APPLIED MICROBIAL AND CELL PHYSIOLOGY

The effects of upaB deletion and the double/triple deletionof upaB, aatA, and aatB genes on pathogenicity of avianpathogenic Escherichia coli

Xiang-kai Zhu-Ge1 & Zi-hao Pan1& Fang Tang1 & Xiang Mao2 & Lin Hu1

&

Shao-hui Wang2 & Bin Xu1& Cheng-ping Lu1

& Hong-jie Fan1& Jian-jun Dai1

Received: 26 May 2015 /Revised: 30 July 2015 /Accepted: 5 August 2015 /Published online: 18 August 2015# Springer-Verlag Berlin Heidelberg 2015

Abstract Autotransporters (ATs) are associated with patho-genesis of Avian Pathogenic Escherichia coli (APEC). Themolecular characterization of APEC ATs can provide insightsabout their relevance to APEC pathogenesis. Here, we char-acterized a conventional autotransporter UpaB in APECDE205B genome. The upaB existed in 41.9 % of 236 APECisolates and was predominantly associatedwith ECORB2 andD. Our studies showed that UpaB mediates the DE205B ad-hesion in DF-1 cells, and enhances autoaggregation and bio-film formation of fimbria-negative E. coli AAEC189(MG1655Δfim) in vitro. Deletion of upaB of DE205B atten-uates the virulence in duckmodel and early colonization in theduck lungs during APEC systemic infection. Furthermore,double and triple deletion of upaB, aatA, and aatB genes cu-

mulatively attenuated DE205B adhesion in DF-1 cells, ac-companying with decreased 50 % lethal dose (LD50) in duckmodel and the early colonization in the duck lungs. However,DE205BΔupaB/ΔaatA/ΔaatB might Bcompensate^ the in-fluence of gene deletion by upregulating the expression offimbrial adhesin genes yqiL, yadN, and vacuolatingautotransporter vat during early colonization of APEC.Finally, we demonstrated that vaccination with recombinantUpaB, AatA, and AatB proteins conferred protection againstcolisepticemia caused by DE205B infection in duck model.

Keywords APEC . Autotransporter UPAB . Pathogenicity .

Double/triple deletion . Vaccination

Electronic supplementary material The online version of this article(doi:10.1007/s00253-015-6925-2) contains supplementary material,which is available to authorized users.

* Jian-jun Daidaijianjun@njau.edu.cn

Xiang-kai Zhu-Gezgxiangkai@163.com

Zi-hao Pan2013207013@njau.edu.cn

Fang Tang850421558@qq.com

Xiang Maoxmao@njau.edu.cn

Lin Hu2013807115@njau.edu.cn

Shao-hui Wang1196820085@qq.com

Appl Microbiol Biotechnol (2015) 99:10639–10654DOI 10.1007/s00253-015-6925-2

Bin Xu1091517919@qq.com

Cheng-ping Lulucp@njau.edu.cn

Hong-jie Fanfhj@njau.edu.cn

1 Key Lab of Animal Bacteriology, Ministry of Agriculture, NanjingAgricultural, University, No.1 Weigang road, Nanjing, JiangsuProvince 210095, China

2 Shanghai Veterinary Research Institute, Chinese Academy ofAgricultural Sciences, Shanghai 200241, China

Introduction

Avian pathogenic Escherichia coli (APEC) infection is dev-astating to poultry industries worldwide, resulting in signifi-cant welfare investment and economic losses (Ewers et al.2003). Due to diverse serotypes and ill-defined molecularmechanisms of APEC pathogenesis, there is no effective vac-cine to prevent and combat APEC infection in the poultryindustry. After inhalation of contaminated dust/aerosols,APEC infects avian respiratory tract during its early coloniza-tion, which associates with inflammation and histopathologi-cal changes in lung (airsacculitis and pneumonia) (Dho-Moulin and Fairbrother 1999; Ewers et al. 2003; Horn et al.2012; Mushtaq et al. 2004; Tivendale et al. 2010). APEC canspread in the bloodstream and various internal organs, whichcan cause systemic infection, including pericarditis,perihepatitis, peritonitis, salpingitis, and sepsis (Dho-Moulinand Fairbrother 1999; Ewers et al. 2003; Horn et al. 2012;Mushtaq et al. 2004; Tivendale et al. 2010).

Adhesion of pathogenic bacteria to host cell surface is themost critical process for the successful colonization and infec-tion (Antao et al. 2009b). After inhalation into the respiratorytract, APEC requires adhesins to colonize the lungs, leading tolocal infection at air sacs and the lung. The typical fimbriaeadhesins are well-known factors to promote the colonizationof Gram-negative pathogens during the infection process(Wright and Hultgren 2006). The best-characterized Type Ifimbrial adhesin, Pap fimbrial adhesin and extraintestinalpathogenic E. coli (ExPEC) Adhesin I are involved in theearly phase of APEC infection (Antao et al. 2009a; Antaoet al. 2009b; de Pace et al. 2010; Kariyawasam and Nolan2009). In addition to fimbrial adhesins, autotransporter (AT)adhesins are also involved in APEC adhesion to host cells andcolonization in the avian lung and other extraintestinal niches.Temperature-sensitive hemagglutinin (Tsh) is the first AT pro-tein identified in APEC and mediates APEC adhesion to thelung of chickens (Stathopoulos et al. 1999). Two novel APECATs (AatA and AatB) are also reported to be involved inAPEC early colonization to host lung tissues (Dai et al.2010; Li et al. 2010; Zhuge et al. 2013).

ATs contribute to pathogen interaction with the host celland are associated with adhesion, cytotoxicity, and aggrega-tion activities (Ackermann et al. 2008; Guyer et al. 2000;Henderson and Nataro 2001; Henderson et al. 2004; Li et al.2010; Zhuge et al. 2013). ATs are modular proteins secretedthrough the type V transport pathway and unique in the struc-tural feature (Henderson and Nataro 2001; Yen andStathopoulos 2007). AT proteins possess three conserved do-mains: a signal sequence at N-terminal to initiate transportthrough the inner membrane, a passenger or alpha (α) domainwith diverse functions, and a C-terminal β-translocator do-main which forms a β-barrel embedded in the outer mem-brane to transport the passenger domain to the bacterial

surface (Ackermann et al. 2008; Henderson et al. 2004). TheAT can be divided into conventional AT and the trimeric AT(TAAs) based on their translocator domains. Conventional ATis a monomeric protein and its translocator domain is com-posed of 12 transmembrane β-strands with 250 to 300 aminoacid (aa) residues at C-terminal, while the translocator domainof TAA contains 60–70 aa and forms the trimer in the outermembrane (Ackermann et al. 2008; Henderson et al. 2004).

Recently, many ATs are identified in the genome of theuropathogenic E. coli (UPEC) strain CFT073, and their viru-lence roles in UPEC have been successively unraveled(Allsopp et al. 2012a; Allsopp et al. 2012b; Parham et al.2004). The UpaB in the UPEC strain CFT073 is involved inits early colonization in the bladder and can bind several ex-tracellular matrix (ECM) proteins in vitro (Allsopp et al.2012b). The roles of AT proteins in APEC pathogenesis isyet to be further elucidated. It has been shown that the upaBgene was also present in the APEC DE205B genome. In thisstudy, we performed bioinformatics analysis on this UpaB,including its phylogenetic relationship with other ATs andthe distribution of upaB among APEC isolates. Furthermore,the roles of UpaB in APEC pathogenesis were unraveled in aDF-1 cell and duck infection model. We further evaluated theeffects of upaB, aatA, and aatB deletion on the pathogenicityof DE205B. Finally, we found that vaccination with recombi-nant UpaB, AatA, or AatB protein conferred protectionagainst colisepticemia caused by DE205B infection in a duckmodel, which provides valuable information for vaccinedesign.

