AatA Is a Novel Autotransporter and Virulence Factor of Avian ...

INFECTION AND IMMUNITY, Mar. 2010, p. 898–906 Vol. 78, No. 30019-9567/10/$12.00 doi:10.1128/IAI.00513-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

AatA Is a Novel Autotransporter and Virulence Factor of AvianPathogenic Escherichia coli�

Ganwu Li,1 Yaping Feng,2 Subhashinie Kariyawasam,3 Kelly A. Tivendale,1 Yvonne Wannemuehler,1Fanghong Zhou,1 Catherine M. Logue,4 Cathy L. Miller,1 and Lisa K. Nolan1*

Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, 1802 University Blvd., Iowa State University,Ames, Iowa 500111; Laurence H. Baker Center for Bioinformatics and Biological Statistics, Iowa State University, Ames,

Iowa 500112; Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park,Pennsylvania 168023; and Department of Veterinary and Microbiological Sciences, 1523 Centennial Blvd.,

North Dakota State University, Fargo, North Dakota 581024

Received 7 May 2009/Returned for modification 1 September 2009/Accepted 10 December 2009

Autotransporters (AT) are widespread in Gram-negative bacteria, and many of them are involved invirulence. An open reading frame (APECO1_O1CoBM96) encoding a novel AT was located in the pathogenicityisland of avian pathogenic Escherichia coli (APEC) O1’s virulence plasmid, pAPEC-O1-ColBM. This 3.5-kbAPEC autotransporter gene (aatA) is predicted to encode a 123.7-kDa protein with a 25-amino-acid signalpeptide, an 857-amino-acid passenger domain, and a 284-amino-acid � domain. The three-dimensional struc-ture of AatA was also predicted by the threading method using the I-TASSER online server and then wasrefined using four-body contact potentials. Molecular analysis of AatA revealed that it is translocated to the cellsurface, where it elicits antibody production in infected chickens. Gene prevalence analysis indicated that aatAis strongly associated with E. coli from avian sources but not with E. coli isolated from human hosts. Also, AatAwas shown to enhance adhesion of APEC to chicken embryo fibroblast cells and to contribute to APECvirulence.

The autotransporter (AT) proteins are a large and diversefamily of extracellular virulence proteins of Gram-negativebacteria. All ATs share the same general structure and arecomprised of three domains: an amino-terminal signal peptide;an � or passenger domain, which confers the function of thesecreted protein; and a C-terminal � domain that mediatessecretion through the outer membrane. The cardinal feature ofconventional ATs is a long C-terminal translocator domainconsisting of about 300 amino acids, in contrast to the veryshort C-terminal translocator domain (about 70 amino acids)of trimeric ATs that form highly stable trimers in the outermembrane (8). While all trimeric AT proteins identified so fardisplay adhesive activity mediating bacterial interactions witheither host cells or extracellular matrix (ECM) proteins, theconventional ATs that have been characterized to date havediverse functions, including adhesion, cytotoxicity, and lipaseor protease activity (3, 6, 7, 46, 49, 54).

Temperature-sensitive hemagglutinin (Tsh) was the first ATdescribed in avian pathogenic Escherichia coli (APEC), apathogen which causes extraintestinal infections in turkeys,layers, and broilers (44). This conventional AT, which is en-coded by a virulence plasmid, occurs as a 106-kDa extracellularprotein and a 33-kDa outer membrane protein. Its passengerdomain contains a 7-amino-acid serine protease motif thatincludes the active-site serine (S259), which has also been found

in the secreted domain of IgA1 protease. Although Tsh did notshow any IgA protease activity in vitro (51), it was involved invirulence through mediation of APEC’s adherence to the airsacs of chickens (11). The gene encoding a second serine pro-tease AT, termed the vacuolating autotransporter or Vat, wasidentified in a pathogenicity island (PAI) adjacent to the thrWtRNA gene in APEC (42). Vat has vacuolating cytotoxic ac-tivity similar to that of VacA of Helicobacter pylori and con-tributes to APEC virulence (48). Both tsh and vat are presentin E. coli from avian sources and are also found in E. coliisolated from human hosts. In the present study, we identifiedand characterized a novel AT that is strongly associated withavian E. coli. This AT is encoded by the APEC autotransportergene (aatA), which has been localized to the PAI found in thevirulence plasmid (pAPEC-O1-ColBM; accession numberNC_009837) of APEC O1, the first APEC strain to be com-pletely sequenced (25, 26).

MATERIALS AND METHODS

Bacterial strains, plasmids, media, and growth conditions. The strains andplasmids used in this study are listed in Table 1. Well-characterized collections ofstrains of APEC, E. coli from the feces of apparently healthy chickens (27),human uropathogenic E. coli (UPEC) (47), and human neonatal meningitis-associated E. coli (NMEC) were used for gene prevalence studies (23). Strainswere grouped phylogenetically using multiplex PCR. APEC O1, an O1:K1:H7strain whose genome shares strong similarities with human extraintestinal patho-genic E. coli (ExPEC) genomes, was used to construct mutants and as a positivecontrol in virulence and other functional assays. E. coli DH5� was employed asa negative control. Cells were routinely grown at 37°C in Luria-Bertani broth(LB) supplemented with kanamycin (Km) (50 mg ml�1), chloramphenicol (Cm)(25 mg ml�1), or ampicillin (Amp) (100 mg ml�1), unless otherwise specified.Chicken embryo fibroblast (CEF) cells (ATCC CRL-12203) were maintained inATCC-formulated Dulbecco’s modified Eagle’s medium (DMEM) with 10%fetal bovine serum (FBS).

