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Jan 23, 2009 - AnthePro 5 software revealed the existence of 10 membrane- spanning .... Identification of PfaI domains that are essential to auto- transporting ..... Kolodziejek, A. M., D. J. Sinclair, K. S. Seo, D. R. Schnider, C. F. Deobald,.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2009, p. 4333–4340 0099-2240/09/$08.00⫹0 doi:10.1128/AEM.00159-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 13

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain䌤† Yong-hua Hu,1,2 Chun-sheng Liu,1,2 Jin-hui Hou,3 and Li Sun1* Institute of Oceanology, Chinese Academy of Sciences, Qingdao, People’s Republic of China1; Graduate University of the Chinese Academy of Sciences, Beijing, People’s Republic of China2; and Xuzhou Institute of Technology, Xuzhou, People’s Republic of China3 Received 23 January 2009/Accepted 7 May 2009

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50°C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection. characterized by alternating hydrophobic and hydrophilic residues and ends with either a tryptophan or a phenylalanine (12, 16, 27). The integrity of this end motif seems to be required for protein translocation, as deletion of certain residues in this sequence impairs protein secretion. The secretion process of the autotransporter is initiated by the signal sequence, which directs the translocation of the protein precursor across the inner membrane into the periplasmic space via the Sec system. Once inside the periplasm, the ␤-domain inserts into the outer membrane and adopts the structure of a ␤-barrel through which the passenger domain is translocated to the cell surface, where it may exist as a membrane-anchored protein covalently linked to the ␤-domain or be cleaved from the ␤-domain as a result of proteolysis (7). Since the discovery of the gonococcal immunoglobulin A1 protease (35), the first autotransporter, and especially with the advent of genome sequencing technology, autotransporters have been identified in many bacterial species (18, 19, 28, 33). Functions assigned to autotransporters are mostly associated with bacterial pathogenicity, which includes adhesion and invasion into host cells, biofilm formation, and cytotoxicity (11, 54). In the present study, we identified and analyzed an autotransporter, PfaI, from a pathogenic Pseudomonas fluorescens strain isolated from diseased fish. We found that, like many of the autotransporters identified in other pathogens, PfaI is a virulence factor that is involved in interactions with host cells and modulation of host immune responses via a protease effector. In addition, we found that the autotransporter property

Protein secretion plays important roles in bacterial life, as many of the secreted proteins are involved in biological processes that are fundamental to the survival and environmental adaptations of the cells. Gram-negative bacteria have evolved a number of secretion systems that utilize different secretion apparatus and mechanisms (9). The classical autotransporter secretion pathway belongs to the type V secretion system (8, 12, 13). Compared to other types of secretion mechanisms, the autotransporter system is unique in that all the components that are required for protein translocation are contained within a single polypeptide. Structurally, autotransporters are characterized by three domains: (i) an N-terminal signal sequence that is recognized by the Sec translocon; (ii) a central passenger domain (or ␣-domain) that contains the effector molecule and is highly variable; and (iii) a C-terminal translocation domain (or ␤-/autotransporter domain) that is conserved in length (250 to 300 amino acids) but varies in primary structure (7, 12, 55). In most cases, the ␤-domain contains 12 antiparallel strands of 9 to 12 residues that, upon integration into the outer membrane, form a ␤-barrel conformation (27, 57). Another conserved feature of the ␤-domain is the presence at the C terminus of a sequence motif, (Y/V/I/F/W)-X-(F/W), that is * Corresponding author. Mailing address: Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, People’s Republic of China. Phone and fax: 86-532-82898834. E-mail: [email protected] † Supplemental material for this article may be found at http://aem 䌤 Published ahead of print on 15 May 2009. 4333




TABLE 1. Plasmids used in this study Plasmid

Relevant characteristicsa

p7TS pAT1 pAT2 pAT3 pAT1V pAT2V pAT3V pAT18 pB328 pBR322 pBSAT1 pBS-T

Tcr; suicide plasmid Apr; carrying PfaI autotransporter regions Apr; carrying PfaI autotransporter regions Apr; carrying PfaI autotransporter regions Apr; pAT1 carrying agaV Apr; pAT2 carrying agaV Apr; pAT3 carrying agaV Apr; pAT1 carrying et18 pBR322 with mutated ScaI site Apr Tcr; general cloning vector Apr; carrying the 5⬘ portion of pfa1 Apr; cloning vector

pEPA1 pEPA2 pET258 pJT

Knr; carrying the coding sequence for Pap Knr; carrying the coding sequence for PapM Knr; expression plasmid Tcr; broad-host-range vector


Source or reference 52 This study This study This study This study This study This study This study This study Takara This study Tiangen, Beijing, China This study This study 60 45

Apr, ampicillin resistant; Knr, kanamycin resistant; Tcr, tetracycline resistant.

of PfaI could be exploited for the delivery and surface display of an immunoprotective antigen. MATERIALS AND METHODS Bacterial strains and growth conditions. Bacillus sp. strain B187 (59), Edwardsiella tarda strain TX1 (fish pathogen) (58), and Pseudomonas fluorescens strain TSS have been reported previously (52). Strain TSS was isolated from diseased Japanese flounder and genetically identified by 16S rRNA gene sequence (GenBank accession no. FJ896114) analysis. The pathogenicity of P. fluorescens strain TSS to Japanese flounder has been described previously (52). Escherichia coli strain DH5␣ is a commercial strain from Takara (Dalian, China). All strains were cultured in Luria-Bertani broth (LB) medium (39) at 37°C (for E. coli) or 28°C (for all others). Cell cultures were maintained at 4°C for shortterm storage (1 to 3 days) and at ⫺80°C in culturing medium containing 10 to 15% (vol/vol) glycerol for long-term storage. Appropriate antibiotics were added at the following concentrations: ampicillin, 100 ␮g/ml; kanamycin, 50 ␮g/ml; and tetracycline, 15 ␮g/ml. Plasmid construction. The plasmids and primers used in this study are listed in Table 1 and Table 2, respectively. pEPA1 was created by inserting the coding sequence of Pap (amplified by PCR with primers F15/R13) into pET258 between the NdeI/XhoI sites. To construct pEPA2, overlapping PCRs were performed with primer pairs F15/MR2 and MF2/R13, followed by a fusion PCR with

