of July 13, 2018.This information is current as

Ail Outer Membrane Proteinpseudotuberculosis

YersiniaComplement Inhibitor C4BP to Functional Recruitment of the Human

MeriErica Ginström, Anna M. Blom, Mikael Skurnik and Seppo Derek K. Ho, Rauna Riva, Vesa Kirjavainen, Hanna Jarva,

ol.1103149http://www.jimmunol.org/content/early/2012/03/30/jimmun

published online 30 March 2012J Immunol 

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on July 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Functional Recruitment of the Human Complement InhibitorC4BP to Yersinia pseudotuberculosis Outer Membrane ProteinAil

Derek K. Ho,* Rauna Riva,* Vesa Kirjavainen,* Hanna Jarva,*,† Erica Ginstrom,*

Anna M. Blom,‡ Mikael Skurnik,*,† and Seppo Meri*,†

Ail is a 17-kDa chromosomally encoded outer membrane protein that mediates serum resistance (complement resistance) in the path-

ogenic Yersiniae (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis). In this article, we demonstrate that Y. pseudotuberculosis

Ail from strains PB1, 2812/79, and YPIII/pIB1 (serotypes O:1a, O:1b, and O:3, respectively) can bind the inhibitor of the classical

and lectin pathways of complement, C4b-binding protein (C4BP). Binding was observed irrespective of serotype tested and

independently of YadA, which is the primary C4BP receptor of Y. enterocolitica. Disruption of the ail gene in Y. pseudotuberculosis

resulted in loss of C4BP binding. Cofactor assays revealed that bound C4BP is functional, because bound C4BP in the presence of

factor I cleaved C4b. In the absence of YadA, Ail conferred serum resistance to strains PB1 and YPIII, whereas serum resistance

was observed in strain 2812/79 in the absence of both YadA and Ail, suggesting additional serum resistance factors. Ail from strain

YPIII/pIB1 alone can mediate serum resistance and C4BP binding, because its expression in a serum-sensitive laboratory strain of

Escherichia coli conferred both of these phenotypes. Using a panel of C4BP mutants, each deficient in a single complement control

protein domain, we observed that complement control protein domains 6–8 are important for binding to Ail. Binding of C4BP was

unaffected by increasing heparin or salt concentrations, suggesting primarily nonionic interactions. These results indicate that

Y. pseudotuberculosis Ail recruits C4BP in a functional manner, facilitating resistance to attack from complement. The Journal of

Immunology, 2012, 188: 000–000.

Fora pathogen to successfully infect the host, it must possessmechanisms for resisting innate immune defenses. A keycomponent of innate immunity is the complement system,

a group ∼40 proteins found in the fluid phase and on cell surfaces.Upon encountering a pathogen, complement is immediately acti-vated by one or several routes, all of which converge at the C3 step:the classical pathway (CP), which is usually initiated by Ab–Aginteractions; the lectin pathway (LP), which is initiated by mannan-binding lectin or ficolin recognition of microbial carbohydrates;and the alternative pathway (AP), which is constitutively active atlow levels, acting as a “surveillance” system with broad specificity(1). Successful activation on a microbial surface leads to opsoniza-tion with C3b and its cleavage product iC3b. Complement activation

also generates inflammation via the released anaphylatoxins and,in the case of Gram-negative bacteria, causes direct lysis by themembrane attack complex. To prevent unnecessary consumptionof complement or attack against self cells or tissues, the comple-ment system is tightly regulated by a group of self-surface boundand fluid-phase inhibitors, collectively named complement inhibi-tory proteins (2).C4b-binding protein (C4BP) and factor H (fH) are the primary

fluid-phase inhibitors of the CP/LP and AP, respectively (2). C4BPis a large glycoprotein with a plasma concentration ∼200 mg/ml.The major isoform found in plasma consists of seven identicala-chains and a single b-chain. The a- and b-chains consist of eightand three complement control protein domains (CCPs), respec-tively. C4BP regulates CP- and LP-mediated complement acti-vation by three mechanisms: by acting as a cofactor for factor I(fI)-mediated cleavage and inactivation of C4b, by acceleratingdecay of the C4b2a convertase, and by preventing the assemblyof the convertase by binding to C4b (3). fH is a single-chainglycoprotein consisting of 20 CCPs with a plasma concentration∼500 mg/ml. fH regulates the AP in a manner analogous to C4BP:it acts as a cofactor for fI-mediated cleavage and inactivation ofC3b, accelerates the decay of C3bBb convertases, and preventsconvertase assembly by competing with factor B for binding toC3b. Because a pathogen must evade or resist the detrimentaleffects of complement to establish infection, it is not surprisingthat many pathogenic microbes have evolved the ability to re-cruit and exploit C4BP and fH as a means of protection againstcomplement (4).Within the genus Yersinia, there are three species that cause dis-

ease in humans: Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis.Y. pestis is the causative agent of plague, a serious systemic diseaseusually transmitted by the bite of an infected flea. Y. enterocolitica

*Infection Biology Program, Department of Bacteriology and Immunology, HaartmanInstitute, University of Helsinki, FIN-00014 Helsinki, Finland; †Helsinki UniversityCentral Hospital, FIN-00029 Helsinki, Finland; and ‡Division of Medical ProteinChemistry, Department of Laboratory Medicine, Lund University, S-205 02 Malmo,Sweden

Received for publication November 4, 2011. Accepted for publication March 1, 2012.

This work was supported by the Academy of Finland (project 114075 to M.S.), theSigrid Juselius Foundation, and the Helsinki University Central Hospital Funds.

The nucleotide sequence presented in this article has been submitted to the EuropeanBioinformatics Institute (http://www.ebi.ac.uk/ena/) under accession number FR875289.1.

Address correspondence and reprint requests to Dr. Derek Ho, Department of Bac-teriology and Immunology, Haartman Institute, University of Helsinki, P.O. Box 21,Haartmaninkatu 3, FIN-00014 Helsinki, Finland. E-mail address: derek.ho@helsinki.fi

Abbreviations used in this article: Amp, ampicillin; AP, alternative pathway; C4BP,C4b-binding protein; CCP, complement control protein domain; Clm, chloramphen-icol; CP, classical pathway; fH, factor H; fHbp, factor H-binding protein; fI, factor I;GVB, veronal-buffered saline supplemented with 0.1% gelatin; HIS, heat-inactivatedserum; Km, kanamycin; KO, knockout; L-B, Luria–Bertani; LP, lectin pathway;NHS, normal human serum; OC, outer core.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103149

Published March 30, 2012, doi:10.4049/jimmunol.1103149 by guest on July 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

and Y. pseudotuberculosis are distantly related enteropathogensfound ubiquitously in the environment. Infection by these patho-gens occurs via ingestion of contaminated food or water (5, 6).Although disease caused by the enteropathogenic Yersiniae isusually self-limiting and restricted to the gastrointestinal tract,sequelae, such as reactive arthritis, may develop postinfection(7). Y. pseudotuberculosis is also the causative agent of Far Eastscarlet-like fever, a severe disease clinically resembling scarletfever caused by Group A streptococci (8). Although Y. pseudo-tuberculosis and Y. pestis cause clinically distinct diseases andhave dissimilar modes of transmission, these two pathogens areclosely related genetically. It was estimated that the plague bacillusevolved from Y. pseudotuberculosis as recently as 9,000–40,000 yago (9).The human pathogenic Yersinia species possess a chromosomally