Materials and methods

Bacterial strains, plasmids, and growth conditions

The strains and plasmids used in the studies were listed inTable 1. The APEC strain DE205B (CVCC3991; O2:K1;ST complex 95, ST140; ECOR B2; isolated from a duck)was used as the model strain; The APEC strain collection(n=236) was used to study the distribution of the upaB gene(Wang et al. 2011a; Wang et al. 2011b; Zhuge et al. 2013).E. coli strains DH5α and BL21 (DE3) were used for genecloning and protein expression, respectively. Cells were rou-tinely grown at 37 °C on solid or in liquid Luria Bertani (LB)medium supplemented with the appropriate antibiotics: kana-mycin (Kan) (50 μg/ml), ampicillin (Amp) (100 μg/ml), orchloramphenicol (Cam) (34 μg/ml).

DNA manipulations and protein sequence analysis

The primers used in DNA manipulation were listed inTable S1 (see Table S1 in the supplemental material); the

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UpaB1-F and UpaB1-R were used in upaB gene PCRdetection.

Transcription promoter prediction of the upaB gene wasperformed using two online prediction tools at (http://linux1.sof tberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) and (http://www.fruitfly.org/seq_tools/promoter.html). The protein primary structureanalysis and the potential homology domain prediction werecarried out by BLASTP on the NCBI website, SignalP 4.1Sever, HHpred program, and online MOTIF Search.ClustalW (MEGA 4.0) software was used to explore thephylogenetic relationship of numerous ATs.

Overexpression and purification of UpaB, preparationof antiserum, and immunoblotting

The upaB ORF (79 to 2295 bp) without the signal peptide (1to 78 bp) and the termination codon was amplified with

primers UpaB2-F and UpaB2-R. The PCR product digestedwith SalI/XhoI was ligated into pET28a(+) vector digestedwith the same enzymes (Novagen, Madison, WI). ThepET28a-UpaB was transformed into E. coli BL21 (DE3),and the expression of UpaB protein was carried out at 22 °Cand induced with IPTG at a final concentration of 1 mM. Thefusion protein was purified using a HisTrap high-performancecolumn (GE Healthcare, Shanghai, China) (Zhuge et al. 2013).

To prepare the rabbit anti-UpaB serum, the New Zealandwhite rabbit was immunized with the UpaB fusion proteinsubcutaneously as described previously (Dai et al. 2010;Wang et al. 2011a; Wang et al. 2011b; Zhuge et al. 2013).The same volume of purified UpaB protein (400 μg) wasmixed with Montanide ISA 206 adjuvant (SEPPIC, Lyon,France) to immunize the rabbit. The anti-UpaB serum wascollected after the third immunization. In addition, the hyper-immune duck anti-DE205B serum was obtained from a pre-vious study (Zhuge et al. 2013).

Table 1 Bacterial strains and plasmids used in this study

Strain or plasmid Characteristics References

Strain

DE205B O2:K1; ST complex 95, ST140; phylogroup B2 (Wang et al. 2011a; Wang et al. 2011b;Zhuge et al. 2013)

DE205BΔupaB upaB deletion mutant in DE205B This study

DE205BCΔupaB DE205BΔupaB with plasmid pSTV28-upaB This study

DE205BPΔupaB DE205B9 with control plasmid pSTV28 This study

AAEC189 MG1655Δfim (Blomfield et al. 1991)

AAEC189PS AAEC189 with control plasmid pSTV28 This study

AAEC189PS-upaB AAEC189 with plasmid pSTV28-upaB This study

DE205BΔaatA aatA deletion mutant in DE205B (Wang et al. 2011b)

DE205BΔaatB aatB deletion mutant in DE205B (Zhuge et al. 2013)

DE205BΔupaB/aatA Parallel deletion of upaB and aatA genes in DE205B This study

DE205BΔupaB/aatB Parallel deletion of upaB and aatB genes in DE205B This study

DE205BΔaatA/aatB Parallel deletion of aatA and aatB genes in DE205B This study

DE205BΔupaB/aatA/aatB Triple deletion of aatA, aatB, and upaB genes in DE205B This study

DH5α F-, Δ(lacZYA-argF)U169, recA1, endA1, hsdR17(rk−, mk+),phoA, supE44, λ-

TIANGEN

BL21 (DE3) F-, ompT, hsdS (rB- mB

-) gal, dcm (DE3) TIANGEN

Plasmid

pET28a (+) Kan, F1 origin, His tag Novagen

pET28a-upaB pET28a (+) carrying upaB gene This study

pET28a-aatA pET28a (+) carrying aatA gene (Wang et al. 2011b)

pET28a-aatB pET28a (+) carrying aatB gene (Zhuge et al. 2013)

pMD® 18-T Vector Amp, lacZ Takara

pSTV28 Cm, lacZ Takara

pSTV28-upaB pSTV28 derivative harboring upaB ORF and its putative promoter This study

pKD46 Amp; expresses λ red recombinase (Datsenko and Wanner 2000)

pKD4 kan gene, template plasmid (Datsenko and Wanner 2000)

pCP20 Cm, Amp, yeast Flp recombinase gene, FLP (Datsenko and Wanner 2000)

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For immunoblot assay, protein samples were subjected to12 % SDS-PAGE and transferred to a polyvinylidenedifluoride (PVDF) membrane (Amersham PharmaciaBiotech, Uppsala, Sweden) with a semidry blotting apparatus(TE77; Amersham Pharmacia Biotech). The anti-UpaB andanti-DE205B serum were used as the primary antibodies,and horseradish peroxidase (HRP)-conjugated anti-rabbitIgG or anti-duck IgG as the secondary antibody. The en-hanced chemiluminescence (Amersham Pharmacia Biotech,Piscataway, NJ) was used for immunoblot detection.

Construction of a ΔupaB mutant, and complementationof the ΔupaB mutant and fimbria-negative E. coli strainAAEC189

The gene was deleted based on the method described previ-ously (Datsenko and Wanner 2000). The primers UpaB3-Fand UpaB3-R were used to amplify a kanamycin resistancecassette from plasmid pKD4 (Wang et al. 2011b; Zhuge et al.2013) (Table S1). The finalΔupaB mutant was designated asDE205BΔupaB. For complementation experiments, pSTV28(pACYC184 origin, TaKaRa) was used as the complementedcarrier for upaB operon (Maseda et al. 2011; Yamanaka et al.2008; Yoshimura et al. 2007). The upaB operon (containingits putative promoters) amplified by the primers UpaB4-F andUpaB4-R was subcloned into pSTV28. The complementedstrains were designated as DE205BCΔupaB andAAEC189PS-upaB (Table 1).

Construction of the mutants for parallel and tripledeletion of aatA, aatB, and upaB genes in APEC strainDE205B

The double and triple deletion of upaB, aatA, and aatB wereconstructed using DE205BΔaatA and DE205BΔaatB(Datsenko and Wanner 2000; Wang et al. 2011b; Zhugeet al. 2013). The primers (WSH50F and WSH51R for aatA;ZGXK3F and ZGXK4R for aatB) were used to amplify aresistance cassette (Wang et al. 2011b; Zhuge et al. 2013)(Table S1). The double and triple deletion mutants of aatA,aatB, and upaB genes were shown in Table 1.