* Corresponding author. Mailing address: Department of Veteri-nary Microbiology and Preventive Medicine, College of VeterinaryMedicine, 1802 University Blvd., Iowa State University, Ames, IA50011. Phone: (515) 294-3785. Fax: (515) 294-1401. E-mail: [email protected].

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DNA and genetic manipulations. DNA manipulations and transformationswere performed using standard methods (2). All restriction and DNA-modifyingenzymes were purchased from New England Biolabs, Invitrogen, or AmershamPharmacia and were used according to the suppliers’ recommendations. Recom-binant plasmids, PCR products, and restriction fragments were purified usingplasmid miniprep, PCR cleanup, and gel extraction kits (Qiagen, Valencia, CA)as recommended by the supplier. Transformation of E. coli strains was routinelydone using electroporation. DNA and amino acid sequence analyses were per-formed using DNASTAR Lasergene 8 software to predict conserved domainsand using the search engine at http://blast.ncbi.nlm.nih.gov/Blast.cgi. TheI-TASSER online server was used to predict the three-dimensional (3D) struc-ture of the amino acid (55–57). The sequence from position 825 to position 1265was predicted to be the translocator domain, and the sequence from position 26to position 824 was predicted to be the passenger domain. Five final models ofthe passenger domain were generated using I-TASSER and evaluated further toselect the most appropriate structure based on the energies calculated by four-body contact potentials (17) and the gapless threading method.

PCR was performed to detect the presence of aatA sequences in different E.coli strains. Primers 5�-TGGTAGTGTTTGGGGAGGAG-3� and 5�-GCATTTCCTGCAGACAGGTT-3� were used to amplify the AatA passenger domain.Reactions were carried out using Taq DNA polymerase (New England Biolabs)under the following conditions: 95°C for 1 min, followed by 30 cycles of 94°C for30 s, 54°C for 30 s, and 72°C for 1 min and then extension at 72°C for 1 min.Specific amplification was confirmed using APEC O1 as a positive control and E.coli DH5� as a negative control.

Construction of an AatA expression-inducible plasmid, a �aatA mutant, anda single-copy complement of the �aatA mutant. The PBAD expression system wasused for cloning and arabinose-inducible expression of aatA. The coding se-quence of aatA (the translation start site in the forward primer is indicated belowby italics) was amplified by PCR using genomic DNA of APEC O1 as thetemplate. An Advantage 2 PCR kit was used in these experiments according tothe manufacturer’s directions (Clontech, Mountain View, CA). The primers usedwere forward primer aatAE-F (5�-GCCAGAGCTCAGGAGGAATTCATGAATAAGAATATACGAATTT-3�), which introduced a SacI site (underlined) and aribosome binding site (bold type), and reverse primer aatAE-R (5�-ATCGTCTAGACCCAGCTAACCATGCCTTAT-3�), which introduced an XbaI site (un-derlined). The complete aatA gene was cloned into the expression vectorpBAD18-cm using the SacI and XbaI sites created (19) to obtain pBAD aatA(Table 1). APEC O1 MaatA mutant strains harboring the empty plasmidpBAD18-cm and pBAD aatA were designated APEC O1 p1 and APEC O1 p2,respectively.

aatA was deleted using the method of Datsenko and Wanner (9). The chlor-amphenicol (Cm) resistance cassette in pKD3, flanked by 5� and 3� sequences of

aatA, was amplified from genomic DNA of strain APEC O1 using primersaatAM-F (5�-GTTGATAAAAATGCATCACTAAAGAAAAAACAGTATGAATGTGTAGGCTGGAGCTGCTTCGA-3�) and aatAM-R (5�-TAAACAATATATTGCGAAGAATGTTCATAATGTAAAGAGTCATATGAATATCCTCCTTAG-3�) and was introduced into APEC O1 by homologous recombinationusing � Red recombinase (the underlined portions of the primer sequences areidentical to the flanking regions of the aatA gene). Successful �aatA::Cm muta-tion was confirmed by PCR, using primers flanking the aatA region. The chlor-amphenicol resistance cassette was cured by transforming plasmid pCP20 andselecting for a chloramphenicol-sensitive mutant strain. The �aatA derivative ofAPEC O1 was designated APEC O1 MaatA. The �aatA mutant strain APEC O1MaatA was complemented by single-copy integration of plasmid pGP aatA. TheaatA operon, including its putative promoter, was amplified by PCR using prim-ers 5�-ATCGTCTAGACTCGCCACGGGAATATCTAC-3� and 5�-CTAGGTCGACCCCAGCTAACCATGCCTTAT-3�, and the sequence was confirmed.pGP aatA was constructed by cloning the XbaI-SalI fragment (the underlinedportions of the primer sequences are cut sites) containing the aatA operon intothe same sites of suicide vector pGP704 (37). A strain that was resistant toampicillin and was found to contain a full-length copy of the aatA gene, asconfirmed by PCR, was designated APEC O1 CaatA. APEC O1 CaatA was con-jugated from strain S17/pGP aatA to strain APEC O1 MaatA.

Protein localization and immunofluorescence microscopy. APEC O1, APECO1 MaatA, APEC O1 p1 (with arabinose), and APEC O1 p2 (with arabinose)cells were cultured until the optical density at 600 nm (OD600) was 1 andharvested. Bacterial fractionation was performed as previously described (16).Ten micrograms of protein was examined by electrophoresis on a 10% sodiumdodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gel using standardmethods (2). Immunodetection was performed following transfer to nitrocellu-lose membranes (Protran; Schleicher & Schuell) using a 1:1,000 dilution ofpolyclonal rabbit antiserum raised against the AatA protein. This antiserum wasgenerated by Open Biosytems (Huntsville, AL) using 19 amino acids (DNMISGGYGIKQGGDAISG) of the AatA passenger domain conjugated with keyholelimpet hemocyanin (KLH).