encoded gene, ail, which was shown to mediate serum resistanceor resistance to complement-mediated killing (10–12). Ail belongsto a family of highly conserved 17–19-kDa outer membraneproteins found in the Enterobacteriaceae, including Rck (Salmo-nella typhimurium and enteritidis), PagC (typhoid and nontyphoidSalmonella, Escherichia coli O157:H7), and OmpX (E. coliand Enterobacter cloacae) (13, 14). All of these proteins havevirulence-associated phenotypes. Structural models and the solvedcrystal structure of OmpX (15) predict that these proteins exhibita common topology consisting of eight transmembrane amphi-pathic b-strands and four surface-exposed loops. The regions ofgreatest similarity are the transmembrane domains, whereas thegreatest sequence diversity is found in the surface-exposed loops,which presumably mediate interactions with the host. This ob-servation suggests that these proteins are functionally dissimilar.However, because Ail, Rck (16), and PagC (17) were shown tomediate serum resistance, and recruitment of fH by Rck (18) andboth fH (19) and C4BP (20) by Y. enterocolitica Ail was dem-onstrated (see below), it is possible that other members of thisprotein family confer serum resistance by similar means.The molecular mechanisms of serum resistance in Y. enter-

ocolitica have been extensively characterized. Ail was shown tobind both fH and C4BP; however, these interactions were inhibitedby the presence of LPS outer core (OC) or O-Ag. In contrast, the

autotransporter protein YadA is capable of binding both of theseproteins independently of LPS OC or O-Ag and, therefore, is theprimary molecule mediating serum resistance on the bacterialsurface (19, 20). LPS itself plays a minor role and possibly hasa detrimental effect on serum resistance, perhaps by interferingwith Ail function or by acting as a target for Abs or mannan-binding lectin (21). Double point mutations in the second surface-exposed loop of Ail, D67G/V68G, or D67A/V68R resulted in lossof the serum-resistance phenotype when the respective proteinswere expressed in E. coli DH5a (22). Ail was also shown tomediate serum resistance in both Y. pestis and Y. pseudotuber-culosis, although the precise mechanisms have not been char-acterized (10, 11). Although binding of C4BP to Y. pestis wasdemonstrated (23), it is not known whether Ail is responsible forthis interaction.In this work, we sought to characterize the molecular mechanism

of Ail-mediated serum resistance in Y. pseudotuberculosis. Wedemonstrate that Ail binds human C4BP, regardless of serotypetested, and independently of YadA. Ail-mediated serum resistancein strains PB1 and YPIII is also independent of YadA, whereasin strain 2812/79, serum resistance apparently involves factor(s) inaddition to YadA and Ail. C4BP binding to Ail is functional, becausewe observed cleavage of C4b in the presence of fI. Ail mediatesC4BP binding independently of other Y. pseudotuberculosis fac-tors, because cloning and expression of Ail from strain YPIII/pIB1in E. coli conferred serum resistance and C4BP binding. Bindingof C4BP to Ail is specific and appears to involve CCPs 6–8. Takentogether, these results suggest that C4BP binding is important forAil-mediated serum resistance in Y. pseudotuberculosis.

Materials and MethodsBacterial strains, plasmids, and growth conditions

Bacterial strains and plasmids are shown in Table I. For all experimentsinvolving Y. pseudotuberculosis, bacteria were streaked from frozen stocksonto Luria–Bertani (L-B) (Becton-Dickinson, Sparks, MD) plates. After∼48 h of growth at room temperature, a single colony was picked andinoculated into 5 ml L-B broth and grown overnight at 37˚C with shaking.The following day, bacteria were subcultured into L-B broth and grown foran additional 3–4 h at 37˚C with shaking. E. coli was grown on L-B platesor in broth at 37˚C. Where appropriate, bacterial growth media were

Table I. Bacterial strains and plasmids used in this study

Bacterial Strain or Plasmid Description Reference

Y. pseudotuberculosisPB1 Serotype O:1a, wild-type (42)2812/79 Serotype O:1b, wild-type (43)YPIII/pIB1 Serotype O:3, wild-type (44)PB1-Ail ail::kan, KmR, derivative of PB1 This study2812/79-Ail ail::kan, KmR, derivative of 2812/79 This studyYPIII/pIB1-Ail ail::kan, KmR, derivative of YPIII/pIB1 This studyPB1-c pYV-cured derivative of PB1 (45)2812/79-c pYV-cured derivative of 2812/79 This studyYPIII pYV-cured derivative of YPIII/pIB1 (44)PB1-Ail-c pYV-cured derivative of PB1-Ail This study2812/79-Ail-c pYV-cured derivative of 2812/79-Ail This studyYPIII-Ail pYV-cured derivative of YPIII/pIB1-Ail This study

E. coliBL21(DE3) Standard laboratory strain Invitrogen (Carlsbad, CA)S17-1lpir thi pro hsdR2 hsdM+ recA::RP4-2-Tc::m-Km::Tn7, strR (lpir) (46)

PlasmidpBR322 Standard cloning vector Promega (Madison, WI)pAY43 pBR322 ail+ from strain YPIII (10)pCVD442-ail::KmGB Suicide vector used to knock out the ail gene by allelic exchange This studypSW23T-ail::KmGB Suicide vector used to knock out the ail gene by allelic exchange This studypCVD442 Suicide vector, AmpR (47)pSW23T Suicide vector, ClmR (48)

2 YERSINIA PSEUDOTUBERCULOSIS Ail BINDS C4BP

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

supplemented with chloramphenicol (Clm; 50 mg/ml), ampicillin (Amp;100 mg/ml), or kanamycin (Km; 100mg/ml). Plasmid pAY43 contains theail gene from Y. pseudotuberculosis strain YPIII/pIB1 cloned into pBR322.pAY43 was a kind gift from Dr. Ralph Isberg (Tufts University, Boston,MA) and was described previously (10).

Ail sequences

The accession numbers for the Ail amino acid sequences are as follows:Y. pseudotuberculosis PB1 (YP_001873389), Y. pseudotuberculosis YPIII/pIB1 (AAB36601), Y. pestis KIM10 (NP_668646), and Y. enterocoliticaO:3 (CAE53849). The amino acid sequence for Ail from Y. pseudotuber-culosis 2812/79 was deduced from the ail gene sequence determined froma PCR fragment amplified from the genomic DNA extracted from thisstrain using PCR primers: PB1-Ail-F, 59-GGCCATGCATAGTGCCAAA-ATACGGATTGC-39 and PB1-Ail-R, 59-GGCCATGCATCCGTGAACAA-AAACAGCGTA-39. The sequencing primers were AilORFfw, 59-ATGG-TTTTTATGAATAAGATATTACTGGTC-39 and AilORFrev, 59-TTAGAA-CCGGTAACCCGC-39. The resulting nucleotide sequence was submittedto the European Bioinformatics Institute (http://www.ebi.ac.uk/ena/) andcan be retrieved via accession number FR875289.1. Sequence align-ments were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Construction of strains

ail mutants of Y. pseudotuberculosis strains PB1, 2812/79, and YPIII/pIB1(Table I) were constructed by allelic exchange following the protocoldescribed previously (24). Briefly, a DNA fragment containing a Km-resistance Genblock cassette (KmGB) in between two ail-gene flankingfragments was cloned into pSW23T or pCVD442 to obtain pSW23T-ail::KmGB or pCVD442-ail::KmGB, respectively. The plasmids were mobi-lized from E. coli S17-1lpir into the three Y. pseudotuberculosis wild-typestrains. KmR ClmR or KmR AmpR transconjugants were grown severalpassages with only Km selection and subjected to cycloserine enrichment(pSW23T derivatives) or sucrose selection (pCVD442 derivatives) to obtainstrains that had undergone allelic exchange due to a double-recombinationevent. The deletion of the ail gene from the mutants named as YPIII/pIB1-ail,PB1-ail, and 2812/79-ail was confirmed by PCR analysis. Curing of thevirulence plasmid in Y. pseudotuberculosis was achieved by selecting a fast-growing colony on MgCl2 and Na-oxalate–containing culture-medium plates(25–27).