primers F15/R13; the PCR products were inserted into pET258 between the NdeI/XhoI sites. To create pB328, the ScaI site of pBR322 was mutated as follows: overlapping PCRs were performed with primer pairs ApF8/ApMR1 and ApMF1/ApR7, followed by a fusion PCR with primers ApF8/ApR7; the PCR products were digested with EcoRI/PstI, and the 750-bp DNA fragment was inserted into pBR322 between the EcoRI/PstI sites. To construct pAT1, the 657-bp DNA fragment containing the 5⬘ region and upstream sequence of pfa1 was amplified by PCR with primers F11/R9, and the PCR products were ligated into pB328 and pBS-T at the EcoRV site, resulting in p328AT1 and pBSAT1, respectively; a 2-kb DNA fragment was amplified by PCR from the 3⬘ region of pfa1 using primers F12/R12, and the PCR products were ligated into pBS-T at the EcoRV site, resulting in pBSAT2, which was digested with ScaI, and the 2-kb DNA fragment was ligated into p328AT1 at the EcoRV site, yielding pAT1. To construct pAT2 and pAT3, pBSAT2 was digested with EcoRV/ScaI and SmaI/ ScaI, respectively, and the resulting 1,890- and 1,420-bp DNA fragments were inserted into p328AT1 at the EcoRV site. pAT1V, pAT2V, and pAT3V were constructed by inserting agaV (amplified by PCR with primers UAF13/UAR14) into pAT1, pAT2, and pAT3 at the ScaI site. To construct pAT18, the coding sequence of the processed Et18 preceded by an 8-glycine linker was amplified by PCR with primers 18F9/18R11; the PCR products were inserted into pAT1 at the ScaI site. Cloning of pfa1. pfa1 was cloned by using in vivo-induced antigen technology as described by Kim et al. (20). Briefly, for the construction of an E. coli strain BL21(DE3)-based inducible expression library, 0.5- to 3-kb Sau3A1 fragments of strain TSS genomic DNA were ligated into pET28abc (Novagen, United States), and the ligation mixture was electroporated into electrocompetent DH5␣. The transformants were used for the preparation of a plasmid library, which was subsequently introduced into BL21(DE3) (Tiangen, Beijing, China) by transformation. To prepare P. fluorescens strain TSS antisera, adult rabbits were immunized with TSS, and antisera were pooled and absorbed with TSS whole cells and cell lysate. As a negative control, sera from phosphate-buffered saline (PBS)immunized rabbits were pooled and treated similarly. For the screening of in vivo-expressed genes, the genomic expression library was plated on LB agar plates, and by using nitrocellulose disks, the colonies that appeared on the plates were replica plated onto LB agar plates supplemented with isopropyl-␤-D-thiogalactopyranoside (IPTG). After incubation at 37°C, the colonies that emerged were lysed by exposure to chloroform vapor. The nitrocellulose disks were blotted with TSS antisera. Positive colonies were immunoblotted with the control sera, and the resulting negative colonies were blotted again with TSS antisera. Plasmids were extracted from positive colonies, and the TSS DNA inserts in the plasmids were identified by DNA sequencing. One of the inserts contains pfa1 truncated at the 3⬘ end. The complete gene of pfa1 was obtained by genome walking as described previously (60). Construction of a pfa1 null strain, TSM. To construct P. fluorescens strain TSM, an internal 766-bp DNA fragment (positions 1354 to 2119) of pfa1 was

TABLE 2. Primers used in this study Primer

Sequence (5⬘ 3 3⬘)a

18F9...........................................................................................GATATCGGTGGTGGTGGTGGTGGTGGTGGTTGCGTCGCCGCCGT (EcoRV) 18R11 ........................................................................................GATATCGTGGTGGTGGTGGTGGTGCTTCAGCAGCGAGAACG (EcoRV) ApF8..........................................................................................AGATCACTGAATTCTTGAAGACGAAAGG (EcoRI) ApMF1......................................................................................CTTGGTTGAATACTCACCAGTCACAGAA ApMR1 .....................................................................................TGGTGAGTATTCAACCAAGTCATTCTGAG ApR7 .........................................................................................TATCTCGAGCCAATGCTTAATCAGTGA F6...............................................................................................TAAGTAAGTAAGCCGGATATCGAAGGCA F11.............................................................................................CGCGGCGTTAACATTTAAATTGATGACGCACATATCCA (SwaI) F12.............................................................................................AGTACTACGCTGACCGGTGGC (ScaI) F15.............................................................................................GCATATGTCCGGCCTGGACCG (NdeI) MF2 ...........................................................................................GGCACCGGAGCTCTGGCGTTG MR2 ..........................................................................................CAGAGCTCCGGTGCCGTTGGCATT R9 ..............................................................................................CCCGGGATATCAGAGTACTATTGACCTGAGCTTCC (SmaI, EcoRV, ScaI) R12 ............................................................................................CAGTACTATTTAAATTAGAACAGGAAGGTAAACCCG (ScaI, SwaI) R13 ............................................................................................CTCGAGGCCAACCGGCGTGT (XhoI) R14 ............................................................................................GTACTATTTAAATAGCTGTTCTGGCCTTCCA (ScaI, SwaI) RTF33 .......................................................................................GCAACGCAATCGCACCC RTR10.......................................................................................GCTGTCCTGAACGCCGATT UAF13 ......................................................................................CCCGGGAGAAGACTGGCGAGAAAT (SmaI) UAR14......................................................................................CCCGGGTTGTATAAATTTCAATCGCTGGT (SmaI) a

Underlined nucleotides are restriction sites of the enzymes indicated in the brackets at the ends.