Immunofluorescence microscopy analysis was performed as follows. Bacteriawere grown at 37°C in LB in the presence of 0.2% arabinose until the OD600 was1. Cells were pelleted by centrifugation and fixed with 3% paraformaldehyde for10 min. Cells were saturated for 15 min with 0.5% bovine serum albumin (BSA)before incubation with a 1:1,000 dilution of the primary polyclonal rabbit anti-serum raised against AatA. Cells were next incubated with a 1:500 dilution of thesecondary polyclonal goat anti-rabbit serum coupled to Alexa 488. Cells wereloaded onto 0.1% poly-L-lysine-treated immunofluorescence microscope slides.The slides were fixed with 3% paraformaldehyde, and then Prolong reagentcontaining 10 mg ml�1 of 4�,6-diamidino-2-phenylindole (DAPI) (Invitrogen)

TABLE 1. Bacterial strains and plasmids used in this study

Strain(s) or plasmid Description Reference

StrainsAPEC O1 O1:K1:H7; fyuA sitA chuA irp2 iroN ireA tsh iucD fimC iss ompA vat traT; contains four

plasmids, including pAPEC-O1-ColBM26

APEC O1 MaatA APEC O1 derivative, �aatA This studyS17�pir recA thi pro hsdRM� RP4::2-Tc::Mu::Km Tn7 lysogenized with �pir phage 33S17pGP aatA S17�pir with plasmid pGP704 aatA This studyAPEC O1-CaatA APEC O1 MaatA with plasmid pGP aatA inserted into bacterial chromosome This studyAPEC O1 p1 APEC O1 MaatA with plasmid pBAD18-cm This studyAPEC O1 p2 APEC O1 MaatA with plasmid pBAD aatA This studyAPEC collection 452 APEC strains isolated from lesions of birds clinically diagnosed with colibacillosis 27AFEC collection 106 AFEC strains isolated from feces of apparently healthy birds 27UPEC collection 200 uropathogenic E. coli strains from MeritCare Medical Center in Fargo, ND 47NMEC collection 91 human neonatal meningitis-causing E. coli strains from the cerebrospinal fluid of

newborns in the Netherlands, isolated from 1989 through 199723

PlasmidspGP704 Apr, suicide plasmid 37pBAD18-cm Cmr, expression plasmid with arabinose-inducible promoter 19pKD46 Apr, expresses � Red recombinase 9, 10pKD3 cat gene, template plasmid 9pCP20 Cmr Apr, yeast Flp recombinase gene, FLP 9pGP aatA pGP704 derivative harboring aatA gene This studypBAD aatA pBAD18-cm derivative, aatA gene under the control of PBAD This study

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was added to fix coverslips to the slides. Finally, the slides were observed byepifluorescence microscopy using an Axiovert 200 inverted fluorescence micro-scope (Zeiss). Images were collected digitally using an AxioCam MR colorcamera and Axiovision AC imaging software (Zeiss). Images were prepared forpresentation using Photoshop and Illustrator software (Adobe Systems).

Enzyme-linked immunosorbent assays (ELISA). The positive sera used in thisstudy were obtained from 3-week-old chickens infected with live APEC O1 byintratracheal inoculation (1 � 107 CFU). These birds received a booster inocu-lation (1 � 107 CFU) of APEC O1 1 month later and were euthanized 2 weeksafter the booster was administered. Negative-control sera were obtained fromchickens sham inoculated with phosphate-buffered saline (PBS) (pH 7.2). Foreach group 10 samples were used to detect AatA antibody. Microtiter plates(Maxisorb; Nunc) were coated overnight at 4°C with 1 g of the synthesizedpolypeptide DNMISGGYGIKQGGDAISG of AatA. Bovine serum albumin(BSA) (Sigma) was used as a negative control. Wells were washed twice with TBS(150 mM NaCl, 20 mM Tris; pH 7.5) and then blocked with TBS-2% skim milkfor 1 h. After washing with TBS, chicken antisera were titrated horizontallyacross the plate starting with a 1:4 dilution, and the mixtures were incubated for2 h. After three washes, 100 l of secondary anti-rabbit horseradish peroxidaseantibody in blocking buffer (diluted 1:1,000) was added and incubated for 3 h atroom temperature. Finally, the chicken antibody against AatA was detected byadding 150 l of 1-Strep ABTS [2,2�-azinobis(3-ethylbenzthiazoline-6-sulfonicacid), diammonium salt; Pierce], and the absorbance at 405 nm was determinedwith a plate reader. A sample was considered positive if the A405 was twice thatof the negative control. The ELISA was repeated to determine the anti-AatAantibody titers of positive sera absorbed by whole-cell E. coli antigens made fromAPEC O1 MaatA.