Sera, proteins, and Abs

Normal human serum (NHS)was pooled from blood samples collected from7–10 healthy adult laboratory personnel. All persons who donated bloodfor this study provided a written informed consent, and the study protocolwas approved by the Section for Research of the Helsinki UniversityCentral Hospital Laboratory (project TYH7214). The blood was allowed toclot, and the sera were subsequently harvested, pooled, aliquoted, andstored at270˚C until used. Heat-inactivated serum (HIS) was generated byincubating NHS for 1 h at 56˚C. Purified human C4b and fI were pur-chased from Calbiochem (San Diego, CA). Human C4BP was purifiedaccording to the protocol of Persson and Lindahl (28). BSA and heparinwere purchased from Sigma-Aldrich (St. Louis, MO). Single CCP-deletionmutants of human C4BP were generated and purified as described (29).Monoclonal mouse anti-human C4BP Ab MK104 (29), sheep polyclonalanti-human C4BP antiserum (The Binding Site, Birmingham, U.K.), AlexaFluor-labeled anti-human IgG (Invitrogen, Carlsbad, CA), and FITC-labeled anti-human IgM (Behring, Marburg, Germany) were used as pri-mary Abs in flow cytometry experiments. The appropriate Alexa Fluor-labeled secondary Abs were acquired from Invitrogen. Rabbit antiserumagainst YadA was a kind gift from Dr. Petra Dersch (30), and anti-rabbitHRP Abs (Jackson ImmunoResearch, West Grove, PA) were used in im-munoblotting. Anti-sheep HRPAbs (Jackson ImmunoResearch) were usedfor ELISA.

Flow cytometry

Bacteria, grown as described above, were centrifuged at 10,000 3 g for 3min and resuspended in PBS to a final concentration ∼1 3 109 CFU/ml.Following an additional wash in PBS, an aliquot of the bacterial suspen-sion was added to HIS or NHS or mixed with purified human C4BP orC4BP CCP-deletion mutants in PBS (final concentration specified in eachexperiment) to a final volume of 50 ml. Samples were incubated at 37˚C forthe indicated times, centrifuged, and washed in 50 ml PBS supplementedwith 1% BSA. After the final wash, bacteria were resuspended in 50 mlPBS/1% BSA. Twenty microliters of a 1:100 dilution of the appropriateprimary Ab (diluted in PBS) was added to the bacteria (final volume 70 ml)

and incubated at room temperature for 20 min. After washing in PBS,bacteria were resuspended in 50 ml PBS, to which 20 ml a 1:200 dilution ofthe appropriate Alexa Fluor 488-conjugated secondary Ab was added,followed by incubation at room temperature in the dark for 20 min. Thecells were washed twice, as above, and resuspended in 0.5 ml filtered PBScontaining 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield,PA). Flow cytometric analysis of 10,000 gated events was performed ona FACScan (BD Biosciences, San Jose, CA) or Cyan ADP cytometer(Beckman Coulter, Miami, FL).

Measurement of dissociation constants

Y. pseudotuberculosis YPIII/pIB1, grown as described above, was resus-pended to ∼1 3 108 CFU/ml in PBS. One hundred-microliter aliquotswere allowed to adhere to individual wells of a 96-well Nunc MaxisorpELISA plate (Nunc, Roskilde, Denmark) overnight at 37˚C. After washing,50 ml purified human C4BP (in PBS) was added in doubling dilutions(highest concentration used: 50 nM C4BP) to the adhered bacteria for 30min at room temperature. The wells were washed, and bound C4BP wasdetected with sheep anti-C4BP antiserum, followed by washing and theaddition of anti-sheep HRP Abs. Blank wells and bacteria-only wells (noC4BP added) served as controls. All washing steps were performed withPBS/0.05% Tween-20. Wells were developed with OPD reagent (Dako),and the color reaction was measured at 492 nm in a Labsystems iEMSReader MF plate reader (Labsystems, Helsinki, Finland). The amount ofbound C4BP was quantified in accordance with a standard curve of boundC4BP. Data analysis and Kd determination were performed with GraphPadPrism (GraphPad Software, La Jolla, CA).

Determination of Ail and YadA expression

Whole-cell protein extracts were prepared by resuspending an aliquot ofan L-B culture in Laemmli buffer under nonreducing conditions for 30 minat 37˚C (Y. pseudotuberculosis) or under reducing conditions, followed byboiling for 3 min (E. coli). A lower temperature and nonreducing con-ditions were used for Y. pseudotuberculosis to prevent disruption of YadAmultimers. Extracts were resolved by SDS-PAGE in 12.5% Tris-glycinegels. Bands were subsequently visualized by staining with GelCode BlueSafe Stain (Thermo Scientific, Rockford, IL) or by immunoblotting withthe anti-YadA antiserum.

Serum bactericidal assay

Y. pseudotuberculosis or E. coli BL21(DE3) bacteria, grown as describedabove, were washed and resuspended in PBS to a final concentration of∼1 3 107 CFU/ml. Fifty-microliter portions of the bacterial suspensionswere added to 30 ml PBS and 20 ml NHS or 20 ml HIS (final serumconcentration 20%, final reaction volume 100 ml) and incubated for 30 minat 37˚C. After incubation, the samples were placed on ice to stop furtherbacteriolysis. Serial dilutions of the samples in PBS were plated on L-Bagar plates and incubated overnight at 37˚C in room air (E. coli) or 5%CO2 (Y. pseudotuberculosis). Survival was determined by counting bacterialcolonies the following day.

Cofactor activity for C4b cleavage

Y. pseudotuberculosis bacteria, grown as described above, were resus-pended to a final concentration ∼1 3 109 CFU/ml in PBS/1% BSA. Thebacteria were then incubated with purified human C4BP at a final con-centration of 1 mg/ml. Following a 30-min incubation at 37˚C, the bacteriawere washed five times in 100 ml PBS/1% BSA, with the final wash in PBSalone. After the last wash, bacteria were resuspended in PBS and incubatedwith ∼50,000 cpm [125I]C4b and 1 mg fI (final reaction volume 25 ml).After a 1-h incubation at 37˚C, the samples were centrifuged, and thesupernatants were analyzed by SDS-PAGE under reducing conditions. Thegels were subsequently dried, and the results were visualized by autora-diography from a phosphoimager plate.

Direct C4BP-binding assays

E. coli BL21(DE3)/pBR322 or E. coli BL21(DE3)/pAY43 were grownin L-B medium, as described above. Thereafter, they were washed andresuspended in veronal-buffered saline (142 mM NaCl, 1.8 mM sodiumbarbital, 3.3 mM barbituric acid [pH 7.4–7.6]) supplemented with 0.1%gelatin (GVB) to a final concentration of 1 3 109 CFU/ml. Twenty micro-liters of this solution was incubated with 20 ml [125I]C4BP (∼20,000 cpm/sample) for 30 min at 37˚C with agitation. After incubation, the sampleswere centrifuged through 250 ml 20% sucrose/GVB at 10,000 3 g toseparate free protein from protein bound to the bacteria. The supernatantsand pellets were separated, and radioactivities were measured in a WallacWizard 3’’ gamma counter (GMI, Minneapolis, MN). The ratio of bound

The Journal of Immunology 3

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

to total radioactivity was then determined. Competition assays were per-formed by determining the relative binding of [125I]C4BP in the presence ofincreasing amounts of unlabeled C4BP, heparin, or NaCl. BSAwas excludedfrom these assays as an irrelevant protein competitor to C4BP binding, be-cause BSAwas used as a blocking agent in the primary Ab-incubation stepfor detection of C4BP binding by flow cytometry (see above).