VOL. 75, 2009 generated by PCR with primers F6/R14; the PCR products were inserted into pBSAT1 at the EcoRV site, resulting in pBSAT3, which was digested with SwaI, and the 1,423-bp DNA fragment was inserted into p7TS at the SmaI site, resulting in p7TSP1. p7TSP1 was introduced into E. coli S17-1␭pir (Biomedal, Spain) by transformation. S17-1␭pir/p7TSP1 was conjugated with strain TSS. The transconjugants were selected first on LB plates supplemented with tetracycline and kanamycin and then on LB plates supplemented with 5% sucrose and kanamycin. The colonies that were resistant to sucrose and sensitive to tetracycline were analyzed by PCR; the PCR products were subsequently subjected to DNA sequencing to confirm the deletion in pfa1. Bacterial conjugation. Bacterial conjugation was performed as described previously (58). qRT-PCR. Quantitative real-time reverse transcriptase PCR (qRT-PCR) was carried out in an ABI 7300 real-time detection system (Applied Biosystems) by using a SYBR ExScript qRT-PCR kit (Takara) as described previously (59). Each assay was performed in triplicate with 16S rRNA as control. The primers used for qRT-PCR of pfa1 and 16S rRNA were RTF33/RTR10 (Table 2) and 933F/16SRTR1 (59), respectively. All data are given in terms of relative levels of mRNA, expressed as means plus or minus standard errors of the means (SE). Bacterial cell number determination. Plate count was used to determine the number of viable TSS cells corresponding to an optical density at 600 nm (OD600) of 1, which is ⬃109 CFU. The bacterial cell numbers indicated for all experiments were estimated based on OD600 measurements. Expression of pfa1 under different growth conditions. To examine pfa1 expression under in vivo conditions, Japanese flounder were injected intraperitoneally (i.p.) with 107 CFU of strain TSS. The livers of the fish (five) were taken at 24 h postinfection and used for total RNA preparation with an RNAprep tissue/bacteria kit (Tiangen, Beijing, China). To examine pfa1 expression under in vitro conditions and in relation to growth phase, TSS was cultured in LB medium at 28°C to an OD600 of ⬃1.8 and used for total RNA extraction as described above. To examine pfa1 expression at different temperatures or pH values, TSS was cultured to an OD600 of ⬃1.8 in LB medium at various temperatures or in LB medium with different pH values at 28°C; the cells were then used for total RNA extraction. The same amounts of total RNA from each preparation were used for qRT-PCR. Biofilm assay. Cells were cultured in LB medium to exponential phase and diluted to 105 CFU/ml. The diluted cells were transferred into a 96-well polystyrene plate (Nunc, Denmark) and incubated at 28°C for 24 h without agitation. After the incubation, the unattached cells were removed from the wells by using a pipette, and the wells were washed five times with PBS. The attached cells were treated with Bouin fixative for 1 h and stained with 1% crystal violet solution for 20 min. The unbound dye was removed by rinsing the plate several times with running water. The plate was air dried. The bound dye was eluted in ethanol, and the A570 of eluates was measured. Serum bactericidal activity assay. The serum bactericidal activity assay was performed as described previously (52). Interaction of bacteria with cultured fish cells. Japanese flounder gill cells (FG cells) were cultured and maintained as described previously (47). Examination of interactions between FG cells and TSS/TSM cells was performed according to the method of Kolodziejek et al. (22). Briefly, FG cells were cultured in 96-well cell culture plates to monolayer and mixed with strain TSS or TSM. After incubation at 30°C for 1 h, the plates were washed five times with PBS. To determine the number of bacterial cells associated with the entire FG cell, the washed FG cells were lysed with 200 ␮l of 1% (vol/vol) Triton X-100 in PBS, and 50 ␮l of the lysate was plated on LB agar plates supplemented with kanamycin. After incubation overnight at 30°C, the numbers of colonies that appeared on the plates were counted. To determine the numbers of bacterial cells that had penetrated into FG cells, the above-mentioned washed FG cells were incubated with penicillin and streptomycin (100 U) for 2 h at 30°C to kill the extracellular bacteria. FG cells were lysed and plated as described above. Experimental infection and virulence determination. P. fluorescens strains TSS and TSM were cultured to an OD600 of 0.7 in LB medium and resuspended in PBS to 3 ⫻ 108 CFU/ml. Japanese flounder were divided randomly into three groups (20 fish/group), and each group was injected i.p. with 100 ␮l of TSS or TSM suspension or PBS. The fish were monitored for mortality for 14 days, during which time the fish were maintained at 22°C in aerated seawater that was changed twice daily. Cumulative mortality was calculated at the end of the monitoring period. To examine bacterial dissemination in blood, Japanese flounder were challenged with strain TSS or TSM as described above. Blood was taken from the fish at 6 h postinfection and, after serial dilution, plated in triplicate on LB agar plates supplemented with kanamycin. After incubation at 28°C for 48 h, the colonies that appeared on the plates were enumerated. The genetic nature of