CEF cell adhesion assay. The interaction of APEC O1, APEC O1 MaatA,APEC O1 p1, and APEC O1 p2 with cultured chicken embryo fibroblasts (CEF)was studied essentially as previously described (36). Briefly, CEF were cultureduntil they were confluent, and then the culture medium was removed and thecells were washed once with Eagle minimum essential medium (MEM) withoutfetal bovine serum (FBS). Bacteria were cultured until the OD600 was 0.3, andarabinose was added to a final concentration of 0.2%. Bacterial cells wereincubated for 1 h to induce AatA expression. Then 106 CFU of bacteria, asconfirmed by counting viable cells, was resuspended in MEM with 0.2% arabi-nose (to induce AatA expression) but without FBS, added to the monolayers ofchicken fibroblasts, and incubated for 1 h at 39°C in the presence of 5% CO2 (alonger incubation time resulted in lysis of APEC O1 p2 because of AatA over-expression). Monolayers were washed three times with PBS to remove non-adherent bacteria, and the eukaryotic cells were lysed with 0.1% Triton X-100.The numbers of adherent and internalized bacteria were determined by platecounting of the lysed cell suspensions. The percentage of adherent bacteria wasdetermined by dividing the number of adherent bacteria by the number ofbacteria inoculated. All experiments were performed in triplicate.

Virulence test. To determine the virulence of the bacteria of interest, 1-day-oldchicks were inoculated intratracheally with 0.1 ml of a bacterial suspensioncontaining 5 � 108 CFU ml�1 of APEC O1, APEC O1 MaatA, or APEC O1CaatA in accordance with an Institutional Animal Care and Use Committee-approved protocol. Birds used as negative controls received 0.1 ml of PBS by thesame route. The chicks were monitored for 7 days. Deaths were recorded, andthe survivors were euthanized and examined for macroscopic lesions. Lesionscores for the air sacs and combined lesion scores for the pericardium and liverwere determined as described by Lamarche et al. (29), except that lesions in thecaudal thoracic air sacs were scored from 0 to 3 using the following criteria: 0,normal and clear; 1, mild cloudiness and thickness; 2, moderate cloudiness andthickness accompanied by serous exudates or fibrin spots; and 3, extensive cloud-iness and thickness accompanied by muco-or fibrinopurulent exudates. Bacteriawere reisolated from livers, hearts, air sacs, and brains of the dead birds, andmultiplex PCR (27) was used to confirm that they were the inoculated strain.

Embryo lethality assay (ELA). The lethality of APEC O1, APEC O1 MaatA,and APEC O1 CaatA for chicken embryos was assessed by inoculating overnightwashed bacterial cultures (500 CFU) into the allantoic cavities of 15-day-oldembryonated specific-pathogen-free eggs. Twenty embryos were used for eachorganism tested. E. coli DH5� was used as the negative-control strain. PBS-inoculated and uninoculated embryos were also used as controls. Embryo deathswere recorded after 24 h, and the assay was performed twice.

Statistical analyses. Statistical analyses were performed with the GraphpadSoftware package (GraphPad Software, La Jolla, CA). A one-way analysis ofvariance (ANOVA) was used in the analysis of adherence data, an unpaired t testwas performed for the ELISA results, a Student t test was used to analyze thelesion scores, and a Fisher exact test was used to analyze the results of theembryo lethality assay. For the in vitro assays, mean values were obtained using

a minimum of three independent values. Statistical significance was establishedusing P values of �0.05.

RESULTS

Identification of a novel putative AT encoded by the PAI ofpAPEC-O1-ColBM. Analysis of the sequence of the APECvirulence plasmid pAPEC-O1-ColBM revealed a 116-kb PAI(25). The conserved region of this PAI harbors all knownimportant virulence genes of APEC plasmids, includingetsABC, which are genes encoding a putative ABC transportersystem; sitABCD, which are genes encoding another ABCtransporter system involved in iron and manganese transport;iucABCD and iutA, which are genes encoding the aerobactinsiderophore system; iss, the increased-serum-survival gene;iroBCDEN, which are genes of the salmochelin siderophoresystem; eitABCD, which are genes encoding a putative irontransporter system; and the temperature-sensitive hemaggluti-nin gene tsh. The APEC autotransporter (aatA) gene(APECO1_O1CoBM96) is between tsh and eitABCD and isannotated as a gene encoding a putative adhesin with similarityto the HMM PF03212 protein family. Both upstream anddownstream sequences flanking aatA include insertion se-quences (ISs) and transposases. These flanking mobile ele-ments and the lower G�C content of aatA (42%) than of theAPEC O1 genome (50.5%) and the ColBM plasmid (49.6%)suggest that aatA was acquired by horizontal gene transfer.This 3,498-bp gene encodes a hypothetical 1,166-amino-acidprotein with a theoretical molecular mass of 123.7 kDa. Usingthe SignalP 3.0 signal peptide prediction software (http://www.cbs.dtu.dk/services/SignalP/), the signal sequence was identi-fied as a sequence that is 25 amino acids long and has apotential cleavage site between residues A25 and Q26 (Fig. 1A).Amino acid sequence analysis of APECO1_O1CoBM96 usingthe protein motif search function of DNASTAR Lasegene 8showed that the C-terminal amino acids 883 to 1166 are pre-dicted to form the � domain of an AT. The C terminus showssimilarity to various outer membrane proteins and � domainsof ATs (Fig. 1A). Analysis of the probable membrane topologyof the protein revealed that hydrophobic amino acids accountfor more than 50% of this domain and that there are severaldifferent stretches of hydrophobic residues with intermittenthydrophilic residues, which together form the antiparallel �sheets and the connecting loops of the outer membrane �barrel of the translocator. The putative passenger (�) domainwas predicted to be from amino acid 26 to amino acid 882, andit shared no significant amino acid sequence identity with the �domain of any other characterized AT. A low level of aminoacid sequence similarity in the � domain was restricted to tworegions in an AidA-I adhesin-like protein from E. coliO157:H7 strain Sakai (20) (Fig. 1A). The 3D structure of AatAwas predicted using the I-TASSER online server (55–57). Thetranslocator domain has 12 antiparallel strands that form a �barrel with a hydrophilic core inside. As shown in Fig. 1B, thelength and diameter of the hydrophilic core are around 30 Åand 25 Å, respectively, as determined by measuring the dis-tances of the C-alpha atoms between Glu1095 and Met933 andbetween Ser1016 and Ala1119, respectively. If side chain atomswere considered, the diameters should be smaller. For thepassenger domain, the I-TASSER online server generated five