ResultsSerum bactericidal assays

Yang et al. (10) demonstrated that ail inactivation in Y. pseudo-tuberculosis strain YPIII (pYV cured) resulted in serum sensitivitycompared with the parent strain, and complementation with plasmid-expressed ail (pAY43) restored serum resistance. We assessed thevalidity of this finding with the strains used in the current study.Inactivation of ail in strain YPIII/pIB1 resulted in the loss of se-rum resistance (Fig. 1A), suggesting that Ail is a critical serum-resistance mediator in this strain, regardless of the presence ofYadA (Fig. 1B). In strain PB1, serum sensitivity was observed inthe ail strain only in the absence of pYV, suggesting the possibilitythat YadA can also mediate serum resistance (Fig. 1). However,strain 2812/79 was serum resistant, even in the absence of both Ailand YadA (Fig. 1). These results indicate that Ail can mediateserum resistance in strains PB1 and YPIII/pIB1, and additionalserum-resistance factors are present in strain 2812/79.

Y. pseudotuberculosis Ail binds C4BP independently of YadA

Previous studies characterizing the mechanism(s) of serum resis-tance in Y. enterocolitica revealed that the Ail and YadA proteinsbind both C4BP and fH (19, 20). Ail can only bind these proteinsin the absence of LPS OC and O-Ag, whereas YadA binds re-gardless of the LPS state. Comparison of the Ail sequences fromY. enterocolitica and the three Y. pseudotuberculosis strains usedin this study indicated that the majority of sequence conservationis observed in the transmembrane regions, whereas the surface-exposed loops exhibit the greatest diversity (Fig. 2). The Ailproteins from Y. pseudotuberculosis are almost identical except fortwo residues, indicated in Fig. 2. It is also worth noting that themature Ail protein from Y. pestis KIM10 is identical to Ail fromY. pseudotuberculosis strain PB1.

Despite the sequence diversity observed between the Ail proteinsof the enteropathogenic Yersiniae, we considered the possibilitythat Y. pseudotuberculosis Ail could also bind C4BP. Using HISas a source of C4BP, we observed by FACS analysis that allY. pseudotuberculosis strains tested can bind C4BP, and inactivationof ail reduced binding (strains PB1 and 2812/79) or eliminatedbinding (strain YPIII/pIB1). C4BP binding was observed regard-less of YadA expression. Moreover, in strains 2812/79 and YPIII,curing of the pYV plasmid did not reduce binding but actuallyenhanced it (Fig. 3A). Using purified C4BP, we observed dose-dependent and saturable binding to the wild-type strains, whereasno C4BP binding was observed on the ail mutants, even at thehighest C4BP concentration used (Fig. 3B). The fact that purifiedC4BP did not bind to the ail mutants of strains PB1 and 2812/79suggests that the residual C4BP binding in HIS observed on thesestrains may be due to other serum proteins tethering C4BP to thebacterial surface. We further characterized this interaction bytesting the binding of dilutions of purified C4BP to whole cellsof immobilized strain YPIII/pIB1. Based on a standard curve ofbound C4BP, the calculated apparent Kd was 4.53 1029 M (Fig. 3C).Taken together, these results demonstrate that Y. pseudotuberculosisAil can bind C4BP with high affinity and independently of theserotype tested or the presence of YadA.

C4BP bound to Ail exhibits functional activity

To determine whether C4BP bound to Ail is functional, we useda cofactor assay to test the ability of bound C4BP to cleave C4b.In this assay, wild-type Y. pseudotuberculosis (Ail expressing) orail-negative bacteria are incubated with purified C4BP, followedby the addition of C4b and fI. As shown in Fig. 4, incubation ofpurified C4BP with C4b and fI results in the generation of the C4dand C4c (∼45 and 25 kDa, respectively) cleavage products, alongwith the consequent disappearance of the C4b a9-chain. Similarresults were also observed when wild-type bacteria were in-cubated with C4BP, washed, and then incubated with C4b and fI.No cleavage was observed in the absence of fI, suggesting thatthe bacteria do not possess intrinsic proteolytic activity againstC4b. No cleavage was observed in the absence of Ail expression.

FIGURE 1. Serum bactericidal assay and de-

termination of YadA and Ail expression in Y.

pseudotuberculosis strains. (A) Serum bactericidal

assay. Approximately 1 3 107 CFU/ml of a mid-

log phase broth culture of Y. pseudotuberculosis

was incubated with HIS or NHS (final concen-

tration 20%) in PBS for 30 min at 37˚C. The re-

action tubes were then placed on ice, serially

diluted, and plated on L-B agar. Following an

overnight incubation at 37˚C/5% CO2, bacterial

survival was enumerated by colony counting. Data

are shown as mean of three experiments6 SD. (B)

Expression of YadA and Ail. Bacteria grown as in

(A) were solubilized with Laemmli buffer under

nonreducing conditions and resolved by SDS-

PAGE. The top portion of the gel was excised and

transferred to nitrocellulose. YadA expression was

detected by immunoblotting with rabbit anti-YadA

antiserum. The remainder of the gel was stained

with GelCode Blue reagent. Molecular mass

markers (in kDa) are shown. Position of Ail is

indicated by the arrow. ail; ail2, YadA+ strain; ail/

pYV2, ail2, yadA2 strain; pYV2, Ail+, yadA2

strain; wt, wild-type strain.

4 YERSINIA PSEUDOTUBERCULOSIS Ail BINDS C4BP

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Collectively, these results show that C4BP bound to Ail has co-factor activity for fI-mediated cleavage of C4b.

IgG and IgM binding

Although C4BP bound to Ail can cleave C4b in the presence offI, we decided to test the possibility that differences in Ab binding(IgG and IgM are activators of the CP) may exist between theAil-expressing and -nonexpressing strains and that this may ac-count for differences in serum sensitivity. For these experiments,we used pYV-cured strains, to eliminate the possible confoundingeffect of YadA. As shown in Fig. 5, all strains tested bound IgG,although the presence of Ail resulted in a clear increase in IgGbinding, particularly with strain YPIII. Similar results were ob-served with IgM, although the IgM binding overall was minimalcompared with IgG. The increased Ab binding to the Ail-expressingstrains may be due to nonbactericidal Abs binding to Ail or thepossibility that Ail itself exposes additional epitopes on the outermembrane, perhaps due to interactions with LPS. These resultssuggest that the CP is activated on the bacterial surface. De-spite increased Ab binding in the presence of Ail, the bacteriaremain serum resistant, suggesting that Ail provides defense againstthe CP.

Expression of Ail in E. coli confers C4BP binding and serumresistance

To rule out the possibility that other components of the Y. pseu-dotuberculosis outer membrane, in addition to Ail, may contributeto serum resistance and/or C4BP binding, we decided to test theability of Ail to mediate serum resistance in a serum sensitive,non-C4BP–binding background. The ail gene from strain YPIII/pIB1cloned into plasmid pBR322 (pAY43) (10) was expressed in thelaboratory E. coli strain BL21(DE3). Ail expression was verifiedby GelCode staining of whole-cell extracts (Fig. 6A). As shown inFig. 6B, under the experimental conditions used, bacteria express-ing the empty vector pBR322 were completely killed, whereasbacteria expressing Ail exhibited complete survival. In additionto serum resistance, Ail expression conferred C4BP-binding ability(Fig. 6C). Collectively, these results indicate that Ail can mediateserum resistance and C4BP binding in a heterologous, serum-sensitive host, independently of any other Y. pseudotuberculosis-specific factors.

