INTRODUCTION

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Aeromonas bacteria (aeromonads) are ubiquitous water-borne organisms that are also found in many foods. Strains of some Aeromonas species (primarily A. hydrophila HG1, A. veronii bv. sobria HG8/10, and A. caviae HG4) cause human gastroenteritis ("summer diarrhea"), particularly in children. They also cause more serious infections, such as septicemia and meningitis, in immunocompromised individuals (21, 25). Recently, aeromonads have been linked to cases of hemolytic-uremic syndrome (7, 9). Disease-associated strains possess a number of significant virulence determinants, including the ability to produce type IV pilus adhesins (14-16, 20, 30, 31) and the pore-forming toxin "aerolysin" (18, 50). Many Aeromonas strains grow at refrigeration temperatures, increasing concern about food-borne transmission (25, 26). Yet relatively little is known of the pathogenic mechanisms of Aeromonas species, and it is not possible to identify virulent strains definitively.

Colonization of the intestinal tract is likely to be a critical step in the disease process. Type IV pilus adhesins are essential for the colonization of the intestine by enteropathogens, such as Vibrio cholerae, enterotoxigenic Escherichia coli, and enteropathogenic E. coli (47). Our past studies have shown that gastroenteritis-associated Aeromonas species have the potential to express at least two distinct families of type IV pili (5). The predominant pilus type expressed on fecal isolates of A. veronii bv. sobria and A. caviae grown under standard in vitro conditions is the bundle-forming pilus (Bfp) (30, 31). Bfp pili have also been isolated from a strain of A. hydrophila, but fecal isolates of this species are often heavily piliated and express numerous type I pili (17). Bfp pili exhibit N-terminal sequence homology with the mannose-sensitive hemagglutinin pilus of V. cholerae. They have been purified from all Aeromonas species associated with diarrhea, but as yet this pilus type has not been genetically characterized (29). A second type IV Aeromonas pilus (Tap) was identified following the cloning of a biogenesis gene cluster (tapABCD) from a strain of A. hydrophila (strain Ah65) (42). Subsequent cloning of the tap cluster from a Bfp-positive strain of A. veronii bv. sobria (strain BC88) proved definitively that this cluster encoded a pilus type distinct from the purified Bfp pilus family. Tap pili differ from Bfp pili in their N-terminal sequences and molecular weights. They exhibit highest homology with the type IV pili of Pseudomonas aeruginosa and pathogenic Neisseria species (5). The tapA gene encodes the subunit protein. The tapB and tapC genes are probably involved in pilus biogenesis (49). The protein encoded by tapD is a type IV prepilin peptidase/N-methyltransferase which is responsible for the processing of several components of the general secretion pathway which mediate secretion of extracellular proteins, including aerolysin. TapD cleaves the 6-amino-acid leader peptide from prepilin and catalyzes the methylation of the N-terminal residue (19, 42, 47). Similar pilus gene clusters have been identified in V. cholerae (pil cluster), P. aeruginosa, and several other gram-negative bacteria. For many of these organisms, the encoded type IV pili have been shown to be important virulence factors, but for V. cholerae epithelial cell adherence or intestinal colonization functions could not be attributed to the pilus structure encoded by the pil cluster (10).