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potential models. We used the four-body contact potentialapproach, which was developed in 2007 to identify the likelynative structure of AatA from thousands of computer-gener-ated models (17). The energy scores for five models weredetermined to be 196.6, 197.9, 185.4, 103.3, and 253.4. Model4 was chosen as the best representation since it had the lowestenergy score, which made it the most stable conformation. The3D structure of the passenger domain is shown above thetranslocator domain in Fig. 1B. The parallel strands form an80-Å rod-like shape in the middle of the passenger domain,and the loops form the C-terminal head. The predicted struc-ture of the passenger domain has some similarity to the struc-ture of the heme binding protein (Hbp), which is the passenger

domain of an AT hemoglobin protease from pathogenic E. coli(40).

AatA is translocated to the cell surface. Our structural pre-dictions for AatA suggested that AatA might be a novel con-ventional AT. It is known that the passenger domain of ATs iseither secreted into the bacterial medium or displayed at thebacterial cell surface. To localize the passenger domain, anaatA isogenic mutant (APEC O1 MaatA) of APEC O1 wasgenerated using the method of Datsenko and Wanner (9). Thesecreted proteins of mutant strain APEC O1 MaatA and thewild type were compared by performing SDS-PAGE. No dif-ferences between the protein patterns of the two strains weredetected, nor were any differences found in the membraneproteins (data not shown). We reasoned that this failure todetect AatA might be due to a low level of expression of AatA.To circumvent this possible problem, we constructed an induc-ible expression plasmid containing aatA in which aatA is underthe control of an arabinose-inducible (pBAD) promoter. Inthis pBAD aatA plasmid, one ribosome binding site was addedupstream of the translation start site, while the structural gene(from the translation start ATG) of the aatA mutant was ex-actly the same as the wild-type structural gene (see Materialsand Methods). Thus, the AatA protein expressed from thisplasmid would be the same in terms of localization and func-tion. Protein analysis of the inducible expression mutant re-vealed a band at the expected molecular mass (120 kDa) in theouter membrane preparations of the APEC O1 p2 strain whichwas absent from the preparations of the negative-control strainAPEC O1 p1 when it was induced by arabinose (Fig. 2A). Toverify the induced protein band of APEC O1 p2, we raisedpolyclonal rabbit antiserum against a polypeptide fragment

FIG. 1. (A) Diagram showing the conserved domains in AatA. Thesignal peptide, passenger domain, and translocator domains are indi-cated; the conserved domains that are similar to protein familyPRKD9707 and known AT AidA-I domains are also indicated.(B) Predicted 3D structure of AatA. Coils, strands, and loops aregreen, yellow, and magenta, respectively,. The translocator and pas-senger domains are indicated. The hydrophilic core of the translocatordomain is approximately 30 Å long and has a diameter of approxi-mately 25 Å; the parallel strands form an 80-Å rod-like shape in themiddle of the passenger domain, and the loops form the C-terminalhead.

FIG. 2. Characterization of AatA. (A) SDS-PAGE analysis dem-onstrating the localization of AatA in APEC O1. Protein samples 2, 3,and 4 were obtained from the membrane protein preparation, andprotein samples 5 and 6 were obtained from the outer membranepreparation. Lane 1, protein ladder; lane 2, APEC O1 wild-type strain;lane 3, APEC O1 MaatA; lane 4, APEC O1 p2 induced by arabinose;lane 5, APEC O1 p1 induced by arabinose; lane 6, APEC O1 p2induced by arabinose. The arrow indicates the position of AatA.(B) Western blot analysis. Outer membrane protein preparations (lane1, induced APEC O1 p2; lane 2, induced APEC O1 p1) and membranepreparations (lane 3, induced APEC O1 p2; lane 4, APEC O1 MaatA;lane 5, wild-type strain APEC O1) were probed with anti-AatA serum.



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synthesized using the theoretical amino acid sequence ofAatA. This band reacted specifically with antibodies directedagainst the polypeptide (DNMISGGYGIKQGGDAISG) inthe passenger domain of AatA (Fig. 2B). To demonstrate thesurface localization of AatA, we performed immunofluores-cence microscopy. AatA antiserum reacted with intact APECO1 p2 cells expressing AatA. The surface of individual bacteriawas labeled in a mottled ring-like staining pattern, confirmingthat the N-terminal region of AatA was effectively translocatedto the cell surface (Fig. 3). In contrast, no evidence of AatAproduction was observed on the surfaces of APEC O1 andAPEC O1 MaatA cells (Fig. 3).

Prevalence of aatA. The widespread occurrence of vat inExPEC strains prompted us to investigate the prevalence ofaatA in a well-characterized collection of E. coli isolates ofavian and human origin. For this purpose, a pair of primers wasdesigned to amplify the �-domain region of aatA. An aatAfragment of the correct size was amplified from 182 of 452APEC strains (40.3%) and 52 of 106 avian fecal commensal E.coli (AFEC) strains (49.1%), but only 4 of 200 UPEC strains(2.0%) and 8 of 91 NMEC strains (8.8%) were positive. Thus,aatA is significantly more likely to be present in E. coli strainsfrom avian sources (P � 0.001). Further analysis revealed that69.6% (93/135) of phylogenetic group D E. coli strains in ourAPEC strain collection were aatA�, while only 24.1% of APECstrains belonging to phylogenetic group A, 31.5% of APECstrains belonging to phylogenetic group B1, and 32.5% ofAPEC strains belonging to phylogenetic group B2 were posi-tive for aatA. Furthermore, 51.6% of the PCR-positive APECstrains belonged to phylogenetic group D, while 22.0%, 12.6%,

and 13.8% of the PCR-positive APEC strains belonged togroups A, B1, and B2, respectively.