Bacterial fractionation and components separation,and immunodetection

Bacterial fractionation was performed with the method previ-ously described (Jiang et al. 2014; Vigil et al. 2012). Briefly,100-ml bacteria culture was harvested by centrifugation,washed twice with PBS, and resuspended in 5 ml PBS. Thecells were lysed by sonication (Sinosource UltrasonicProcessor, Guangzhou) and centrifuged (8000×g) to removethe cell debris. The lysate was then centrifuged at 300,000×gfor 60 min at 4 °C; the precipitation (containing the cell

membrane) and the supernatant (containing the soluble peri-plasmic and cytoplasmic proteins) were collected. Underroom temperature, the precipitation was washed with PBStwice, and then incubated with 1 % Triton X-100 for 30 min(Vigil et al. 2012). The membrane fraction was centrifuged at150,000×g for 45 min. The supernatant (containing the innermembrane proteins) and the pellet (the outer membrane pro-teins) were collected and stored at −70 °C. The proteins insupernatant were concentrated by Millipore centrifugal filterunit (10 kDa cut-off). The proteins in the supernatant wereconcentrated by ammonium sulfate precipitation and dialyzedtwice in PBS at 4 °C. The presence of UpaB in the culturesupernatant, outer membrane proteins, inner membrane pro-teins, and cytoplasmic/periplasmic were analyzed by Westernblot.

Immunofluorescent imaging for UpaB surface localization

Immunofluorescence analysis for UpaB localization was per-formed as previously described (Li et al. 2010). Bacteria cellswere harvested by centrifugation and incubated with 3% para-formaldehyde for 10 min, and then blocked in 0.5 % BSA for15 min. The bacteria were treated with 1:500 diluted anti-UpaB serum at 37 °C for 2 h, and then washed three timeswith PBS. The bacteria were incubated with goat anti-rabbitIgG-TRITC (1:500 dilution; EarthOx) for 1 h at 37 °C, andwashed with PBS three times. The bacteria were then incubat-ed with DAPI (10 mg/ml, Invitrogen) for 15 min. Bacterialsuspension (10 μL) was added onto immunofluorescence mi-croscope slide and treated with 3 % paraformaldehyde. Thefluorescence was observed by a Zeiss LSM-510 META con-focal laser scanning microscope.

DF-1 cells adhesion and invasion assays

To measure the adhesion ability of the wild-type strain, genedeletion strains, or complemented strain, the monolayer cellswere infected with bacteria at a multiplicity of infection of 100for 3 h (Li et al. 2010; Zhuge et al. 2013). The cells werewashed four times with PBS, and treated with 0.1 % TritonX-100 to lyse the cells. The total number of adherent bacteriawas determined through plate counting of the serially dilutedcell suspension. For the number of invasive bacteria (cell in-vasion), the monolayer cell was infected with the bacteria for3 h, and then incubated with DMEM containing gentamicin(100 μg/mL) for 1 h to kill the adherent bacteria (Wang et al.2011a). The cells were washed three times by PBS and lysedwith 0.1 % Triton X-100. The number of invasive bacteria wasmeasured by plate counting of the serially diluted cell suspen-sion. DF-1 cells only incubated with DMEM were used asnegative control in all assays. These experiments were carriedout in triplicate.

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The adhesion inhibition experiment was carried out (Wanget al. 2011b; Zhuge et al. 2013). Briefly, the bacteria wereincubated with UpaB antiserum before infecting DF-1 mono-layer cell. The adhesion assays were preformed as describedabove. The experiments were carried out in triplicate.

Animal experiments

To determine the virulence of wild-type, deletion, andcomplemented strains in the animal, the 7-day-old HBK-Q-SPF ducks or 8-week-old ICR mice were used (Zhuge et al.2013). The virulence difference among these strains was eval-uated by using 50 % lethal dose (LD50). For duck model ofcolisepticemia, each of these bacteria was diluted 10 times byPBS, and the 0.2 ml bacterial suspension or PBS (the negativecontrol) was inoculated intratracheally (n=10 per group). Themortality was calculated at the 7th day after postinfection, andthe LD50 for strains were calculated by the Spearman-Karbermethod (Fung andWong 1989). The LD50 of the mouse sepsismodel for each strain was also measured through intraperito-neal infection model (Zhu Ge et al. 2014). All mortality ex-periments were preformed three times.

The duck model was also used to evaluate the colonizationcapabilities for each bacterium during systemic infections.The 7-day-old duck was infected with bacteria at 1.0×108 CFU. The ducks was euthanized and dissected at 24 hpostinfection. The number of bacteria colonizing in the lungsand brains were determined as follows: organ samples wereobtained from infected ducks. The samples were weighed,suspended in PBS (1 ml/g), and homogenized. The numberof bacteria in the homogenized organs was measured by platecounting of the serially diluted cell suspension (Wang et al.2011a; Wang et al. 2011b; Zhuge et al. 2013).

Autoaggregation assay

The autoaggregation assay was performed according to theprevious study (Alamuri et al. 2010; Wang et al. 2011b).Briefly, bacteria were grown to logarithmic phase (finalOD600nm=1.5) and then incubated on ice. The assays werecarried out by testing OD600nm of the upper part of the culturesat regular time intervals. The wild-type, mutant strains, andAAEC189/MG1655 variant strains were tested for theautoaggregation capacity. The experiments were carried outthree times. Data are expressed as means of results for threeindependent tests, and statistical significance analysis was per-formed using Student’s t test.

Biofilm formation assay

The 96-well plate was used to detect biofilm formation foreach derivative strain (Wang et al. 2011a; Zhuge et al.2013). LB medium containing 5 g liter−1 of glucose was used

to dilute the cultured bacteria. Each well of the plate was filledwith 200-μl diluted bacteria, and the DMEM alone was usedas negative control. The plates were cultured at 37 °C for 24 h.The medium in each well was discarded and washed withPBS. The cells were then stained with 200 μl of 1 % (wt/vol) crystal violet (Sigma) for 1 h. The wells were washedthree times by PBS and dried for 1 h; 200 μl ethanol andacetone solution (80:20) were used to dissolve the bound cells,and the absorbance of the solution in each well was deter-mined by an enzyme-linked immunosorbent assay reader(Bio-Radmicroplate reader, model 550) at 595 nm. The exper-iments were carried out three times.

qRT-PCR

The quantitative real-time reverse transcription-PCR (qRT-PCR) was used to explore the difference of upaB mRNAlevels in vitro and in vivo. RNA was isolated from culturedbacteria using an E.Z.N.A. bacterial RNAkit (Omega Bio-Tek, Beijing, China). The bacterial RNA was obtained fromthe tissue and blood of the infected duck according to thefollowing procedure. Ducks were euthanized and dissectedafter 24 h postinfection. In order to isolate bacterial RNA ininfected lung tissues, the tissues were homogenized in the ice-cold solution containing 0.2 M sucrose and 0.01 % SDS. Thehomogenate was centrifuged for 20 min at 250 rpm, and thesupernatant was filtered by a 5-μm filter to remove tissuefragment. The suspension was incubated with lysate buffer(1 % Triton X-100 and NP-40) for 10 min and centrifugedfor 10 min at 5000 rpm. The precipitation was then washedthree times with 1 % Triton X-100. For gene expression ofbacteria infected with DF-1 cells in vitro, the infected DF-1cells were treated with lysate buffer for 3 min, and centrifugedfor 5 min at 5000 rpm. The precipitate was washed three timesby ice-cold lysate buffer. The total RNAwas isolated, and thenprocessed with MICROBEnrichTM Kit (Ambion; catalog no.AM1901) to remove the host RNA (Kansal et al. 2013).Following the procedure of depletion of the bacterial rRNA,t h e r em a i n d e r t o t a l RNA w a s t r e a t e d w i t hMICROBExpressTM (Ambion; catalog no. AM1905)(Kansal et al. 2013).