Mapping of the Ail binding site on C4BP

To determine which domain of the C4BP a-chain is responsible forthe interaction with Ail, we used a panel of C4BP mutants, eachdeficient in a single a-chain CCP (29). The amount of boundprotein was determined by FACS analysis. As shown in Fig. 7A,we observed that the N-terminal and central CCPs 1–5 were notinvolved in the Ail interaction, because the deletion of these CCPsdid not reduce binding compared with purified wild-type C4BP.However, the deletion of CCPs 6–8 resulted in reduced binding toAil. These results were observed regardless of the serotype tested.Because it is possible that the polyclonal antiserum used in thesestudies may preferentially detect the C-terminal CCPs, whichwould result in reduced detection when these CCPs are absent, wedecided to verify these results with a mAb specific to CCP1. Asshown in Fig. 7B, using CCP1-specific mAb MK104, we observedthat deletion of CCPs 6–8 also resulted in reduced binding to Ail,corroborating the results observed with the polyclonal antiserum.The CCP1 deletion mutant was included as a negative control forMK104. These results suggest that C4BP CCPs 6–8 are responsiblefor the Ail interaction.

C4BP-binding–competition assays

To further characterize the interaction of Ail with C4BP, we usedunlabeled C4BP, heparin, or NaCl as competitors to the binding ofradiolabeled C4BP. We first confirmed that binding of [125I]C4BPto bacteria was associated with Ail expression. As shown in Fig. 8,E. coli BL21(DE3) expressing Ail bound [125I]C4BP, whereasbacteria expressing the empty vector did not. Increasing concen-trations of unlabeled C4BP can compete with [125I]C4BP binding,indicating that the C4BP–Ail interaction is specific (Fig. 8B).Increasing concentrations of heparin or NaCl had no effect on thebinding (Fig. 8C, 8D), suggesting that the interaction with Ail isprimarily nonionic. Because the heparin binding sites on C4BP arelocated on CCPs 1–3 (29, 31), the inability of heparin to competewith the interaction is consistent with the observation that CCPs6–8 are important for Ail binding (Fig. 7).

DiscussionAny successful animal pathogen must possess mechanisms forevading or minimizing the detrimental effects of complement. In

FIGURE 2. Alignment of Ail proteins from pathogenic Yersiniae. Underlined regions indicate the predicted four extracellular loops. The signal sequence

cleavage site is marked with an arrow. Amino acid positions marked with an arrowhead indicate nonconserved residues between the Ail proteins of

Y. pseudotuberculosis. Alignment was performed using ClustalW2. KIM10, Y. pestis strain KIM10; YptbO1a, Y. pseudotuberculosis serotype O:1a strain

PB1; YptbO1b, Y. pseudotuberculosis serotype O:1b strain 2812/79; YptbO3, Y. pseudotuberculosis serotype O:3 strain YPIII/pIB1; YeO3, Y. enterocolitica

serotype O:3.

The Journal of Immunology 5

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

this work, we demonstrated that the Ail outer membrane proteinfrom Y. pseudotuberculosis can bind the human complement in-hibitor C4BP, regardless of the serotype tested and independentlyof YadA. C4BP bound to Ail was functional, because we observedcleavage of C4b in the presence of fI. Ail can mediate serum re-sistance and C4BP binding, independently of other Y. pseudotu-berculosis-specific factors, because cloning and expression of Ailin E. coli conferred these phenotypes. This binding is specific,mediated by nonionic interactions, and involves CCPs 6–8 of theC4BP a-chain.

In contrast to Y. pestis, both Y. enterocolitica and Y. pseudotu-berculosis carry a functional copy of the yadA gene, which enc-odes for the trimeric autotransporter protein YadA. Because YadAwas shown to mediate serum resistance in Y. enterocolitica, atleast in part by binding C4BP (20), it is possible that YadA fromY. pseudotuberculosis could also bind this protein. In contrast toY. enterocolitica, binding of C4BP to Y. pseudotuberculosis wasdependent on Ail expression, regardless of the presence or absenceof YadA (Figs. 1B, 3). These results are not surprising because thetwo YadA proteins display ∼75% identity with each other (datanot shown). Moreover, YadA may interfere with C4BP binding toAil in Y. pseudotuberculosis, because we observed enhancedC4BP binding in the absence of this protein in strains 2812/79 andYPIII (Fig. 2A). However, we cannot rule out the possibility thatother proteins encoded by the pYV virulence plasmid, such asouter membrane proteins involved in the type three secretionsystem machinery, may be involved.Although we did not observe C4BP binding to YadA, the data

presented in this article suggest the possibility that YadA is able tomediate serum resistance in Y. pseudotuberculosis, independent ofAil. The loss of Ail was not sufficient to confer serum sensitivityto strains PB1 and 2812/79, suggesting a role for YadA in serumresistance. Although loss of Ail in strain YPIII/pIB1 resulted incomplete serum sensitivity, despite the presence of YadA, thisresult could be explained by the observation that YadA appears tobe expressed in this strain at lower levels compared with the othertwo strains used in this study (Fig. 1B). Strain 2812/79 was serumresistant, even in the absence of Ail and YadA, suggesting thatadditional serum resistance factors are present in this strain, suchas specific LPS structures or uncharacterized outer membraneproteins.Similar to the Ail proteins in the enteropathogenic Yersiniae, the

Ail protein from Y. pestis was shown to mediate serum resistance(11). Although C4BP binding to Y. pestis was demonstrated (23),it remains to be shown whether Ail is responsible for this inter-action. Because Y. pestis and Y. pseudotuberculosis are closelyrelated genetically, it is not surprising that a high similarity be-tween the Ail proteins from these species is observed (Fig. 2).Sequence alignment with all currently available Y. pestis Ailproteins in the databases reveals that the mature Ail proteins fromY. pestis strains KIM10, CO92, Antiqua, and Orientalis F1991016are 100% identical to the mature Ail protein from Y. pseudotu-berculosis PB1 (Fig. 2, data not shown). The Ail proteins fromother Y. pestis strains possess several nonconserved residuescompared with Y. pseudotuberculosis Ail, primarily in surface-

FIGURE 3. Y. pseudotuberculosis Ail binds C4BP. (A) Ail binding to

C4BP is independent of serotype and presence of YadA. Approximately

1 3 108 CFU of bacteria grown to mid-log phase were washed in PBS and

subsequently mock treated (PBS only) or incubated with 20% HIS for 30

min at 37˚C. Bound C4BP was detected with monoclonal anti-human

C4BP Ab MK104 and flow cytometry. Data are shown as mean fluores-

cence intensity (MFI) values from three separate experiments 6 SD. (B)

C4BP binding to Ail is dose dependent and saturable. Same as in (A), using

increasing amounts of purified human C4BP in lieu of HIS. Bound C4BP

was detected with a sheep anti-C4BP antiserum, followed by flow

cytometry. Data are shown as mean of three experiments 6 SD. Numeric

background fluorescence values of PBS-only–treated bacteria (0 mg/ml

C4BP added) are indicated in the figure as mean of three MFI values from

three separate experiments 6 SD. (C) Kd of C4BP binding to Ail. Strain

YPIII/pIB1 was immobilized on microtiter wells and incubated with

varying amounts of purified C4BP, followed by detection with sheep anti-

C4BP antiserum and anti-sheep HRP Abs. Bound C4BP was quantified in

accordance with a standard curve of C4BP. Nonlinear fitting analysis and

Kd determination were performed with GraphPad Prism. Data are shown as

the mean of three experiments performed in duplicate wells 6 SD.

FIGURE 4. Cofactor assay for C4b cleavage. Bacteria grown as in Fig. 2

were incubated with purified C4BP (1 mg/ml) for 30 min at 37˚C and

washed, followed by addition of fI (1 mg/reaction) and [125I]C4b (50,000

cpm). After a 1-h incubation at 37˚C, samples were analyzed with reducing

SDS-PAGE and visualized by autoradiography. The a9-, b-, and g-chains

of C4b and the C4d and C4c cleavage products are indicated by arrows and

arrowheads, respectively. Representative data are shown.