these colonies was verified by PCR analysis using primers specific to TSS and TSM. The PCR products were randomly selected for DNA sequencing. Respiratory burst assay. Two groups (five fish/group) of Japanese flounder were injected i.p. with the same dose (107 CFU) of TSS or TSM. Blood was collected from the caudal veins of the fish at different time points after the infection and used for respiratory burst assay, which was performed exactly according to the method of Kumar et al. (24). Purification and reconstitution of recombinant proteins. For the purification of recombinant Pap and the mutant Pap, named PapM, E. coli BL21(DE3) harboring pEPA1 and pEPA2 was grown in LB medium to an OD600 of 0.7, followed by the addition of 1 mM IPTG to the culture. After an additional 5 h of growth, recombinant proteins were purified under denaturing conditions by using nickel-nitrilotriacetic acid columns (GE Healthcare, United States) as recommended by the manufacturer. The proteins were then reconstituted as follows: the proteins were incubated with Ni-nitrilotriacetic acid-agarose (Qiagen, United States) in the lysis buffer (100 mM NaH2PO4, 10 mM Tris-Cl, and 8 M urea [pH 8.0]) for 30 min at 20°C and then dialyzed against the reconstitution buffer containing 50 mM Tris-Cl, 200 mM KCl, 10 mM MgCl2, 10 ␮M ZnCl2, 5 mM ␤-mercaptoethanol, 1 mM EDTA, 20% glycerol, and decreasing concentrations of urea (4, 2, 1, 0.5, 0.25, and 0 M). Reconstituted proteins were eluted in elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole [pH 8.0]) and dialyzed overnight against 2 liters of PBS. The proteins were concentrated by using Amicon Ultra centrifugal filter devices (Millipore, United States). Purification of recombinant Et18 was described previously (14). Protease activity assay. The protease activity of purified recombinant Pap and PapM was analyzed in the assay buffer with azocasein as the substrate, as described previously (59). One unit of enzyme activity was defined as an increase of A350 of 0.001 over the activity of the control (azocasein incubated with PBS). The effects of Na⫹, Ca2⫹, Mg2⫹, and Zn2⫹ were determined by adding each of the metals to the assay buffer at 1 and 10 mM. Antiserum. Antiserum to recombinant Pap was prepared by injecting adult rats (⬃242 g) subcutaneously with 60 ␮g of the purified recombinant protein. The animals were boosted, and the blood was collected as described previously (46). The titer and specificity of the antiserum were determined by enzyme-linked immunosorbent assay (ELISA) and Western and immunoblotting analysis as described previously (45). Western and immunoblotting analysis. Cells were grown in LB medium to an OD600 of 1. Extracellular proteins and those of the periplasm, cytoplasm, and outer membrane were prepared as described previously (5, 45, 59). The supernatant proteins were concentrated ⬃300 times by using Amicon Ultra centrifugal filter devices (Millipore). The proteins were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting was performed as described previously (45, 59) using anti-Pap or anti-His antibodies (Tiangen, Beijing, China). Cytotoxic effect of Pap on FG cells. An assay of the cytotoxic effect of Pap on FG cells was performed according to the method of Alamuri and Mobley (2). In brief, FG cells were cultured to confluence in 96-well plates. Different concentrations of Pap or PapM or PBS were added to the cells. After incubation at 20°C for various times, the cells were used either for the determination of viability using an MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] cell proliferation and cytotoxicity assay kit (Beyotime, Beijing, China) or for microscopic observation. For the latter purpose, the cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS, stained with Giemsa solution (Solarbio, Beijing, China), and then destained with 10% methanol. The plate was dried and examined under an inverted microscope. Whole-cell ELISA. Whole-cell ELISA was performed as described previously (45). Briefly, 107 CFU of bacterial cells or PBS (the control) was added to a 96-well ELISA plate. After being blocked with bovine serum albumin, the cells were treated with mouse anti-His antibody and then with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Bios, Beijing, China). The plate was read at 450 nm, and positive readings were defined as at least twice the value for the control. Vaccination. This was performed as described previously (59). Briefly, E. coli strains DH5␣/pAT18 and DH5␣/pAT1 and Bacillus sp. strain B187 were cultured in LB medium and resuspended in PBS to 2 ⫻ 108 CFU/ml. Purified recombinant Et18 was added to the B187 suspension at 200 ␮g/ml (named Et18-B187 mix). Healthy Japanese flounder (⬃12 g) were divided randomly into four groups (30 fish/group) designated A to D, and the fish in these groups were injected i.p. with 100 ␮l of DH5␣/pAT18, DH5␣/pAT1, Et18-B187 mix, and PBS, respectively. At the 20th day postimmunization, groups A, B, and D were boosted with the same amount of bacterial cells or PBS as was used in the initial immunization; group C was boosted with 20 ␮g of Et18 suspended in PBS without B187 cells. The fish were challenged via i.p. injection with 5 ⫻ 106 CFU




FIG. 1. qRT-PCR analysis of pfa1 expression in relation to growth phase (A) and growth conditions (B and C). (A) qRT-PCR was performed with total RNA extracted from P. fluorescens strain TSS cultured to different densities in LB medium. (B and C) qRT-PCR analyses were performed with total RNA extracted from TSS grown in LB medium of different pH values (B) or from TSS grown in LB medium at different temperatures (C). In all panels, the pfa1 mRNA level was normalized to that of the 16S rRNA. Data are the means of three assays and presented as the means ⫾ SE. **, P ⬍ 0.01; *, P ⬍ 0.05.

of E. tarda TX1 at the 14th day postboost and monitored for mortality for 14 days. The relative percent of survival (RPS) of the fish was calculated according to the method of Amend (3): RPS ⫽ {1 ⫺ (% mortality in vaccinated fish/% mortality in control fish)} ⫻ 100. Statistical analysis. All statistical analyses were performed using SPSS 15.0 software (SPSS, Inc., United States). Differences in the cytotoxic effect of Pap and the interaction between Pap and FG cells were analyzed using one-way analysis of variance; the vaccination data were analyzed using the chi-square test. All other statistical analyses were performed using the Student t test. In all cases, the significance level was defined as a P value of ⬍0.05. Database search and in silico analysis. The protein database search was conducted using the BLAST programs at the NCBI. Structural analysis was performed using the MOTIF, SMART, and NCBI Conserved Domain Search servers. The secondary structure of the autotransporter domain of PfaI was predicted by using AnthePro, version 5. Signal peptide search was performed by using SignalP, version 3.0. Nucleotide sequence accession number. The nucleotide sequence of pfa1 has been deposited in the GenBank database under the accession number FJ539022.