The primers for qRT-PCR were listed in Table S1. TheΔΔCT method and expression of the housekeeping genednaE was used to determine the upaB expression level(Livak and Schmittgen 2001). The dnaE transcription levelis consistent during APEC infection, and the positive genewas defined if it is highly expressed than dnaE, otherwisenegative gene (Wang et al. 2014; Zhuge et al. 2013). To fur-ther gain insights into the effect of aatA, aatB, and upaB genesdeletion on expression profile of virulence genes, qRT-PCRwas used to measure the expression of selected virulencegenes (Table S1) between wild-type and mutant strains

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in vitro and in vivo. The experiments were carried out threetimes.

Challenge studies for vaccination against recombinantUpaB, AatA, and AatB proteins

The immunization and challenge assay was performed (Baoet al. 2013). Briefly, 7-day-old ducks were divided into fivegroups, and subcutaneously immunized with purified 200 μgUpaB protein, AatA protein (Wang et al. 2011b), AatB protein(Zhuge et al. 2013), the combination of these three proteins(100 μg for each protein), or PBS (negative control) mixedwith same volume of ISA 206 adjuvant. The same immuniza-tion process was carried out again 10 days later. Aweek afterthe second immunization, the blood was obtained randomlyfrom the ducks in each group from toe venous. The serumsobtained from the ducks were stored at −20 °C.

The serum IgG antibodies against UpaB, AatA, and AatBwere measured using indirect ELISA (Wang et al. 2014).Briefly, the plates were overnight coated at 4 °C with purifiedrecombinant proteins (0.5 μg/well), respectively. The antise-rums were added to ELISA plate wells for 2 h. The boundimmunoglobulin was tested using horseradish peroxidase(HRP)-conjugated anti-duck IgG (KPL, Gaithersburg, MD,USA) (1:4000). The accurate titers were defined as the lastwell producing a 2.1-fold ratio value above negative serum.After the success of vaccination against recombinant proteinswas confirmed with ELISA (10 days after the second immu-nization), 20 ducks per each group were challengedintratracheally with wild-type DE205B (5.0×108 CFU). Thewild-type DE205B colonization capabilities in duck lungs infour vaccination groups and the control group were also eval-uated at 24 h after challenged intratracheally (5.0×108 CFU).

Statistical analyses

Statistical analyses were performed by GraphPad softwarepackage (GraphPad Software, La Jolla, CA). One-way analy-sis of variance (ANOVA) was used for analysis of the adhe-sion assay and biofilm formation assay data in vitro, and two-way ANOVA was performed on the qRT-PCR results. Theanimal infection study analysis was performed using the non-parametric Mann-WhitneyU test. The mean values are shownin the figures. Statistical significance was established at a Pvalue of <0.05.

Nucleotide sequence accession number

The sequence of the AT upaB gene in the DE205B strain wassubmitted to GenBank, and the accession number isKP729058.

Results

The molecular characterization of an autotransporterUpaB from APEC strain DE205B

The upaB gene, first identified in the UPEC strain CFT073(Allsopp et al. 2012a; Allsopp et al. 2012b; Parham et al.2004), was also present in the DE205B genome (c0426).The predicted upaB ORF in the DE205B genome was2298 bp in length. Analysis on 250 bp upstream of upaB startcodon suggested that a putative transcription promoter waslocated at −40 bp (P=0.79).

Bioinformatic analysis of the UpaB sequence showed thatUpaB contained four domains: a signal peptide (amino acid 1to amino acid 26), the passenger domain (amino acid 27 toamino acid 450), the α-helical linker domain (amino acid 451to amino acid 450), and theβ-barrel translocator domain (ami-no acid 496 to amino acid 765) (Fig. 1). The C-terminustranslocator domain shared similarities with β-domains ofvarious autotransporters as suggested by BLASTP andHHpred programs. Based on protein homology predictionby HHpred program (database: pfamA 27.0), the passengerdomain shared a low level of sequence similarities with threeprotein structures: Peptidase S6 IgA endopeptidase (Otto et al.1998; Pohlner et al. 1987); Pertactin-like passenger domain(Emsley et al. 1996); and DUF4353 (domain of unknownfunction: pfamA).

The phylogenetic analysis of UpaB with the other 68 ATsshowed that it was clustered within the conventional ATgroup. This AT group also contains TibA (adhesin/invasin ofenterotoxigenic E. coli), Pertactin precursor (adhesin ofBordetella pertussis), and the other 9 ATs (Fig. S1) (Mooiet al. 1998; Sherlock et al. 2005). The amino acid tripletarginine-glycine-aspartic acid (RGD) motif is commonly in-volved in adherence to host cells (Mooi et al. 1998; Sherlocket al. 2005). Nine of these 11 ATs contain RGD cell attach-ment sequence in passenger domain, except for UpaB andTibA (Mooi et al. 1998; Sherlock et al. 2005). Even sharingclose relationships in phylogenetic analysis, UpaB shared lowidentities with TibA and Pertactin precursor, approximately 31and 29 %, respectively.

Prevalence of the upaB gene among APEC isolates

The prevalence of the upaB gene among the 236 APECstrains was determined via PCR, which showed 41.9 % ofthe 236 strains had upaB gene. The phylogenetic relation-ship of the APEC isolates containing the upaB gene wasdetermined: group A, 20/96 (20.8 %); group B1, 18/51(35.3 %); group B2, 30/40 (75.0 %); and group D, 31/49 (63.3 %). These results suggested that the upaB genewas widely distributed among APEC isolates and predom-inantly associated with group ECOR B2 and D.

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Furthermore, all APEC predominantly serotypes (O1:K1and O2:K1) strains (total 16 strains in group B2) had aupaB gene.

UpaB enhanced autoaggregation and biofilm formationof fimbria-negative E. coli AAEC189 (MG1655Δfim)

The previous phenotypic characterization showed that de-letion of aatA and aatB genes did not affect growth kinet-ics of mutant DE205B strains (Wang et al. 2011b; Zhugeet al. 2013). The growth kinetics of wild-type DE205Band strains lacking aatA, aatB, or upaB gene were similarwhen cultured in LB medium. Furthermore, the transfor-mation of the complemented plasmid into gene deletionstrains did not affect growth kinetics of correspondingmutants.