6 YERSINIA PSEUDOTUBERCULOSIS Ail BINDS C4BP

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

exposed loops 2 and 3, although it is worth noting that there areat most 2 or 3 aa substitutions between strains. This degree ofsequence conservation is greater compared with Y. enterocoliticaAil. Based on the results presented in this article, we anticipatethat Y. pestis Ail can also bind C4BP. Although Y. pestis lacks bothYadA (32) and LPS O-Ag (33), it is nevertheless possible that Y.pestis-specific surface components, such as the F1 capsule, couldinterfere with the putative Ail–C4BP interaction.The CP is likely of key importance in the bactericidal activity of

serum against Y. pseudotuberculosis. As shown in Fig. 1A, incu-bation in 20% NHS leads to complete killing of the ail knockout(KO) and ail KO/pYV2 strains of YPIII, whereas the wild-typestrain is not killed. Furthermore, there is no defect in IgG bindingin the presence of Ail (Fig. 5), suggesting that this initial step ofCP activation takes place on Ail-bearing strains. In contrast, al-though killing is observed with the ail KO strain of YPIII/pIB1 in40% Mg2+/EGTA serum (AP-only serum) compared with thewild-type strain, the amount of killing is reduced by several ordersof magnitude compared with 20% NHS (data not shown). Thissuggests that the AP itself is poorly activated by Y. pseudotuber-culosis. Thus, interactions between the AP and Y. pseudotuber-culosis will require further studies.The ability to inhibit multiple steps in the complement pathway

likely increases the chances of a pathogen to escape complementattack. Preliminary investigations in our laboratory showed thatstrain YPIII/pIB1 can also bind fH, and binding is lost in theabsence of Ail expression (data not shown). This binding is likely

of lower affinity compared with C4BP, because FACS analysisrevealed that .50 mg/ml of fH is required for saturation as op-posed to the ∼5 mg/ml required for C4BP (Fig. 3B, data notshown). Given the possibility that multiple complement-resistancemechanisms are mediated by Ail, further studies are needed todefine the precise role and contribution of C4BP binding.Although Ail from strain 2812/79 was functionally active (Fig.

4), this strain was serum resistant, even in the absence of YadAand Ail (Fig. 1A). Using GelCode staining of whole-cell proteinextracts, we did not observe any additional bands that were absentin the other two strains tested (Fig. 1B, data not shown), althoughit is possible that other outer membrane proteins, which areexpressed at levels below the GelCode detection threshold, maymediate serum resistance. LPS may also play a role in the serumresistance of this strain, perhaps by shielding C4b or C3b targetsor by preventing membrane attack complex insertion and function.Our studies characterizing the Ail–C4BP interaction revealed

that the a-chain C-terminal CCPs 6–8 of C4BP may be involvedin mediating binding, and increasing NaCl and heparin concen-trations had no effect on binding, suggesting mainly nonionicinteractions. These results are in contrast with those observed withY. enterocolitica Ail, where both increasing NaCl and heparinconcentrations were able to compete with the C4BP interaction(20), suggesting that ionic interactions mediate C4BP binding.CCPs 1–3 appear to be involved in the C4BP interaction with Y.enterocolitica Ail, because this is the location of the heparinbinding site on C4BP (29). Because a binding assay using indi-

FIGURE 5. IgG and IgM binding. Bacteria grown and prepared as in Fig. 3A were mock treated (PBS only) or incubated with 20% HIS for 10 min at

37˚C. Bound IgG (left panel) and IgM (right panel) were detected by anti-human IgG and IgM Abs, followed by flow cytometry. Note difference in scale

between IgG and IgM panels. Data are shown as mean fluorescence intensity (MFI) values from three separate experiments 6 SD.

FIGURE 6. Expression of Ail in E. coli BL21(DE3) is associated with serum resistance and C4BP binding. (A) Verification of Ail expression. E. coli

BL21(DE3) was transformed with either empty vector (pBR322) or pAY43. An aliquot of an overnight L-B culture was solubilized in Laemmli buffer under

reducing conditions, resolved by SDS-PAGE, and visualized by GelCode staining. Position of Ail is indicated by the arrow. Molecular mass markers are

shown. (B) Ail expression in E. coli BL21(DE3) is associated with serum resistance. Approximately 13 107 CFU of BL21(DE3)/pBR322 and BL21(DE3)/

pAY43 from an overnight L-B culture were washed in PBS, followed by incubation with either 20% HIS or NHS for 30 min at 37˚C. Bacterial survival was

enumerated by plating and counting colonies the following day. Data from an experiment performed three times are shown and expressed as means 6 SD.

(C) Ail expression confers C4BP binding. Bacteria grown as in (B) were either mock treated (PBS only) or incubated with 20% HIS for 30 min at 37˚C.

Bound C4BP was detected with mAb MK104 and flow cytometry. Data are shown as mean fluorescence intensity (MFI) values from three separate

experiments 6 SD.

The Journal of Immunology 7

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

vidual C4BP CCP-deletion mutants was not performed in thatstudy, it remains possible that other CCPs are involved in binding.Given the lack of sequence similarity in the surface-exposed loopsof the Ail proteins of the enteropathogenic Yersiniae (Fig. 2),these contrasting results are not surprising. However, the obser-vation that Y. pseudotuberculosis Ail binds CCPs 6–8 is consistentwith previous observations demonstrating that the N- or C-terminal, but not central CCPs, are the preferred binding sites ofother bacterial pathogens that recruit C4BP (34–39). Nevertheless,the fact that Ail, from genetically distant Yersinia species withdiverse exposed-loop sequences, can bind C4BP at two differentsites indicates the importance of C4BP binding for complementevasion and suggests that the two interactions evolved separately.

Previous investigations on Y. enterocolitica Ail and PagC fromSalmonella enterica Choleraesuis revealed the presence of twocritical amino acid residues at the C-terminal end of the secondsurface-exposed loop. Ail double-point mutants D67G/V68G orD67A/V68R resulted in loss of the serum-resistance phenotypewhen expressed in E. coli DH5a (22). The analogous mutation inPagC, E89S/V90R, also resulted in loss of the serum-resistancephenotype when expressed in E. coli (17). Based on these results,we hypothesized that the analogous mutation in Y. pseudotuber-culosis Ail would result in the loss of C4BP binding, accompaniedby restored serum sensitivity. However, using site-directed muta-genesis, we were unable to successfully isolate viable trans-formants expressing mutant Ail, suggesting the possibility thatmutations in this region may generate a protein that fails totranslocate to the outer membrane or may be toxic to the host cell.However, based on the results from the studies performed on Y.enterocolitica Ail and S. enterica Choleraesuis PagC, we antici-pate that the analogous mutation in Y. pseudotuberculosis Ail, ifexpressed and translocated properly, would abrogate the serum-resistance phenotype, presumably due to loss of C4BP binding.Creation of vaccines that induce generation of Abs that spe-

cifically disrupt bacterial interactions with complement inhibitorsis an attractive preventive measure, as exemplified by the fH-binding protein (fHbp)-containing meningococcal vaccines cur-rently under development (40). fHbp confers serum resistance tomeningococci by recruiting fH, which subsequently inhibits AP-mediated complement activation or amplification on the bacterialsurface. Abs against fHbp would be expected to have a two-tieredeffect: Abs bound to fHbp can initiate the CP, while simulta-neously blocking fH recruitment, thus preventing the bacteriafrom exploiting fH as a means of defense against complementattack (41). Having demonstrated in this study that Y. pseudotu-berculosis C4BP can bind Ail, it is worth considering the possi-bility that Abs against Ail would be bactericidal by the mechanismdescribed above, although it is possible that YadA or other surfacefactors could mediate serum resistance in the absence of Ailfunction. Given the fact that the Ail protein in several Y. pestisstrains is 100% identical to the Ail protein found in Y. pseudotu-berculosis PB1, it is plausible that bactericidal Abs against Ailcould provide protection against both species. Although we an-ticipate that Ail would also bind C4BP in a Y. pestis background,this interaction has not been verified experimentally.The three Y. pseudotuberculosis strains used in this study rep-

resent different serotypes, each expressing unique Ail molecules(Fig. 2). Although we were able to demonstrate functional C4BPbinding to Ail in each background tested, we cannot conclude that