RESULTS Sequence characterization of PfaI. pfa1 codes for a putative protein of 1,242 amino acids. BLAST sequence analyses indicated that the closest homologues of PfaI are two outer membrane autotransporters from the P. fluorescens strain Pf0-1 and the Serratia proteamaculans strain 568 (GenBank accession nos. ABA74424 and ABV43830, respectively), which share approximately 37% overall sequence identity with PfaI. Bioinformatics analyses using SignalP 3.0, SMART sequence analysis tools, and the NCBI Conserved Domain Search server identified three domains in PfaI: a Sec-dependent signal peptide formed by the N-terminal 43 residues, a passenger domain (residues 209 to 553), and an autotransporter beta domain (residues 970 to 1234). The passenger domain contains a putative protease (named Pap, for “PfaI protease”) of the MEROPS peptidase family S8. Pap possesses a serine active site (residues 231 to 242) and an aspartic acid active site (residues 505 to 515) that are typical for proteases of the subtilase family. Structural analysis of the beta domain using the AnthePro 5 software revealed the existence of 10 membranespanning antiparallel ␤-sheets corresponding to those that form the outer membrane ␤-barrels of autotransporters. The beta domain of PfaI terminates with the sequence NAGFT

FIG. 2. SDS-PAGE (A) and Western immunoblotting (B) analysis of Pap in the supernatants of P. fluorescens strains TSS and TSM. (A) Equal amounts of TSS and TSM supernatants were analyzed by SDS-PAGE. The TSS-unique protein is indicated by an arrow. (B) TSS and TSM supernatants were resolved by SDS-PAGE as described for panel A; the proteins were transferred to a nitrocellulose membrane and blotted with anti-Pap antibodies. Lanes 1, protein markers.

FLF, which conforms to the consensus sequence motif (Y/V/ I/F/W)-X-(F/W) that has been discovered in most autotransporters. Taken together, these structural features identify PfaI as probably a novel protein of the autotransporter family. Expression of pfa1 is modulated by growth phase and growth conditions. Since PfaI appeared among the antigenic proteins whose production levels were heightened during infection, we determined the expression levels of pfa1 under in vivo (i.e., during infection) and in vitro (i.e., cultured in LB medium at 28°C) conditions. For this purpose, Japanese flounder were infected with TSS, and pfa1 expression in the liver of the infected fish was determined by qRT-PCR. The results showed that pfa1 expression in vivo was 13-fold higher than that in vitro. We next examined the potential effects of other growth conditions, i.e., pH, temperature, and growth phase, on pfa1 expression by qRT-PCR. The results showed that pfa1 expression increased with cell density and reached a maximum at an OD600 of 2 (Fig. 1A). High pH (pH 9) increased the expression of pfa1 to a level 5.7-fold higher than that seen at pH 7, whereas low pH (pH 5) had no effect on pfa1 expression (Fig. 1B). Compared to pfa1 expression at 28°C, a low temperature (18°C) significantly enhanced pfa1 expression, whereas a sublethal high temperature (35°C) had no significant effect on pfa1 expression (Fig. 1C). Construction and characterization of a pfa1 null mutant. To investigate the functional importance of pfa1, the gene was mutated by markerless in-frame deletion. SDS-PAGE analysis of the supernatant proteins of strain TSS and the pfa1 null mutant, TSM, showed that they differed mainly in one protein, which appeared in the supernatant of TSS but was absent in that of TSM (Fig. 2A). This TSS-unique protein exhibits a molecular weight that matches the predicted molecular mass of Pap (37.6 kDa). Western immunoblotting analysis showed that the 37.6-kDa protein could react with anti-Pap antibodies but not with the preimmune serum (Fig. 2B and data not shown). Taken together, these results suggest that pfa1 very likely encodes an active autotransporter that can deliver its effector protease, Pap, out of the cell as an extracellular protein. Mutation of pfa1 has multiple effects. (i) Effect on growth and biofilm production. Since P. fluorescens TSS is a fish pathogen, we compared TSS and the mutant strain TSM for the capacities that are known to be associated with pathogenicity, which include biofilm formation, serum resistance, in-

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FIG. 3. Respiratory burst activities of blood neutrophils of Japanese flounder after infection by P. fluorescens strains TSS and TSM. Two groups of Japanese flounder were infected with the same dose of TSS or TSM for various times. Blood was collected from the infected fish at the indicated time points and used for respiratory burst activity assay. Data are presented as the means ⫾ SE. **, P ⬍ 0.01; *, P ⬍ 0.05.