There were no differences in autoaggregation and biofilmformation among DE205B, DE205BΔupaB , andDE205BCΔupaB (data not shown). Allsopp et al. shows thatUpaB did not contribute to autoaggregation and biofilm for-mation in wild-type UPEC CFT073 and E. coli OS56(MG1655Δflu) (Allsopp et al. 2012b). In order to investigatewhether UpaB mediated E. coli autoaggregation and biofilmformation were blocked by type 1 fimbriae, UpaB wasexpressed in K-12 strains MG1655 and AAEC189(MG1655Δfim) containing complemented plasmidpSTV28-upaB. As shown in Fig. 2a, after 7 h, the maximalautoaggregation of AAEC189PS-upaB reduced 36 % in theuppermost layer of the culture (assayed by OD600nm) com-pared to the control strain AAEC189 (P<0.01). However,maximal autoaggregation between MG1655PS-upaB andthe corresponding control strain did not differ significantly(Fig. 2a). Similarly, as shown in Fig. 2b, biofilm formationof strain AAEC189PS-upaB was enhanced (14.3 %,P<0.01), compared with the negative control AAEC189PS,and there was no difference between MG1655PS-upaB andMG1655PS (data not shown). These results suggested thatUpaB enhanced the autoaggregation and biofilm formationof AAEC189 (MG1655Δfim) in vitro, which might beblocked by type 1 fimbriae in wild-type DE205B andMG1655.

Expression and purification of recombinant UpaB,and detection of the expression and surface localizationof UpaB among wild-type DE205B, mutant,and complemented strains

The fusion protein UpaB (82.2 kDa) was successfullyexpressed in E. coli (Fig. 3a), and the rabbit anti-UpaB serumwas prepared. Anti-UpaB serum was used to detect the pres-ence of UpaB in vitro, which showed that UpaB were presentin wild-type DE205B and complemented DE205BCΔupaB,but not in mutant strain DE205BΔupaB and negative controlstrain DE205BPSΔupaB (Fig. 3b). The previous study sug-gested that UpaB was located at the cell surface of UPECCFT073 (Allsopp et al. 2012b). Our immunoblotting resultalso showed that UpaB was only present in the outer mem-brane fraction of DE205B (Fig. 3c). The immunofluorescenceanalysis further demonstrated the mottled ring-like stainingpattern on the surface of DE205B and complementedDE205BCΔupaB, confirming the surface localization ofUpaB in DE205B (Figure 3d).

UpaB stimulated the duck to produce antibodiesduring infection, and was increasingly expressedduring infection in vitro and in vivo

To identify whether the duck produces antibody against UpaBduring infection, the duck hyperimmune anti-DE205B serumwas used to detect the purified UpaB protein. As shown inFig. 3e, the purified UpaB protein could be detected inWestern blot analysis, indicating that UpaB stimulates an an-tibody response during DE205B infection in duck.

qRT-PCR was used to measure the RNA level of upaB(relative to dnaE) during the infection in vitro and in vivo.The upaB transcription levels at different conditions werelower than the expression of the housekeeping gene dnaE.Our result indicated the upaB transcription level increased4.7-fold in infected DF-1 cells when compared with the con-trol (P<0.01) (Fig. 4a). The upaB mRNA level was upregu-lated by 12.4-fold in lung tissues of the infected ducks(P<0.01) (Fig. 4b). However, the upaB mRNA level wasnot significantly different between the DE205B isolated fromthe blood of infected ducks and LB culture (P≥0.05) (Fig. 4b).

Fig. 1 Schematic illustration of the UpaB conserved domains. The signalpeptide (1 to 26 aa), the passenger domain (27 to 450 aa), the α-helicallinker domain (451 to 450 aa), and the β-barrel translocator domain (496

to 765 aa) were shown. The passenger domain shares a low level of aminoacid sequence homology with three protein structures: peptidase S6 IgAendopeptidase, pertactin-like passenger domain, and DUF4353

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The upregulation of the upaB mRNA level in infected DF-1cell and duck lungs suggested that UpaB might be associatedwith the pathogenicity of APEC DE205B.

UpaB mediates the APEC adhesion to DF-1 cells in vitro

It was reported that UpaB could bind several extracellularmatrix (ECM) proteins in vitro (Allsopp et al. 2012b). In orderto determine whether UpaBmediated the APEC adhesion andinvasion, we conducted the adhesion and invasion assays inDF-1 cells. DF-1 cells were infected with wild-type DE205B,gene deletion, or complemented mutant strains. As shown inFig. 5a, DE205BΔupaB showed 41.4 % reduction of adhe-sion in DF-1 cells compared with the wild-type DE205B(P < 0.01). Furthermore, the adhesion capacity ofcomplemented DE205BPΔupaB mutant was restored to thesimilar level of wild-type DE205B (P<0.01). Meanwhile, ourdata indicated that the invasion capability of mutant strainDE205BΔupaB was similar to that of DE205B andDE205BCΔupaB strains (data not shown). Since there wasno invasion domain in UpaB, we deduced that UpaB mightnot be associated with APEC invasion.

The adhesion inhibition experiment was preformed addi-tionally to confirm that UpaB mediated the APEC adhesion.The monolayer DF-1 cells were infected with DE205B pre-incubated with UpaB antiserum, and pre-immune serum-pretreated DE205B was served as the negative control. Asshown in Fig. 5b, the adhesion abilities of DE205B andcomplemented DE205BPΔupaB strains were significantly

reduced by 32.0 and 30.7 % after the pretreatment withUpaB antiserum (P<0.01), respectively. These results sug-gested that UpaB might mediate the APEC adhesion in DF-1 cells, since upaB deletion attenuated the capability ofDE205B adhesion in vitro.

Deletion of upaB from DE205B attenuates the virulencein duck model and early colonization of the duck lungsduring APEC systemic infection

To investigate the effect of upaB deletion on the virulence ofDE205B, the LD50 of wild-type, gene deletion, andcomplemented strains were determined in the duck and mousemodels. As shown in Table 2, the LD50 value ofDE205BΔupaB was 3.26×106 CFU/duck, which attenuatedthe virulence by 6.4-fold compared with LD50 of wild-typeDE205B (5.08×105 CFU/duck). The virulence of thecomplemented DE205BPSΔupaB (6.32×105 CFU/duck)was restored to the similar level of DE205B. UpaB deletiondid not attenuate the virulence and bacterial survival in mousesepsis model (data not shown). These tests illustrated thatUpaB acted as an important virulence factor during APECnatural infection route, and upaB deletion reduced the fitnessin duck model.

To determine the effect for upaB deletion on DE205B col-onization in vivo, the systemic infection experiment was car-ried out to evaluate the number of bacteria colonized in dif-ferent duck organs. All three strains caused the early coloni-zation in duck lungs. As shown in Fig. 5c, DE205BΔupaB

Fig. 2 UpaB enhanced autoaggregation and biofilm formation offimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. aAutoaggregation mediated in AAEC189PS-upaB, MG1655PS-upaB,and control strains. After 7 h, the maximal autoaggregation ofAAEC189PS-upaB (the inset in panel a) reduced by 36 % as assayedby optical density (OD600nm) of the uppermost layer of the culturecompared to the control strain (the inset in panel b) (P<0.01). Statistical

significance analysis was performed using Student’s t test (*P<0.01). bBiofilm formation mediated in AAEC189PS-upaB, MG1655PS-upaB,and control strains. The biofilm formation of strain AAEC189PS-upaBenhanced by 14.3 % compared with the negative control strainAAEC189PS (P<0.01). Statistical significance analysis was carried outusing one-way ANOVA (***P<0.01)