FIGURE 7. CCPs 6–8 of C4BP mediate the interaction with Ail. (A) Bacteria grown as in Fig. 3Awere washed with PBS and mock treated (PBS only) or

incubated with 5 mg/ml of purified human C4BP or a C4BP single CCP-deletion mutant. Following incubation at 37˚C for 30 min, bacteria were washed,

and bound C4BP was detected with a sheep anti-C4BP antiserum, followed by flow cytometry. The mean fluorescence intensity value obtained from wild-

type C4BP bound to Ail was set at 100%, and the relative binding of the C4BP mutants was determined. Data are shown as mean of three individual

experiments 6 SD. (B) Same as in (A), using mAb MK104 to detect bound C4BP or C4BP mutants. C4BP, Purified wild-type C4BP; M, PBS-only

treatment; D1–8, C4BP single CCP-deletion mutants.

FIGURE 8. C4BP binding to Ail is specific and unaffected by increasing

heparin or salt concentrations. Direct binding of [125I]C4BP to Ail was

performed by incubating ∼1 3 108 CFU of BL21(DE3)/pBR322 or BL21

(DE3)/pAY43 in GVB with [125I]C4BP (∼20,000 cpm/sample). The ra-

tio of bound to total radioactivity was determined (percentage binding).

Data are expressed as means of two independent experiments performed

in duplicate 6 SD. (A) Expression of Ail correlates with binding to

[125I]C4BP. (B) Binding of C4BP to Ail is specific, because increasing

amounts of unlabeled C4BP can compete with binding to [125I]C4BP.

Increasing amounts of heparin (C) or NaCl (D) do not affect the C4BP–Ail

interaction. (B–D) Percentage of relative binding indicates the amount

of [125I]C4BP binding in the presence of competitor compared with the

absence of competitor (set to 100%).

8 YERSINIA PSEUDOTUBERCULOSIS Ail BINDS C4BP

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Ail will mediate similar phenotypes in the other 18 Y. pseudo-tuberculosis serotypes. Unique Ail molecules may exist withindifferent strains of the same serotype, or serotype-specific O-Agstructures may modulate and/or interfere with C4BP binding toAil, as observed in Y. enterocolitica.In the current study, we demonstrated that the Ail outer mem-

brane protein from Y. pseudotuberculosis can bind C4BP in afunctional manner, independently of the bacterial serotype testedand YadA expression. These results suggest that Y. pseudotuber-culosis can escape both the CP and LP of complement via specificrecruitment of C4BP by Ail. Because the Ail protein from severalY. pestis strains is identical to the Ail protein in Y. pseudotuber-culosis PB1, the results from these studies will also be relevant tocomplement interactions with Y. pestis.

AcknowledgmentsWe thank Monika Rybak and Tiina Kasanen for technical assistance in con-

structing the ail mutant strains, Hanna Jarvinen for purification of human

C4BP, and Marjatta Ahonen for general technical support.

DisclosuresThe authors have no financial conflicts of interest.

References1. Walport, M. J. 2001. Complement. First of two parts. N. Engl. J. Med. 344:

1058–1066.2. Morgan, B. P., and C. L. Harris. 1999. Complement Regulatory Proteins. Aca-

demic Press, San Diego, CA.3. Blom, A. M., B. O. Villoutreix, and B. Dahlback. 2004. Complement inhibitor

C4b-binding protein-friend or foe in the innate immune system? Mol. Immunol.40: 1333–1346.

4. Lambris, J. D., D. Ricklin, and B. V. Geisbrecht. 2008. Complement evasion byhuman pathogens. Nat. Rev. Microbiol. 6: 132–142.

5. Naktin, J., and K. G. Beavis. 1999. Yersinia enterocolitica and Yersinia pseu-dotuberculosis. Clin. Lab. Med. 19: 523–536, vi.

6. Rimhanen-Finne, R., T. Niskanen, S. Hallanvuo, P. Makary, K. Haukka,S. Pajunen, A. Siitonen, R. Ristolainen, H. Poyry, J. Ollgren, and M. Kuusi.2009. Yersinia pseudotuberculosis causing a large outbreak associated withcarrots in Finland, 2006. Epidemiol. Infect. 137: 342–347.

7. Ternhag, A., A. Torner, A. Svensson, K. Ekdahl, and J. Giesecke. 2008. Short-and long-term effects of bacterial gastrointestinal infections. Emerg. Infect. Dis.14: 143–148.

8. Eppinger, M., M. J. Rosovitz, W. F. Fricke, D. A. Rasko, G. Kokorina,C. Fayolle, L. E. Lindler, E. Carniel, and J. Ravel. 2007. The complete genomesequence of Yersinia pseudotuberculosis IP31758, the causative agent of Far Eastscarlet-like fever. PLoS Genet. 3: e142.

9. Achtman, M., K. Zurth, G. Morelli, G. Torrea, A. Guiyoule, and E. Carniel.1999. Yersinia pestis, the cause of plague, is a recently emerged clone of Yersiniapseudotuberculosis. Proc. Natl. Acad. Sci. USA 96: 14043–14048.

10. Yang, Y., J. J. Merriam, J. P. Mueller, and R. R. Isberg. 1996. The psa locus isresponsible for thermoinducible binding of Yersinia pseudotuberculosis to cul-tured cells. Infect. Immun. 64: 2483–2489.

11. Bartra, S. S., K. L. Styer, D. M. O’Bryant, M. L. Nilles, B. J. Hinnebusch,A. Aballay, and G. V. Plano. 2008. Resistance of Yersinia pestis to complement-dependent killing is mediated by the Ail outer membrane protein. Infect. Immun.76: 612–622.

12. Bliska, J. B., and S. Falkow. 1992. Bacterial resistance to complement killingmediated by the Ail protein of Yersinia enterocolitica. Proc. Natl. Acad. Sci.USA 89: 3561–3565.

13. Heffernan, E. J., J. Harwood, J. Fierer, and D. Guiney. 1992. The Salmonellatyphimurium virulence plasmid complement resistance gene rck is homologousto a family of virulence-related outer membrane protein genes, including pagCand ail. J. Bacteriol. 174: 84–91.

14. Konczy, P., K. Ziebell, M. Mascarenhas, A. Choi, C. Michaud, A. M. Kropinski,T. S. Whittam, M. Wickham, B. Finlay, and M. A. Karmali. 2008. Genomic Oisland 122, locus for enterocyte effacement, and the evolution of virulentverocytotoxin-producing Escherichia coli. J. Bacteriol. 190: 5832–5840.

15. Vogt, J., and G. E. Schulz. 1999. The structure of the outer membrane proteinOmpX from Escherichia coli reveals possible mechanisms of virulence. Struc-ture 7: 1301–1309.

16. Heffernan, E. J., S. Reed, J. Hackett, J. Fierer, C. Roudier, and D. Guiney. 1992.Mechanism of resistance to complement-mediated killing of bacteria encoded bythe Salmonella typhimurium virulence plasmid gene rck. J. Clin. Invest. 90: 953–964.

17. Nishio, M., N. Okada, T. Miki, T. Haneda, and H. Danbara. 2005. Identificationof the outer-membrane protein PagC required for the serum resistance phe-notype in Salmonella enterica serovar Choleraesuis. Microbiology 151: 863–873.