teraction with host cells, and dissemination in host blood. The results of growth studies showed that the growth profile of TSM in nutrient medium (LB) was similar to that of TSS, but the biofilm growth of TSM on a polystyrene surface was significantly slower (2.1-fold less) than that of TSS. (ii) Effect on serum resistance. For many pathogens, resistance against host serum killing constitutes part of the virulence mechanism. TSS exhibits apparent serum resistance, as 69% of the cells survived after incubation with Japanese flounder serum. In contrast, only 17% of TSM cells survived after the same treatment, which is significantly lower than for TSS. The presence of Mg2⫹-EGTA, which inhibits the classical pathway of complement activation by removing Ca2⫹, increased the survival rates of TSS and TSM to 84.8 and 72.5%, respectively. These results suggest that PfaI is required for blocking the activation of the classical complement pathway. (iii) Effect on interaction with cultured host cells. To examine whether PfaI played any role in interaction with host cells, cultured FG cells were incubated with TSS or TSM, and the bacterial cells associated with the host cells were enumerated. The results showed that 47.4 and 12.5% of TSS and TSM cells, respectively, were recovered from the entire (i.e., from the surface and the intracellular milieu) FG cells, while 16.2 and 2% of TSS and TSM cells, respectively, were recovered from the intracellular milieu of FG cells. Hence, TSM is significantly impaired in the ability to adhere to and invade FG cells. (iv) Effect on dissemination in blood and overall bacterial virulence. Blood dissemination analysis showed that, following i.p. injection, the number of TSM cells recovered from the blood was 15-fold lower than the number of TSS cells. Consistently, i.p. injection of 3 ⫻ 107 CFU of TSS into Japanese flounder caused 90% cumulative mortality in a period of 2 weeks, whereas i.p. injection of the same dose of TSM led to 30% cumulative mortality, which is significantly (P ⬍ 0.01) less than that caused by TSS injection. Injection of PBS caused no mortality. These results demonstrate that TSM is significantly attenuated in overall bacterial virulence. (v) Effect on respiratory burst activity of the host phagocytes. Since, as described above, TSM exhibited impaired infectivity compared to that of TSS, we examined whether TSM and TSS induced different host immune responses during infection. For this purpose, Japanese flounder were infected separately with the same dose of TSS and TSM, and the re-



FIG. 4. Effects of pH (A) and temperature (B) on the activity of purified recombinant Pap. (A) The effect of pH was determined in three different buffers: 50 mM citric acid-sodium phosphate (pH 4 to 6; f), 50 mM sodium phosphate (pH 6 to 9; 䉫), and 50 mM glycineNaOH (pH 9 to 11; Œ). (B) The effect of temperature (䉬) was determined in the assay buffer. Thermostability (䡺) was determined by preincubating the enzyme in the assay buffer at the indicated temperature for 1 h before initiating the enzymatic reaction by the addition of azocasein. Data are the means from three independent assays and presented as the means ⫾ SE.

spiratory burst activities of the neutrophils of the infected fish were determined at 4, 8, 12, and 24 h postinfection. The results showed that, at all time points examined, the respiratory burst activities of the neutrophils of TSM-infected fish were significantly higher than those of the TSS-infected fish (Fig. 3). Analysis of the protease activity of the purified recombinant Pap. To examine the potential protease activity of Pap, the coding sequences of wild-type Pap and the mutant Pap (named PapM), bearing an in-frame deletion of part of the serine active site (S507 to S514), were expressed in E. coli cells. Recombinant Pap and PapM were purified and appeared as single bands of the expected molecular mass (⬃37 kDa) following SDS-PAGE analysis (see Fig. S1 in the supplemental material). Protease activity analyses based on the A350 showed that incubation of azocasein with 1 ␮l of Pap and PapM caused absorbance increases of 0.025 and 0.0004, respectively, over the level in the control, which correspond to protease activities of 25 and 0.4 U/␮l, respectively. The protease activity of Pap was abolished in the presence of the serine protease inhibitor phenylmethanesulfonyl fluoride. These results suggest that Pap is a serine protease whose activity requires the integrity of the serine active site. Effects of pH, temperature, and metal ions on the protease activity of recombinant Pap. Enzymatic analyses showed that the optimal pH and temperature of recombinant Pap were 8.0 and 50°C, respectively. The protein exhibits more than 60% of the maximum activity over a pH range of 6 to 10 and a temperature range of 30 to 80°C (Fig. 4). Thermostability analysis showed that recombinant Pap was stable over a temperature range of 10 to 80°C and retained 76% of the maximum activity after incubation at 80°C for 1 h. The presence of Ca2⫹ (10 mM) caused a 21% increase in the activity of Pap. Na⫹, Mg2⫹, and Zn2⫹ had no apparent effect on the protease activity of Pap. Cytotoxicity analysis of the purified recombinant Pap. Cytotoxicity analyses showed that the presence of Pap significantly reduced the viability of FG cells in a time- and dosedependent fashion, whereas the presence of the proteolytically defective PapM had no apparent effect on FG cells (Fig. 5A and data not shown). The number of viable FG cells decreased linearly after treatment with 0.5 to 4 ␮g of Pap. Consistently, microscopic observation showed that incubation of FG cells




FIG. 6. Subcellular localization of the His-tagged AT1-Et18 chimera in E. coli DH5␣/pAT18. Proteins in the outer membrane (OM), cytoplasm (CP), periplasm (PP), and supernatant (SN) were prepared from DH5␣/pAT18 cells and analyzed by SDS-PAGE. As a negative control, outer membrane proteins of DH5␣/pAT1 (lane 6) were also subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane and blotted with anti-His antibody. NC, negative control. Lane 1, protein markers.