AatA elicited an antibody response in chickens. For AatA tocontribute to APEC virulence, it must be expressed in the host.We could not detect AatA in APEC O1 when it was culturedin vitro. In order to determine if it was expressed in vivo, wedeveloped an ELISA to measure anti-AatA generated inAPEC-infected chickens. The ELISA results showed thatAatA did elicit generation of an antibody in vivo with a titer ashigh as 27.4 on average, while the titers of the sera from controlchickens were significantly lower (22 on average) (Fig. 4). Torule out the possibility that the increase in the ELISA titer wasdue to greater production of an overall IgG response followinginfection with APEC O1 compared to the group that receivedonly PBS, we preabsorbed anti-AatA antiserum with whole-cell E. coli antigens made from the mutant strain (APEC O1MaatA) and performed the ELISA again. No significant differ-ence between preabsorbed and nonabsorbed sera was detected(Fig. 4). Thus, the increase in the ELISA titer was not causedby the background effect. These results provide indirect evi-dence that AatA is expressed in vivo, where it is available tocontribute to the pathogenesis of colibacillosis.

AatA expression enhances APEC O1 adherence to CEFcells. AatA showed some amino acid sequence similarity tocertain regions of AidA-I, an adhesin that contributes to au-toaggregation, biofilm formation, and adherence and that isassociated with virulence in many Gram-negative pathogens.Therefore, we investigated whether AatA could mediate auto-aggregation, biofilm formation, and cell adherence. No differ-ences in autoaggregation and biofilm formation between the

FIG. 3. Surface localization of the AatA passenger domain. Immunofluorescence assays with AatA from wild-type strain APEC O1, mutantstrain APEC O1 MaatA, and APEC O1 p2 were performed in the presence of 0.2% arabinose. Bacteria were fixed and incubated with anti-AatAserum, and this was followed by incubation with a secondary polyclonal goat anti-rabbit serum coupled to Alexa 488 (A, D, and H) or DAPI (B,E, and I). (C, F, and J) Merged images. (G) Magnification of the area in the box in panel F. Scale bars � 10 m.



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APEC O1 �aatA mutant and the wild type were detected, andinduction of aatA expression in APEC O1 p2 (following arabi-nose induction) did not lead to an increased capacity forautoaggregation or biofilm formation. To test whether AatAplays a role in adherence, CEF cell monolayers were infectedwith APEC O1, APEC O1 MaatA, the expression-induciblestrain APEC O1 P2, and the empty plasmid control strainAPEC O1 P1. Enumeration of bacteria adhering to cells didnot reveal significant differences between APEC O1 MaatA andthe wild-type APEC O1 strain in adherence to this cell line.However, APEC O1 p2, following induction of AatA, showed5-fold-greater efficiency in adherence to CEF cells than thewild type, the APEC O1 MaatA mutant, and the empty plasmidcontrol strain APEC O1 P1 (P � 0.005) (Fig. 5). We alsoincluded noninduction (without arabinose) and glucose repres-sion (with 0.2% glucose) controls for APEC O1 p2. No signif-icant difference in adherence capacity among the wild type, themutant strain, APEC O1 p1, and these noninduction and glu-cose repression controls was found (data not shown). Theseresults suggest that the production of AatA by APEC O1significantly enhances the capacity of this strain to adhere toCEF cells.

AatA is necessary for the full virulence of APEC O1. SinceaatA expression enhanced adherence to CEF cells, it was re-garded as a putative virulence gene. To test the contributionsof this gene to APEC virulence, the abilities of APEC O1,APEC O1 MaatA, and the complemented strain APEC O1CaatA to cause disease in chickens were compared. Groups of1-day-old birds were inoculated intratracheally with APEC O1,the isogenic mutant, or the complemented strain. All threestrains were able to invade and infect deeper tissues, to gen-erate gross lesions, and to cause a systemic infection and death.

However, compared with the aatA mutant, the wild-type strain,APEC O1, caused earlier death, a higher level of mortality,and more severe lesions (Table 2). Within 48 h after infection,6 of 12 chicks in the wild-type group had died, and four hadsevere lesions in the heart, liver, and air sacs (slight lesionswere observed in two chicks that died within 24 h after infec-tion). Bacteria could be reisolated from these organs and alsofrom the brains of all dead chickens. After 7 days of infection,all survivors were euthanized. Among the survivors, two chick-ens in the wild-type group were found to have severe lesions inthe heart, liver, and air sacs. On average, chickens in thewild-type group had lesion scores of 1.9 for the air sacs and 2.6for the liver and heart. In contrast, only 4 of 12 chickens in theaatA mutant group died during the 7-day period, and thechickens infected with the mutant had significantly (P � 0.05)lower lesion scores (1.33 for the air sacs and 1.83 for the liverand heart) than the chickens infected with the wild type. TheaatA-complemented derivative APEC O1 CaatA exhibited wild-type levels of virulence (Table 2). Despite some delay in theaverage time to death, the complemented strain caused mor-tality and gross lesions of air sacculitis and pericarditis-peri-hepatitis that were similar to those caused by the wild-typeparent.