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exhibited attenuated colonization capacity in lungs comparedwith DE205B (P<0.01), and the colonization capacity ofcomplemented strain DE205BPSΔupaB in the lungs was al-most restored to the similar level of DE205B (P<0.01).However, no significant colonization capacity differenceswere observed among these three strains in duck blood,

spleen, liver, and kidney tissues (data not shown). As shownin Fig. 5d, there was no statistical difference among DE205B,DE205BΔupaB, and DE205BPSΔupaB for their coloniza-tion in duck brains (P>0.05). The systemic infection experi-ment indicated that UpaB conferred an advantage in the col-onization of the duck respiratory system during DE205B

Fig. 3 a Purification of UpaB expressed in E. coli BL21(DE3). Thepurified fusion protein UpaB (lane 1, 82.2 kDa, and 0.8 mg ml-1),UpaB from the total bacteria extracts (lane 2), and the negative controlBL21(DE3) (line 3). Lane M, protein marker. b Western blot analysis ofUpaB in DE205B (line 1), DE205BΔupaB (line 2), DE205BCΔupaB(line 3), and DE205BPSΔupaB (line 4) whole-cell lysates. c The UpaBwas present in OM (outer membrane proteins) of DE205B cellcomponents. Sup concentrated culture supernatant, IM inner membrane

proteins, Cyt cytoplasmic/periplasmic. d The surface localization ofUpaB in DE205B. DE205B, DE205BCΔupaB, and DE205BΔupaBfor immunofluorescence detection were fixed and incubated with anti-UpaB serum, and then incubated with goat anti-rabbit IgG conjugated toTRITC (2, 5, and 9) or DAPI (1, 4, and 8). 2, 5, and 9Merged images, andthe magnifying image (7) for the box in panel 6. Scale bars=10 μm. eThe immunoblot detection of the purified UpaB protein by thehyperimmune duck anti-DE205B serum

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infection. However, there were no differences among theLD50 and colonization of wild-type, gene deletion, andcomplemented strains in the mouse sepsis model (data notshown), indicating that UpaB did not confer a fitness advan-tage in mouse sepsis model.

Double and triple deletion of upaB, aatA, and aatB genescumulatively attenuated APEC DE205B adhesion to DF-1cells in vitro

ATs UpaB, AatA, and AatB mediated the APEC adhesion inDF-1 cells, and single deletion of upaB, aatA, or aatB geneattenuated APEC adhesion in DF-1 cells in vitro. In addition,deleting upaB, aatA, or aatB gene in DE205B did not affectthe expression of the other two genes. We next studied theeffects of double or triple deletion of upaB, aatA, and aatBgenes (DE205BΔupaB/aatA, DE205BΔupaB/aatB,DE205BΔaatA/aatB, and DE205BΔupaB/aatA/aatB) onthe pathogenicity of APEC DE205B, such as adhesion capac-ity in DF-1 cells, virulence in the duck model, and coloniza-tion in duck lungs. The DE205BΔaatA and DE205BΔaatBconstructs were used (Wang et al. 2011b; Zhuge et al. 2013).

DF-1 cell adhesion assay showed that the individual loss ofupaB, aatA, or aatB gene resulted in the significant reductionin adhesion by approximately 41, 29, and 53 %, respectively(Wang et al. 2011b; Zhuge et al. 2013). As shown in Fig. 6a,the adhesion capabi l i t ies of the double mutants(DE205BΔupaB /aatA , DE205BΔupaB /aatB , andDE205BΔaatA/aatB) were cumulatively impaired (approxi-mately 63, 72, and 67%, respectively), suggesting that doubledeletion of the upaB, aatA, or aatB gene further attenuated theadhesion capability compared to the single deletion mutants

(P<0.01). Furthermore, DE205BΔupaB/aatA/aatB showedapproximately 84 % adhesion capability reduction (P<0.01).These results indicated that double and triple deletion of theupaB, aatA, or aatB gene cumulatively attenuated APECDE205B adhesion in DF-1 cells in vitro.

Double and triple deletion of upaB, aatA, and aatB genescumulatively attenuated APEC DE205B virulenceand early colonization of the duck lungs in vivo

The individual loss of the upaB, aatA, or aatB gene resulted inapproximately 6.4-fold, 7.9-fold, and 10-fold increase ofDE205B LD50 in the duck model (Table 2) (Wang et al.2011b; Zhuge et al. 2013). As shown in Table 2, the LD50 ofmutants (DE205BΔupaB/aatA, DE205BΔupaB/aatB,DE205BΔaatA/aatB, and DE205BΔaatA/aatB/upaB) wereclearly increased approximately 15.5-fold, 19.2-fold, 24.2-fold, and 48.2-fold compared to that of DE205B, respectively.The results indicated that the impact of double/triple genedeletion on LD50 seemed to be cumulative.

The individual loss of the upaB, aatA, or aatB gene causeddeficiency of the colonization capacity in the duck lung (Wanget al. 2011b; Zhuge et al. 2013). To determine whether double/triple deletion of the upaB, aatA, or aatB gene affects APECearly colonization in the duck lung in vivo, the ducks wereinfected intratracheally (1.0×108 CFU). As shown in Fig. 6b,the mutant strains attenuated the colonization capacity in theduck lung by more than an order of magnitude compared toDE205B at 24 h postinfection (P<0.01), suggesting doubleand triple deletion of the upaB, aatA, or aatB gene cumula-tively attenuated APEC early colonization in the duck lungin vivo.

Fig. 4 The expression level ofUpaB in different conditions andin vivo by qRT-PCR. aQuantification of the expressionlevel of UpaB during DE205Binfecting DF-1 cells compared tothe control in vitro. BQuantification of the upaBtranscription level from lungtissues and blood of infectedducks in vivo compared to thecontrol in vitro. Statisticalsignificance analysis wasperformed using two-wayANOVA (***P<0.01)

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Expression profile comparison of virulence factorsbetween wild-type DE205Band DE205BΔupaB/aatA/aatB in vivo

The qRT-PCR was used to measure the effects of aatA, aatB,and upaB deletion on expression profiles of 16 virulencegenes (adhesins, invasins, and outer membrane proteins ofiron uptake system) in vitro and in vivo (Table S1). As shownin Figure S2, the expression levels of several virulence/fitnessfactors were upregulated during the DE205B infection process(P<0.01), but no significant differences were found amongwild-type DE205B, individually knockout strains, and cul-tured in DF-1 cell in vitro and isolated from infected ducksin vivo (data not shown). Furthermore, there were no signifi-cant differences for these genes between wild-type DE205Band DE205BΔupaB/aatA/aatB cultured in DF-1 cell in vitroand isolated from infected blood in vivo (data not shown). As

Fig. 5 UpaB mediated the APECadhesion and early colonizationduring APEC systemic infection.a UpaB mediated the APECadhesion to DF-1 cells, and upaBdeletion attenuated the capabilityof DE205B adherence in vitro. bUpaB antiserum inhibitedsignificantly adhesion of DE205Band complementedDE205BPΔupaB to DF-1 cells.Statistical significance analysiswas performed using one-wayANOVA (***P<0.01). cDeletion of upaB from DE205Battenuates early colonization inthe duck lungs during APECsystemic infection. d Nostatistical difference existed incolonization in duck brainsamong DE205B,DE205BΔupaB, andDE205BPSΔupaB (P>0.05). Anonparametric Mann-Whitney Utest was performed for statisticalsignificance analysis(***P<0.01)

Table 2 The effect of upaB deletion and parallel/triple deletion ofupaB, aatA, and aatB genes on the virulence of DE205B in the duckmodel

Strains used in infection LD50

cfu/duck Changea

DE205B 5.08×105 –

DE205BΔupaB 3.26×106 6.4

DE205BCΔupaB 6.32×105 –

DE205BΔaatA 4.03×106 7.9

DE205BΔaatB 5.08×106 10

DE205BΔupaB/aatA 7.87×106 15.5

DE205BΔupaB/aatB 9.74×106 19.2

DE205BΔaatA/aatB 1.23×107 24.2

DE205BΔupaB/aatA/aatB 2.45×107 48.2

a Relative fold increase compared with the wild-type DE205B

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shown in Fig. 7, during APEC early colonization in ducklungs, the expression levels of fimbrial adhesin genes yqiL,yadN, and vacuolating autotransporter vat were significantlyenhanced in DE205BΔupaB/aatA/aatB by 1.83-fold, 2.23-fold, and 1.75-fold, respectively (P<0.05). The expressionprofile comparison suggested that DE205BΔupaB/aatA/aatB might Bcompensate^ the effect for the cumulative lossesof upaB, aatA, and aatB genes by upregulating the expressionof yqiL, yadN, and vat during APEC early colonization.