FIG. 5. Examination of the cytotoxic effect of purified recombinant Pap on cultured FG cells by cell viability analysis (A) and microscopy (B). (A) FG cells were treated for 0, 30, and 60 min with the same amount of recombinant Pap or PapM. Viabilities of the cells were determined by the MTT method. Data are the means from three independent assays and presented as the means ⫾ SE. *, P ⬍ 0.05; **, P ⬍ 0.001. (B and C) FG cells were treated with Pap (B) or PapM (C) as described for panel A. The cells were stained with Giemsa stain and observed under an inverted microscope. Images were taken at ⫻120 magnification. Bar ⫽ 50 ␮m.

with Pap had a profoundly damaging effect on the cells, which occurred in a time-dependent manner, beginning with disruption of cellular structures at 30 min after Pap treatment and progressing to complete cell lysis at 60 min after the treatment (Fig. 5B). In contrast, FG cells treated with PapM or PBS exhibited no morphological changes during the entire incubation period (Fig. 5C). Together, these results demonstrate that purified recombinant Pap can act as a cytotoxin on FG cells. Identification of PfaI domains that are essential to autotransporting activity. To determine the autotransporter regions that are required for translocator function in PfaI, three PfaI derivatives, AT1, AT2, and AT3, were constructed, each consisting of two segments of PfaI: (i) the N-terminal position 1 to 208 region and (ii) the C-terminal position 534 to 1242 (AT1), 614 to 1242 (AT2), or 771 to 1242 (AT3) region. To analyze the potential autotransporter activities of AT1, AT2, and AT3, we utilized a previously identified extracellular ␤-agarase, AgaV (60). Owing to its ability to degrade agar in LB medium, AgaV can cause the formation of visible craters around AgaV-secreting bacterial colonies on LB agar plates. With this feature, AgaV is an ideal reporter for the study of translocation properties of carrier proteins with secretion capacity. In this study, AgaV devoid of the secretion domain was inserted, as a passenger, into AT1, AT2, and AT3 immediately downstream from the N-terminal position 1 to 208 region (i.e., at the position Pap occupies in PfaI). The plasmids pAT1V,

pAT2V, and pAT3V, which express the coding elements of AgaV fused in-frame to AT1, AT2, and AT3, respectively, were introduced into E. coli DH5␣ cells by transformation. The transformants were plated on LB agar plates, and the plates examined for crater formation. Deep craters were formed around colonies of DH5␣/pAT1V, whereas no craters were formed around the colonies of DH5␣/pAT2V or DH5␣/ pAT3V (see Fig. S2 in the supplemental material). These results suggest that AT1, but not AT2 or AT3, can act as an effective autotransporter and deliver the heterologous passenger AgaV out of the cell. Application of AT1 as an antigen carrier. Since AT1 is an effective transporter of AgaV, we wondered whether it (i.e., AT1) could be used in the transport and surface display of bacterial antigens. We have previously identified an immunoprotective antigen, Et18 (17.8 kDa), from a virulent strain of Edwardsiella tarda (14), which is an important aquaculture pathogen that can infect many cultured marine species. Et18 is a periplasmic protein that, when used as a vaccine in the form of purified recombinant protein, can confer a certain protection (RPS, 61%) upon fish against E. tarda infection (14). To examine whether Et18 could be transported as a passenger by AT1, the mature and C-terminally His-tagged Et18 was inserted in-frame into AT1 between the N-terminal position 1 to 208 region and the C-terminal autotransporter domain. The plasmid pAT18, which expresses the coding element of the AT1-Et18 chimera, was introduced into E. coli DH5␣ by transformation. The subcellular localization of AT1-Et18 in DH5␣/ p〈⌻18 was determined by Western immunoblotting, which showed that the chimeric protein was detected in the outer membrane (Fig. 6). Consistently, whole-cell ELISA showed that DH5␣/p〈⌻18 could react with anti-His antibody (the A450 was 4.5-fold higher than that of the control), whereas DH5␣ harboring the control plasmid pAT1 could not (the A450 was comparable to that of the control). To examine the immunoprotective effect of DH5␣/pAT18, Japanese flounder were vaccinated separately with DH5␣/ pAT18, DH5␣/pAT1, purified recombinant Et18 protein, and PBS. The fish were challenged with the pathogenic E. tarda strain TX1 and monitored for mortality over a period of 2 weeks. The results showed that the cumulative mortality rates of the fish vaccinated with DH5␣/pAT18, Et18, DH5␣/pAT1, and PBS were 16.7, 33.3, 86.7, and 90%, respectively. Hence, the protection efficacy, in terms of RPS, of DH5␣/pAT18 was 81%, which was significantly higher than that of the recombinant Et18 (63%).