18. Ho, D. K., H. Jarva, and S. Meri. 2010. Human complement factor H binds toouter membrane protein Rck of Salmonella. J. Immunol. 185: 1763–1769.

19. Biedzka-Sarek, M., H. Jarva, H. Hyytiainen, S. Meri, and M. Skurnik. 2008.Characterization of complement factor H binding to Yersinia enterocolitica se-rotype O:3. Infect. Immun. 76: 4100–4109.

20. Kirjavainen, V., H. Jarva, M. Biedzka-Sarek, A. M. Blom, M. Skurnik, andS. Meri. 2008. Yersinia enterocolitica serum resistance proteins YadA andail bind the complement regulator C4b-binding protein. PLoS Pathog. 4:e1000140.

21. Biedzka-Sarek, M., R. Venho, and M. Skurnik. 2005. Role of YadA, Ail, andLipopolysaccharide in Serum Resistance of Yersinia enterocolitica Serotype O:3.Infect. Immun. 73: 2232–2244.

22. Miller, V. L., K. B. Beer, G. Heusipp, B. M. Young, and M. R. Wachtel. 2001.Identification of regions of Ail required for the invasion and serum resistancephenotypes. Mol. Microbiol. 41: 1053–1062.

23. Ngampasutadol, J., S. Ram, A. M. Blom, H. Jarva, A. E. Jerse, E. Lien,J. Goguen, S. Gulati, and P. A. Rice. 2005. Human C4b-binding protein selec-tively interacts with Neisseria gonorrhoeae and results in species-specific in-fection. Proc. Natl. Acad. Sci. USA 102: 17142–17147.

24. Bengoechea, J. A., L. Zhang, P. Toivanen, and M. Skurnik. 2002. Regulatorynetwork of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocoliticaincludes cell envelope-dependent signals. Mol. Microbiol. 44: 1045–1062.

25. Gemski, P., J. R. Lazere, and T. Casey. 1980. Plasmid associated with patho-genicity and calcium dependency of Yersinia enterocolitica. Infect. Immun. 27:682–685.

26. Gemski, P., J. R. Lazere, T. Casey, and J. A. Wohlhieter. 1980. Presence ofa virulence-associated plasmid in Yersinia pseudotuberculosis. Infect. Immun.28: 1044–1047.

27. Higuchi, K., and J. L. Smith. 1961. Studies on the nutrition and physiology ofPasteurella pestis. VI. A differential plating medium for the estimation of themutation rate to avirulence. J. Bacteriol. 81: 605–608.

28. Persson, J., and G. Lindahl. 2005. Single-step purification of human C4b-bindingprotein (C4BP) by affinity chromatography on a peptide derived from a strep-tococcal surface protein. J. Immunol. Methods 297: 83–95.

29. Blom, A. M., L. Kask, and B. Dahlback. 2001. Structural requirements forthe complement regulatory activities of C4BP. J. Biol. Chem. 276: 27136–27144.

30. Eitel, J., and P. Dersch. 2002. The YadA protein of Yersinia pseudotuberculosismediates high-efficiency uptake into human cells under environmental con-ditions in which invasin is repressed. Infect. Immun. 70: 4880–4891.

31. Blom, A. M., J. Webb, B. O. Villoutreix, and B. Dahlback. 1999. A cluster ofpositively charged amino acids in the C4BP alpha-chain is crucial for C4bbinding and factor I cofactor function. J. Biol. Chem. 274: 19237–19245.

32. Rosqvist, R., M. Skurnik, and H. Wolf-Watz. 1988. Increased virulence ofYersinia pseudotuberculosis by two independent mutations. Nature 334: 522–524.

33. Skurnik, M., A. Peippo, and E. Ervela. 2000. Characterization of the O-antigengene clusters of Yersinia pseudotuberculosis and the cryptic O-antigen genecluster of Yersinia pestis shows that the plague bacillus is most closely related toand has evolved from Y. pseudotuberculosis serotype O:1b. Mol. Microbiol. 37:316–330.

34. Accardo, P., P. Sanchez-Corral, O. Criado, E. Garcıa, and S. Rodrıguez deCordoba. 1996. Binding of human complement component C4b-binding protein(C4BP) to Streptococcus pyogenes involves the C4b-binding site. J. Immunol.157: 4935–4939.

35. Blom, A. M., K. Berggard, J. H. Webb, G. Lindahl, B. O. Villoutreix, andB. Dahlback. 2000. Human C4b-binding protein has overlapping, but not iden-tical, binding sites for C4b and streptococcal M proteins. J. Immunol. 164: 5328–5336.

36. Ram, S., M. Cullinane, A. M. Blom, S. Gulati, D. P. McQuillen, B. G. Monks,C. O’Connell, R. Boden, C. Elkins, M. K. Pangburn, et al. 2001. Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neis-seria gonorrhoeae. J. Exp. Med. 193: 281–295.

37. Jarva, H., S. Ram, U. Vogel, A. M. Blom, and S. Meri. 2005. Binding of thecomplement inhibitor C4bp to serogroup B Neisseria meningitidis. J. Immunol.174: 6299–6307.

38. Prasadarao, N. V., A. M. Blom, B. O. Villoutreix, and L. C. Linsangan. 2002. Anovel interaction of outer membrane protein A with C4b binding proteinmediates serum resistance of Escherichia coli K1. J. Immunol. 169: 6352–6360.

39. Hallstrom, T., H. Jarva, K. Riesbeck, and A. M. Blom. 2007. Interaction withC4b-binding protein contributes to nontypeable Haemophilus influenzae serumresistance. J. Immunol. 178: 6359–6366.

40. Granoff, D. M. 2010. Review of meningococcal group B vaccines. Clin. Infect.Dis. 50(Suppl. 2): S54–S65.

41. Beernink, P. T., J. A. Welsch, M. Bar-Lev, O. Koeberling, M. Comanducci, andD. M. Granoff. 2008. Fine antigenic specificity and cooperative bactericidalactivity of monoclonal antibodies directed at the meningococcal vaccine can-didate factor h-binding protein. Infect. Immun. 76: 4232–4240.

42. Porat, R., W.R. McCabe, and R.R. Brubaker. 1995. Lipopolysaccharide-associated resistance to killing of yersiniae by complement. Innate Immunity2: 91–97.

The Journal of Immunology 9

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

43. Skurnik, M. 1984. Lack of correlation between the presence of plasmids andfimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis. J. Appl.Bacteriol. 56: 355–363.

44. Bolin, I., L. Norlander, and H. Wolf-Watz. 1982. Temperature-inducibleouter membrane protein of Yersinia pseudotuberculosis and Yersinia enter-ocolitica is associated with the virulence plasmid. Infect. Immun. 37: 506–512.

45. Kukkonen, M., M. Suomalainen, P. Kyllonen, K. Lahteenmaki, H. Lang,R. Virkola, I. M. Helander, O. Holst, and T. K. Korhonen. 2004. Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonellaenterica. Mol. Microbiol. 51: 215–225.

46. de Lorenzo, V., and K. N. Timmis. 1994. Analysis and construction of stablephenotypes in gram-negative bacteria with Tn5- and Tn10-derived mini-transposons. Methods Enzymol. 235: 386–405.

47. Donnenberg, M. S., and J. B. Kaper. 1991. Construction of an eae deletionmutant of enteropathogenic Escherichia coli by using a positive-selection suicidevector. Infect. Immun. 59: 4310–4317.

48. Demarre, G., A. M. Guerout, C. Matsumoto-Mashimo, D. A. Rowe-Magnus,P. Marliere, and D. Mazel. 2005. A new family of mobilizable suicide plasmidsbased on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPalpha)conjugative machineries and their cognate Escherichia coli host strains. Res.Microbiol. 156: 245–255.

10 YERSINIA PSEUDOTUBERCULOSIS Ail BINDS C4BP

by guest on July 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from