Vaccinations against recombinant UpaB, AatA, and AatBproteins conferred protection against colisepticemiacaused by DE205B infection in the duck model

We further investigated whether vaccination with recombinantUpaB, AatA, and AatB proteins conferred protection againstcolisepticemia in the duck model. As shown in Fig. 8a,

antibody titers of 10 ducks randomly selected from each vac-cination group were significantly enhanced and reached to theaverage of 104, suggesting recombinant UpaB, AatA, andAatB proteins could stimulate high-level IgG antibodies in aduck. After challenged intratracheally with wild-typeDE205B, the mortality/survival rates among four vaccinationgroups (20 ducks for each group) were assessed and comparedwith the PBS control group. As shown in Fig. 8b, the protec-tive efficacy of three individual protein-immunized groupswas not significantly enhanced compared to the control group.The survival curves indicated the death peak of the duck im-munized with UpaB, AatA, or AatB protein was postponedcompared to the control group (P<0.01). Furthermore, immu-nization with three proteins showed 35 % survival rate (pro-tective efficacy) (P<0.01). As shown in Fig. 8c, the ducksimmunized with UpaB, AatA, or AatB attenuated theDE205B colonization capacity in the lungs (P<0.01).

Fig. 6 The effect for double and triple deletion of upaB, aatA, and aatBgenes on APEC adhesion and early colonization. a Parallel and tripledeletion of upaB, aatA, and aatB genes cumulatively attenuated APECDE205B adhesion to DF-1 cells in vitro. Statistical significance analysiswas performed using one-way ANOVA (***P<0.01). b Parallel and

triple deletion of upaB, aatA, and aatB genes cumulatively attenuatedearly colonization of the duck lungs in vivo. A nonparametric Mann-Whitney U test was performed for statistical significance analysis(***P<0.01)

Fig. 7 Expression profile ofvirulence factors yqiL, yadN, andvat between wild-type DE205Band DE205BΔupaB/aatA/aatB.qRT-PCR was used to measurethe expression of these genesbetween wild-type DE205B andDE205B ΔupaB/aatA/aatBin vitro and in vivo. Statisticalsignificance analysis wasperformed using two-wayANOVA (*P<0.05)

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Furthermore, the ducks vaccinated with three proteins furtherattenuated the DE205B colonization in duck lungs (P<0.01).The challenge results indicted that vaccination with recombi-nant UpaB, AatA, and AatB proteins conferred protection

against colisepticemia in the duck model caused by DE205Binfection.

Discussion

APEC requires adhesins to colonize on avian air sacs afterinhalation into the respiratory tract (Antao et al. 2009b).Several typical fimbriae adhesins, including the type I fimbrialadhesin, Pap fimbrial adhesin, and ExPEC Adhesin I, are in-volved in the early colonization of APEC infection (Antaoet al. 2009a; Antao et al. 2009b; de Pace et al. 2010;Kariyawasam and Nolan 2009). Except for the fimbriaeadhesins, non-fimbrial ATs also contribute to adhesion to thecell and tissue during the early stage of infection, includingTsh, AatA, and AatB (Dai et al. 2010; Li et al. 2010;Stathopoulos et al. 1999; Zhuge et al. 2013).

Comparative genomic analysis shows that APEC strainscontain multiple AT-encoding genes (Zhu Ge et al. 2014). Inthis study, the molecular characterization of DE205B UpaBsuggested that it is a typical conventional AT, like Ag43 andAatA (Henderson et al. 2004; Li et al. 2010). The convention-al ATs are secreted to bacterial surface or extracellular milieuvia type V transport system (Henderson and Nataro 2001; Yenand Stathopoulos 2007). Like Ag43, AatA and tsh, UpaB islocated on the surface of APEC DE205B, which is consistentwith surface localization of UpaB in UPEC CFT073 (Allsoppet al. 2012b). The upaB mRNA level was significantly en-hanced during DE205B infection in DF-1 cell and duck lungscompared to the culture in LB, suggesting that UpaB might beinvolved in APEC pathogenesis The upregulation of UpaBexpression caters to the view that the pathogens need to up-regulate their virulence/fitness genes expression to facilitateits survival and pathogenicity during infection (Cossart 2011;Hagan et al. 2010; Hey et al. 2013; Kansal et al. 2013).

Several strategies were preformed to unravel the virulenceroles of UpaB during APEC infection, including adhesionassay and system infection experiment. UpaB mediated theAPEC adhesion in DF-1, and its deletion attenuated the capa-bility of DE205B adherence in vitro. Moreover, our systemicinfection experiment indicated UpaB acted as an importantvirulence factor in a natural infection route, and upaB deletionreduced fitness in the duck model. ATs are modular proteinswith a general head-stalk-anchor architecture, in which mem-brane anchors (β-translocator domain) are highly conservedand displaying a β-barrel embedded in the outer membrane.However, the head and stalk (passenger domains) possess thediverse motifs and functions and vary in length (Hendersonand Nataro 2001; Henderson et al. 2004). UpaB shared lowidentities with TibA and Pertactin precursor, although phylo-genetic analysis suggested the close relationship betweenthem (Mooi et al. 1998; Sherlock et al. 2005). UnlikePertactin and other 9 ATs using RGD as the cell attachment

Fig. 8 Vaccinations against recombinant UpaB, AatA, andAatB proteinsconferred protection against colisepticemia in duck model caused byDE205B infection. a The titers for serum IgG antibody against rUpaB,rAatA, and rAatB were measured using indirect ELISA. b The protectiveefficacy of four protein-immunized groups. Survival was determined after7 days postinfection. **P<0.01, the control PBS-immunized group. cVaccinations against recombinant UpaB, AatA, and AatB proteinsattenuates early colonization of the duck lungs during APEC systemicinfection

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motif, we speculated that UpaB and TibA might utilize othercell attachment sequences to adherence to host cells, whichremains to be further elucidated.