VOL. 75, 2009


DISCUSSION As mentioned earlier, most of the autotransporters with known functions are found to be implicated in bacterial pathogenicity (14). Since the effector molecules of autotransporters are either surface exposed on the outer membrane or secreted into the extracellular milieu, autotransporters contribute to virulence mainly by direct interaction with host cells and host defense systems (2, 6, 10, 25, 41, 48, 49, 53). In our study, we found that PfaI is required for interactions with cultured host cells. Since Pap appears as a soluble protein in TSS and recombinant Pap displayed a clear cytotoxic effect on FG cells, Pap probably acts as a cytotoxin, rather than an adhesin, during the course of TSS infection. This hypothesis agrees with the observation that mutation of pfa1 had a more profound effect on invasion into than attachment to FG cells. It is possible that Pap, being a protease, may function in the degradation of certain surface proteins of the host cell or extracellular matrix components and thus facilitate bacterial invasion. Likewise, PfaI-mediated resistance against serum bactericidal activity, which is an important innate immune response against bacterial infection, could be due to proteolysis of some complement factors by Pap, though it is also possible that Pap may inactivate the complement elements by negative interaction, as is found in the cases of some adhesin autotransporters (1, 26, 31, 38, 42). Like serum bactericidal activity, respiratory burst is another key innate defense mechanism by which phagocytes destroy invading pathogens. The observation that PfaI is implicated in modulation of these immune responses and that mutation of pfa1 significantly reduced the infectivity of TSS indicates that PfaI is an essential virulence factor of TSS. In line with this conclusion, pfa1 expression was augmented during infection and when the temperature was at 18°C, which approximates the in vivo temperature of the host animals (Japanese flounder), which are maintained at 18 to 22°C in most farms in north China. In the autotransporter secretion system, the fate of the passenger, once exported out of the cell, varies among different autotransporters. In some cases, the secreted passengers undergo no further processing, while in others the passengers are cleaved from the rest of the autotransporter domains as part of the maturation process (7). In the case where the passenger is a protease, the cleavage can be catalyzed by the passenger protease itself or by external peptidases of the host, as is found in the serine protease autotransporters of Enterobacteriaceae (11, 34, 40, 44, 46). The cleaved passenger molecule may either remain attached to the cell surface via noncovalent interactions or be released into the environment as a soluble protein. In our study, we found that Pap, the passenger protease of PfaI, is secreted into the culture supernatant of TSS, which suggests that Pap must have gone through certain processing steps involving proteolysis. Considering the facts that most autotransporter passengers with autocatalytic properties belong to MEROPS peptidase families S6/S8 and that Pap is classified, by its structural features, as an S8 family protease, it is possible that Pap may catalyze its own maturation with or without the facilitation of host cell factors. In support of this hypothesis, Et18 delivered by AT1, which lacks Pap, remained surface anchored. The results of recent studies have indicated that protein secretion via the autotransporter pathway is not as self-sufficient as had been initially thought and that other cellular com-


ponents, such as the outer membrane protein Omp85 (30, 32, 43, 50, 51, 56, 57) and the periplasmic chaperone Skp (36, 51), that are external to the autotransporter polypeptide are involved in the translocation process of many autotransporters. Nevertheless, the autotransporter is still the simplest form of protein secretion system and, perhaps, the least host specific. The simplicity of the autotransporter secretion system, together with the modular structure of the protein, renders it serviceable in several ways, one of which is in the development of surface display systems, in which the passenger domain of the autotransporter is replaced by a heterologous protein that, as a surrogate of the native passenger, is translocated to the cell surface by the ␤-domain of the autotransporter. Autotransporter-based surface display, or autodisplay, has been applied to the presentation on the bacterial surface of a wide range of molecules, including enzymes, antibody fragments, peptides, and antigens (reviewed in references 4, 15, and 17). In this study, we found that, like the E. coli autotransporter AIDA-I, which has been shown to be able to display various antigenic determinants (21, 23, 29, 37, 49), the PfaI derivative, AT1, could translocate the heterologous antigen Et18, in the place of Pap, to the cell surface. Compared to Et18 in the form of purified recombinant protein, Et18 displayed by AT1 is a more effective vaccine, which could be due to the possibilities that (i) compared to the soluble protein, Et18 covalently linked to the membrane-anchored AT1 is more stable; (ii) Et18, when presented on the cell surface, may assume a higher-order structure that is more readily recognized by the host immune system; and (iii) the live bacterial carrier may function as an adjuvant and facilitate the induction or/and heighten the intensity of the specific immune response induced by Et18. ACKNOWLEDGMENTS This work was supported by grant 40576071 from the National Natural Science Foundation of China (NSFC), grant 2006CB101807 from the National Basic Research Program of China, and grant 2008AA092501 from the 863 High Technology Project. REFERENCES 1. Ackermann, N., M. Tiller, G. Anding, A. Roggenkamp, and J. Heesemann. 2008. Contribution of trimeric autotransporter C-terminal domains of oligomeric coiled-coil adhesin (Oca) family members YadA, UspA1, EibA, and Hia to translocation of the YadA passenger domain and virulence of Yersinia enterocolitica. J. Bacteriol. 190:5031–5043. 2. Alamuri, P., and H. L. Mobley. 2008. A novel autotransporter of uropathogenic Proteus mirabilis is both a cytotoxin and an agglutinin. Mol. Microbiol. 68:997–1017. 3. Amend, D. F. 1981. Potency testing of fish vaccines. Dev. Biol. Stand. 49: 447–454. 4. Buddenborg, C., D. Daudel, S. Liebrecht, L. Greune, V. Humberg, and M. A. Schmidt. 2008. Development of a tripartite vector system for live oral immunization using a gram-negative probiotic carrier. Int. J. Med. Microbiol. 298:105–114. 5. Chen, Z., B. Peng, S. Wang, and X. Peng. 2004. Rapid screening of highly efficient vaccine candidates by immunoproteomics. Proteomics 4:3203–3213. 6. Conners, R., D. J. Hill, E. Borodina, C. Agnew, S. J. Daniell, N. M. Burton, R. B. Sessions, A. R. Clarke, L. E. Catto, D. Lammie, T. Wess, R. L. Brady, and M. Virji. 2008. The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable trimeric coiled-coil. EMBO J. 27: 1779–1789. 7. Dautin, N., and H. D. Bernstein. 2007. Protein secretion in gram-negative bacteria via the autotransporter pathway. Annu. Rev. Microbiol. 61:89–112. 8. Desvaux, M., N. J. Parham, and I. R. Henderson. 2004. Type V protein secretion: simplicity gone awry? Curr. Issues Mol. Biol. 6:111–124. 9. Economou, A., P. J. Christie, R. C. Fernandez, T. Palmer, G. V. Plano, and A. P. Pugsley. 2006. Secretion by numbers: protein traffic in prokaryotes. Mol. Microbiol. 62:308–319. 10. Felek, S., M. B. Lawrenz, and E. S. Krukonis. 2008. The Yersinia pestis




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