Since no statistically significant differences in mortality werenoted in the chicken tests, we performed a chicken embryo

FIG. 4. Antibody titers for infected and noninfected chickens. Serafrom 10 chickens infected with APEC O1 (group 1) and from 10chickens inoculated with PBS (control) (group 2) and preabsorbedsera obtained using whole-cell E. coli antigens from the mutant strain(APEC O1 MaatA) (group 3) were used to detect antibody againstAatA with an ELISA. There were significant differences (P � 0.01)among the antibody titers for groups 1 and 2 and for groups 2 and 3,but no significant differences were detected for groups 1 and 3.

FIG. 5. Adherence assay. The capacities of wild-type strain APECO1, mutant strain APEC O1 MaatA, APEC O1 p1 induced by arabi-nose, and strain APEC O1 p2 induced by arabinose to adhere to CEFcells were compared. The bars indicate the averages of three indepen-dent experiments, and the error bars indicate the standard errors. Thedifferences in adherence capacity were significant (P � 0.05).

TABLE 2. Mortality and gross lesion scores for infected chickens

Strain% Mortality

(no. that died/no.tested)

Lesion scores (mean SEM)a

Air sacsb Liver and heartc

APEC O1 50 (6/12) 1.91 0.4 A 2.6 0.2 AAPEC O1 MaatA 33.3 (4/12) 1.33 0.3 AB 1.83 0.3 ABAPEC O1 CaatA 50 (6/12) 2.25 0.6 B 2.50 0.2 B

a Values followed by the same letter are significantly different (P � 0.05) fromeach other within a lesion group (i.e., air sacs or liver and heart).

b Mean lesion scores for air sacculitis in both caudal thoracic air sacs.c Combined lesion scores for pericarditis and perihepatitis.



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lethality assay (ELA). The results of the ELA showed that themortality rates for embryos with APEC O1 MaatA and wild-type strain APEC O1 were 20% (4/20) and 65% (13/20), re-spectively. No deaths occurred in the uninoculated group or inthe groups inoculated with the negative-control strain DH5�or PBS. The mortality rate for the complemented strain wassimilar to that for the wild-type parent (60%, 12/20). Thedifference in mortality between the wild type and the mutantwas significant (P � 0.05).

DISCUSSION

The vast majority of APEC strains harbor large virulenceplasmids that encode both known and unknown phenotypesthat contribute to virulence (27). In this study, we described anovel AT, AatA, encoded by the PAI of APEC O1’s virulenceplasmid, pAPEC-O1-ColBM, which mediates adherence tochicken fibroblasts and contributes to virulence. Gram-nega-tive bacteria have evolved several specialized secretion path-ways. The simplest and most widespread of these transportpathways is the AT or type V pathway. All ATs have the samegeneral structure and are comprised of three domains: anamino-terminal leader peptide; an � or passenger domain,which confers the function of a secreted protein; and a car-boxy-terminal domain (� domain) that mediates secretionthrough the outer membrane. AatA is predicted to have a �domain consisting of 284 amino acids, suggesting that it is aconventional AT, not a trimeric AT. The primary differencebetween it and a trimeric AT is that its C terminus, which isabout 70 amino acids long, is sufficient for translocating thepassenger domain across the outer membrane. In contrast, thetranslocator domains of conventional ATs consist of 300amino acids. For conventional ATs, like Pet (13), EspP (5),and Tsh (51), the passenger domain may be processed andreleased into the extracellular milieu, or it may remain incontact with the bacterial surface via a noncovalent interactionwith the � domain after cleavage (e.g., Ag43 [41] and AIDA-I[4]). In the case of AatA, neither autoproteolytic cleavage ofthe passenger domain nor cleavage by a membrane-boundprotease was observed. However, we demonstrated that theintact 120-kDa protein is present in the outer membrane frac-tion but not in cell supernatant (data not shown). It is unclearwhether cleavage requires special conditions, and how cleav-age of the passenger domain from the translocation unit occursis still not known (21).

Since its discovery in the late 1980s, the AT family has beenexpanded continuously (1, 31, 54). AatA is the third AT fromAPEC identified and the second AT which is encoded in thePAI of APEC O1’s virulence plasmid. Flanking aatA are twomobile genetic elements, IS2 and IS629, suggesting that thisgene was acquired horizontally. The gene encoding Tsh (tem-perature-sensitive hemagglutinin), the first serine protease ATof the Enterobacteriaceae (SPATE) described for APEC, is alsoin pAPEC-O1-ColBM’s PAI. This gene occurs in 50% to63.2%, of APEC strains but is rarely found in commensal E.coli strains (15, 45). tsh has also been found in UPEC strains,and its prevalence ranges from 4.0% to 4.5% (15, 45). Vat,another AT first described in APEC, is closely related to Tshand shares 78% identity at the amino acid level, and the genesencoding these proteins have regions where there are high

levels of nucleotide identity. Vat is also encoded by a genelocated in a PAI. The vat gene is widespread in APEC (39.8%)and human ExPEC strains, including UPEC (54.5%) andNMEC (50%) strains (15). Unlike vat, aatA has been associ-ated predominantly with avian E. coli strains, including APECstrains (40.3%) and AFEC strains (49.1%), but not with hu-man ExPEC strains, and it occurs in only 2% of UPEC strainsand 8.8% of NMEC strains. For this reason, we named theprotein avian E. coli AT (AatA). The host specificity of thisgene suggests that it may play a significant role in the patho-genesis of disease caused by APEC. Also, the similar pre-valence of aatA in APEC and AFEC should not be taken as anindication that aatA is not a virulence gene. Ewers et al. (14)recently showed that AFEC strains exhibiting characteristicstypical of APEC were capable of causing disease in immuno-competent chickens, meaning that the prevalence data cannotrule out the possibility that aatA is involved in APEC patho-genesis.