Like TibA adhesin/invasin, our study shows that UpaBpromoted autoaggregation and biofilm formation of type 1fimbriae-negative E. coli strain AAEC189 (MG1655Δfim)in vitro, which is a convenient model for the observation ofautoaggregation and biofilm formation. Allsopp et al. hasshowed that UpaB does not contribute to autoaggregationand biofilm formation in wild-type UPEC CFT073 andE. coli OS56 (MG1655Δflu) (flu gene encoding Ag43 pro-tein), which rules out the possibility that this phenotype isinterfered with biofilm formation-related Ag43 (Allsoppet al. 2012b). Therefore, the type 1 fimbriae instead of Ag43shielded the DE205BUpaB interaction with wild-type E. coli,which might cause the phenotype changes duringautoaggregation and biofilm formation. In addition to theautoaggregation and biofilm formation assay, the adhesionassay, invasion assay, and the early colonization capacity wereperformed in the duck model to evaluate the virulence be-tween non-v i ru len t MG1655/AAEC189 and thecomplemented strains of pSTV28-UpaB to overexpressUpaB. However, the data showed that there were no signifi-cant difference between MG1655/AAEC189 and thecomplemented strains of pSTV28-UpaB (data not shown).Compared to highly virulent APEC strains, we speculated thatthe pathogenicity of non-virulent E. coli might not be en-hanced by overexpressing single virulence factor.

APEC andUPEC are two typical extraintestinal pathogenicE. coli (ExPEC) pathotypes. The common ExPEC-specificvirulence factors, such as Type I, Pap, and ExPEC AdhesinI, contribute to bacterial adaptation and colonization in extra-intestinal niches during infection (Antao et al. 2009a; Antaoet al. 2009b; de Pace et al. 2010; Kariyawasam and Nolan2009; Zhu Ge et al. 2014). Our studies confirmed that UpaBconfers an advantage in the early colonization in the duck lungduring DE205B infection, like its role in early colonization ofCFT073 in the bladder (Allsopp et al. 2012b). TwoAPECATs(AatA and AatB) mediate the APEC adhesion in DF-1 cellsand are involved in APEC early colonization in host lungtissues (Dai et al. 2010; Li et al. 2010; Zhuge et al. 2013).The double and triple deletion of upaB, aatA, and aatB genescumulatively attenuated bacteria adhesion in DF-1 cells, theearly colonization in duck lungs, and the LD50. The cumula-tive effect on APEC pathogenicity was also observed whenα-hemolysin (hlyI and hlyII) determinants were lost on UPECpathogenicity in a murine model of ascending urinary tractinfection (UTI) (Nagy et al. 2006). During adhesion to epithe-lial cells and other non-phagocytosis cells in avian lung, hostimmune system can suppress and clear out APEC, associatedwith inflammation in the lung, and only highly pathogenicAPEC can spread in the bloodstream and cause multi-systemic fatal infection (Antao et al. 2009a; Antao et al.

2008; Antao et al. 2009b; Horn et al. 2012). The mutantDE205B strains with single, double, or triple deletion ofUpaB, AatA, and AatB might be relatively easy to be sup-pressed and cleared out by the host immune system (Antaoet al. 2009a; Antao et al. 2008; de Pace et al. 2010; Horn et al.2012), accompanying with significant reduced infection in theduck lung and decreased LD50 in duck model. Owing to theexpression of other fimbrial adhesins, DE205B infection wasnot completely abolished (Antao et al. 2009a; de Pace et al.2010; Horn et al. 2012).

The influence on the expression levels of virulence factorshave been reported in several adhesion/invasion-related mu-tants of APEC/ExPEC (Cortes et al. 2008; Crepin et al. 2008;de Pace et al. 2010). The recent reports suggested pathogensregulate their gene expression patterns to Bcompensate^ forthe lack of important virulence/fitness factors during the in-fection (Cortes et al. 2008; Crepin et al. 2008; de Pace et al.2010). The expression profile comparison showed the expres-sion levels of fimbrial adhesin genes (yqiL and yadN) andvacuolating autotransporter vat were significantly enhancedin the mutant DE205BΔupaB/aatA/aatB during infection inthe duck lung. The ExPEC Adhesin I (yqiL gene product) andYad adhesin (yadN gene product), the common virulence fac-tors of APEC and UPEC, are involved in urinary tract infec-tion and the early colonization of APEC (Antao et al. 2009a;Dziva et al. 2013; Spurbeck et al. 2011). The expression of Yqiand Yad fimbriaes is coordinated during UPEC infection(Spurbeck et al. 2011). The vacuolating autotransporter Vatis an AT toxin involved in the early colonization of APEC,which is secreted through the type V transport pathway(Parreira and Gyles 2003). The expression profile suggestedthat higher expression of two fimbrial adhesins (ExPECAdhesin I and Yad adhesin) and Vat toxin might be able tocompensate for the loss of three non-fimbrial AT adhesinsduring APEC early colonization.

Epidemiological studies demonstrate that APEC isolates,especially highly virulent APEC, belong to ECOR B2 andD, while intestinal pathogenic and commensal E. coli of thegut flora belong to group A and B1 (Dho-Moulin andFairbrother 1999; Ewers et al. 2003; Zhu Ge et al. 2014).Our studies show that upaB, aatA, and aatB genes are pre-dominantly associated with ECOR B2 and D, and especiallyhighly virulent APEC O1:K1 and O2:K1 isolates, whichcause severe avian colisepticemia and has zoonotic potentialto cause septicemia and meningitis in neonatal rats (Ewerset al. 2003; Johnson et al. 2007; Zhu Ge et al. 2014). TheUpaB, AatA, and AatB might be considered as APEC/ExPEC-specific virulence factors, which validated their po-tential as pathogen-specific vaccine candidates (Wieser et al.2010).

It has been shown that UpaB, AatA, and AatB spontane-ously stimulated a low-level antibody response in ducks dur-ing DE205B infection, and the antiserum of these protein can

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significantly inhibit the adhesion of DE205B in vitro (Li et al.2010; Wang et al. 2011b; Zhuge et al. 2013). In this study, wedetermined that recombinant UpaB, AatA, and AatB proteinscould stimulate high-level IgG antibodies in ducks, and thechallenge study and colonization assay showed that vaccina-tion using recombinant proteins conferred protection againstcolisepticemia caused by DE205B infection in the duck mod-el. Therefore, vaccinations using recombinant UpaB, AatA,and AatB proteins might be the valuable candidates for thepathogen-specific vaccine design. Due to limited protectionprovided by single antigen in the duck model, Wieser et al.introduced multiepitope subunit vaccine to provide protectionagainst ExPEC infection (Wieser et al. 2010). Therefore,UpaB, AatA, and AatB proteins would also be novel targetsto develop the multiepitope subunit vaccines against APECinfection.

In conclusion, our studies provided insights that UpaB me-diates the DE205B adhesion in DF-1 cells and the early colo-nization in duck model, and enhances autoaggregation andbiofilm formation of fimbria-negative E. coli AAEC189(MG1655Δfim) in vitro. Furthermore, double and triple dele-tion of upaB, aatA, and aatB genes cumulatively attenuatedDE205B adhesion in DF-1 cells, accompanying with de-creased LD50 in duck model and the early colonization inthe duck lung. Additionally, we demonstrated that vaccinationwith recombinant UpaB, AatA, and AatB proteins conferredprotection against colisepticemia caused byDE205B infectionin the duck model.

Compliance with ethical standards

Funding This work was supported by The Fund of Priority AcademicProgram Development of Jiangsu Higher Education Institutions (PAPD)and the Chinese Special Fund for Agro-scientific Research in the PublicInterest (201403054).

Conflict of interest The authors declare that they have no competinginterests.

Ethical approval All animal experimental protocols were approved bythe Laboratory Animal Monitoring Committee of Jiangsu Province,China.

This article does not contain any studies with human participantsperformed by any of the authors.

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