shakingfor 1 h at room temperature and then added to each columnfollowing manufacturer instructions Unbound protein wasremoved by washing three times with PBS and immunopre-cipitated proteins were eluted To evaluate the formation ofthe complex AafAfibronectinintegrin 12057251205731 we performedthe same procedure described above We incubated 5120583gmLof AafA-dsc 10 120583gmL of purified fibronectin and 5120583gmLof purified integrin 12057251205731 protein for 1 h at room temperatureFractions recovered from control column and column withanti-AafA antibodies were analyzed by Dot blot

25 Dot Blot Nitrocellulose membrane was washed withPBS and then coated with 10 120583L of fractions recoveredfrom control column and column with anti-AafA antibodiesThe membrane was blocked with BSA 1 for 1 h at roomtemperature and the presence of AafA-dsc fibronectin orintegrin 12057251205731 proteins was determined using anti-AafA anti-fibronectin (Sigma) or anti-integrin 1205725 (Santa Cruz Biotech-nology) Membranes were washed three times with PBS-Tween 005 and anti-rabbit HRP was added Peroxidaseactivity associated was detected by the addition of TMBsubstrate solution directly on membrane The resulting dotswere scanned and signal intensity was quantified using UN-SCAN-IT 61 software

26 Adherence Assays Epithelial cells grown in 96-wellplates were incubated for 30minwith 100120583Lwell ofDMEM-F12 only or supplemented with fibronectin (1 120583gwell) anti-bodies anti-integrin 12057251205731 that block the interaction withRGD site in fibronectin (Abcam) or anti-integrin 1205725 (Santa


EAEC EHEC ETEC00

05

10

15

BSA

lowast

lowast

lowast

OD450

nm

Integrin 12057251205731

(a)

BSA00

02

04

06

08

042

lowast

lowast

OD450

nm

042aafA

Integrin 12057251205731

(b)






2 Later




3 Results





00

05

10

15

BSA

lowast

lowast

lowast

lowast

OD450

nm

Integrin 12057251205731

(a)


00

01

02

03

04

05


lowast

lowast

OD450

nm

(b)

00

02

04

06lowast

lowast lowast

OD450

nm

BSA

Inte

grin12057251205731

Inte

grin12057251205731

Fn

BSA

Fn

(c)

00

02

04

06

08

10 lowast

OD450

nm


042042aafA

(d)










4 Discussion






00

02

04

06

BSA

lowast


OD450

nm

(a)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

E1E2

E1 E2

120572-AafA

120572-Fn

(b)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

25

E3E4

E3 E4

Integrin 1205725

120572-AafA

120572-Fn

120572-1205725

(c)



10

15

20

25

30 lowastlowast

lowast

Con

trol

Fn

Fn120572

-12057251205731

(RG

D)

Fn120572

-12057251205731

(1205725

)

104

CFU

wel

l


4

5

6

7

8

ControlFn

lowast

lowast


106

CFU

wel

l







Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


EAEC EHEC ETEC00

05

10

15

BSA

lowast

lowast

lowast

OD450

nm

Integrin 12057251205731

(a)

BSA00

02

04

06

08

042

lowast

lowast

OD450

nm

042aafA

Integrin 12057251205731

(b)






2 Later




3 Results





00

05

10

15

BSA

lowast

lowast

lowast

lowast

OD450

nm

Integrin 12057251205731

(a)


00

01

02

03

04

05


lowast

lowast

OD450

nm

(b)

00

02

04

06lowast

lowast lowast

OD450

nm

BSA

Inte

grin12057251205731

Inte

grin12057251205731

Fn

BSA

Fn

(c)

00

02

04

06

08

10 lowast

OD450

nm


042042aafA

(d)










4 Discussion






00

02

04

06

BSA

lowast


OD450

nm

(a)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

E1E2

E1 E2

120572-AafA

120572-Fn

(b)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

25

E3E4

E3 E4

Integrin 1205725

120572-AafA

120572-Fn

120572-1205725

(c)



10

15

20

25

30 lowastlowast

lowast

Con

trol

Fn

Fn120572

-12057251205731

(RG

D)

Fn120572

-12057251205731

(1205725

)

104

CFU

wel

l


4

5

6

7

8

ControlFn

lowast

lowast


106

CFU

wel

l







Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



00

05

10

15

BSA

lowast

lowast

lowast

lowast

OD450

nm

Integrin 12057251205731

(a)


00

01

02

03

04

05


lowast

lowast

OD450

nm

(b)

00

02

04

06lowast

lowast lowast

OD450

nm

BSA

Inte

grin12057251205731

Inte

grin12057251205731

Fn

BSA

Fn

(c)

00

02

04

06

08

10 lowast

OD450

nm


042042aafA

(d)










4 Discussion






00

02

04

06

BSA

lowast


OD450

nm

(a)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

E1E2

E1 E2

120572-AafA

120572-Fn

(b)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

25

E3E4

E3 E4

Integrin 1205725

120572-AafA

120572-Fn

120572-1205725

(c)



10

15

20

25

30 lowastlowast

lowast

Con

trol

Fn

Fn120572

-12057251205731

(RG

D)

Fn120572

-12057251205731

(1205725

)

104

CFU

wel

l


4

5

6

7

8

ControlFn

lowast

lowast


106

CFU

wel

l







Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






4 Discussion






00

02

04

06

BSA

lowast


OD450

nm

(a)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

E1E2

E1 E2

120572-AafA

120572-Fn

(b)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

25

E3E4

E3 E4

Integrin 1205725

120572-AafA

120572-Fn

120572-1205725

(c)



10

15

20

25

30 lowastlowast

lowast

Con

trol

Fn

Fn120572

-12057251205731

(RG

D)

Fn120572

-12057251205731

(1205725

)

104

CFU

wel

l


4

5

6

7

8

ControlFn

lowast

lowast


106

CFU

wel

l







Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


00

02

04

06

BSA

lowast


OD450

nm

(a)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

E1E2

E1 E2

120572-AafA

120572-Fn

(b)

Sign

al in

tens

ity

AafA Fn0

5

10

15

20

25

E3E4

E3 E4

Integrin 1205725

120572-AafA

120572-Fn

120572-1205725

(c)



10

15

20

25

30 lowastlowast

lowast

Con

trol

Fn

Fn120572

-12057251205731

(RG

D)

Fn120572

-12057251205731

(1205725

)

104

CFU

wel

l


4

5

6

7

8

ControlFn

lowast

lowast


106

CFU

wel

l







Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


10

15

20

25

30 lowastlowast

lowast

Con

trol

Fn

Fn120572

-12057251205731

(RG

D)

Fn120572

-12057251205731

(1205725

)

104

CFU

wel

l


4

5

6

7

8

ControlFn

lowast

lowast


106

CFU

wel

l







Acknowledgments


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article participation of integrin 5 1 in the fibronectin

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