Several ATs encoded in genomic islands have previouslybeen shown to be phylogenetically distributed (45). vat wassignificantly linked with phylogenetic group B2, as nearly allstrains classified as group B2 strains, irrespective of their ori-gins, were vat positive. In contrast, the conjugative virulenceplasmid containing tsh has wider distribution among all strainsregardless of phylogenetic group. Interestingly, aatA, which isalso located on a plasmid, is significantly associated with iso-lates assigned to phylogenetic group D; 70% of APEC strainsbelonging to phylogenetic group D are aatA�, and more thanone-half of all strains that were aatA� belonged to phyloge-netic group D. Similar to the group B2 strains, E. coli strainsbelonging to phylogenetic group D are experimentally andepidemiologically associated with extraintestinal infections (24,43). A possible explanation for this phylogenetic position-spe-cific distribution is that the plasmid containing aatA is limitedlargely to certain groups of strains due to incompatibility issuesand selective pressure for retention of other plasmids (45).

Responding to changes in their circumstances, bacteriamodulate their patterns of gene expression by upregulatinggenes that are specifically required for survival. Thus, it is likelythat bacterial genes that are upregulated during infection orunder conditions simulating infection compared to routine cul-ture are candidate virulence genes. AatA could not be detectedwhen the wild-type APEC O1 strain was cultured in LB. How-ever, antibodies against AatA were detected in infected chick-ens, suggesting that AatA might be involved in the pathogen-esis of colibacillosis. Similarly, a UPEC-specific trimeric AT,UpaG, was shown to have limited expression in vitro (54). Also,unpublished microarray data collected in our lab demonstratedthat aatA was upregulated more than 10-fold when the organ-ism was cultured in chicken serum, suggesting that aatA is an invivo-induced gene (34, 50). Further sequence analysis of aatArevealed that the intergenic region between aatA and its up-stream gene is very A-T rich, with several putative H-NS nu-cleation sites containing six or more matches with the 10-bpH-NS consensus sequence (data not shown) (30). These fea-tures coincide with features reminiscent of H-NS-repressedloci (10). The H-NS protein is an abundant global repressorthat controls genes related to pathogenicity and stress re-sponses. The gene encoding it is acquired by horizontal genetransfer and is found in PAIs (18, 22, 32, 35, 39, 52). H-NS



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silence may be relieved in vivo by a mechanism that is not yetclear (38). Thus, the possibility that a failure to detect AatAexpression in vitro might be due to H-NS protein silencingwarrants further study.

Unlike trimeric ATs, known conventional ATs function ascytotoxins, enterotoxins, immunoglobin proteases, mucinases,heme-binding proteins, and adhesins in E. coli and otherGram-negative bacteria (3, 6, 7, 46, 49, 54). AatA shares lowamino acid sequence similarity in the � domain with two re-gions identified in an AidA-I adhesin-like protein from E. coliO157:H7 that contributes to a diffuse adhesion phenotype (4).Like AidA-I, AatA acts as an adhesin. Induction of expressionof AatA was shown to enhance adhesion of APEC to CEFcells, although no difference in adherence between the wildtype and the aatA deletion mutant was found. The lack of anadherence phenotype in the wild type may be simply due tosilencing of expression of AatA in vitro, which is consistent withour SDS-PAGE and Western blot results. Tsh, a conventionalAT, contributes to the early stages of infection, including col-onization of air sacs (11), and EspP, an AT of E. coli O157:H7,has been shown to influence intestinal colonization of calves(12). Unlike AIDA-I, AatA did not function in autoaggrega-tion or biofilm formation. The functional differences may dueto the structural difference in the passenger domain of theseATs, since in silico analyses and empirical data from circulardichroism spectra suggest that the passenger domain ofAIDA-I has a significant amount of �-strand structure (28).

Comparison of the pathogenicities of the aatA mutant strain,the wild-type strain, and the complemented strain revealedsignificant differences in organ lesions. The wild-type and com-plemented strains caused earlier and more deaths and moresevere lesions in internal organs than the aatA mutant. Also,the results of the ELA showed that there were significantdifferences in mortality between the wild type and the mutantgroups. These results, together with the results of the adher-ence assay, suggest that AatA may make a significant contri-bution to APEC virulence through bacterial adherence to hosttissues. The fact that the aatA deletion mutant still causeddeath and lesions suggests that multiple factors contribute toadherence. Thus, deletion of only one adhesin does not appearto totally abolish the virulence of APEC O1. In our preliminarytests (data not shown) subcutaneous inoculation (1 � 106

CFU/chicken) of 1-day-old chickens was used to assess viru-lence. In this case, major differences in mortality and lesionscores between strains were not found (data not shown), indi-cating that this route of infection could not be used to detectdifferences in the capacities of strains to colonize the tracheaand lung, probably because the bacteria were able to bypassthe respiratory tract completely (53). Therefore, the differencebetween the results obtained with these two models also indi-cates that AatA may play a role in the early stages of infection,such as colonization of the trachea and lung.

ACKNOWLEDGMENTS

This work was supported by the Iowa Livestock Health AdvisoryCouncil (ILHAC) and the USDA NRICGP Microbial FunctionalGenomics Program (grant 20083560418805).

We thank Paul M. Mangiamele for technical assistance and KathyMou, Ashraf Hussein, and Musafiri Karama for their indispensablehelp with the animal tests.

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