While there is increasing evidence that the Aeromonas Bfp pili are intestinal colonization factors (29), the significance of Tap pili for Aeromonas virulence is unknown. The aim of this study was to investigate the role of Tap pili in the pathogenesis of Aeromonas infection. The adhesive abilities of isogenic tapA mutant strains of A. hydrophila Ah65 and the dysenteric isolate of A. veronii bv. sobria, strain BC88, to HEp-2 cells were compared with those of the respective wild-type strains. The latter strain and its tapA mutant were also compared for their ability to adhere to intestinal cells and tissue and to produce diarrhea and/or colonize animals (rabbits and infant mice). Wild-type and selected tapA and tapD mutant strains were used to examine TapA expression using polyclonal antisera raised against His-Tag-TapA fusion proteins.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Additional details of the Aeromonas strains examined are given in the Results section. Aeromonas strains were grown (37°C, 18 to 24 h) from storage on tryptone soy agar (TSA) supplemented with 6.0 g of yeast extract L21 (TSAY; Oxoid, Basingstoke, United Kingdom) per liter. For genetic manipulations, Aeromonas veronii bv. sobria strains were grown in brain heart infusion broth (BHIB; Oxoid), tryptone soy broth (TSB; Oxoid), or tryptone soy broth containing yeast extract (TSBY; Oxoid), as described above. A. hydrophila Ah65N was routinely grown at 22°C in TSB or BHIB. Transconjugants were grown on nutrient broth agar (NBA; Oxoid) or brain heart infusion agar (BHIA; Oxoid). E. coli was grown in Luria-Bertani (LB) medium (45). For Escherichia coli, the following antibiotic concentrations (in micrograms/milliliter) were used: spectinomycin, 50; carbenicillin, 100; ampicillin, 150; and chloramphenicol, 30. For Aeromonas, the antibiotic concentrations (in micrograms per milliliter) were: spectinomycin, 50; ampicillin, 150; chloramphenicol, 2.5; and nalidixic acid, 5. Isopropyl--D-thiogalactopyranoside (IPTG) was used at a final concentration of 1 mM. For exotoxin assays, Aeromonas spp. were grown in TSBY, at 37°C, for 24 h with shaking. For rabbit pathogenicity and mouse colonization experiments, Aeromonas wild-type and tapA mutant strains, and the E. coli K-12 surgical control strain EC101, were grown from stored cultures on BHIA at 35°C for 24 h. Log-phase cultures were then prepared in BHIB at 35°C for 18 h (static).

DNA preparation and manipulations. For small-scale plasmid preparations, E. coli DH5 served as the host strain, and the alkaline lysis procedure was followed (6). Restriction endonuclease digestion, ligation, and transformation and DNA electrophoresis were performed as described by Sambrook et al. (45). Plasmids were introduced from E. coli S17-1pir into A. hydrophila and A. veronii bv. sobria by conjugation.

Construction of tap mutant strains. A map of the tap gene cluster of A. hydrophila Ah65 is shown in Fig. 1. Ah65 strains with mutations in tapA and tapD (Ah65N-A18 and Ah65N-D33.2, respectively) were constructed. These were prepared by allelic exchange of the wild-type copies of these genes with  interposon-disrupted copies encoding spectinomycin-streptomycin resistance. To create the tapA mutation, the  interposon from pUC19 was cloned as a SmaI fragment into a blunted PstI site within tapA resulting in pCP1180. A 3.1-kb SalI-XbaI fragment [both sites originating from the pBluescript II SK() polylinker] from pCP1180 carrying the tapA gene was then inserted into pJQ200KS digested with the same enzymes, generating pCP1182.

View larger version (6K): [in this window] [in a new window]   FIG. 1.   The tap gene cluster of A. hydrophila Ah65 is encoded on a 5.5-kb DNA fragment as shown. Arrows indicate the direction of transcription of the open reading frames. Restriction sites of interest are indicated.

In vitro characterization of the tapA mutant strain of A. veronii bv. sobria BC88. Bacterium-free broth supernatants from the wild-type and tapA mutant strains of A. veronii bv. sobria strain BC88 were examined for hemolytic activity against rabbit red blood cells, cytotoxic activity for Vero cells, and enterotoxic activity in suckling mice as described elsewhere (26, 32). The hemolysin titer was recorded as the last broth dilution showing 50% hemolysis, while the cytotoxin titer was recorded as the last broth dilution causing 50% of the cells to round up or die.

Construction of expression plasmids. To construct a tapA-overexpressing plasmid of A. hydrophila strain Ah65, a KpnI site was created upstream of the coding region (GGAACC changed to GGTACC at positions 202 to 207 of the tapABCD sequence; EMBL-GenBank-DDBJ Data Libraries accession number U20255) by PCR using Kpn-A (5'-CAC TTC CCA GGT ACC AAG GAC AAA A-3') and T3 (5'-ATT AAC CCT CAC TAA AG-3') as primers and pCP1065 as a template. The 0.79-kb product was digested with KpnI, producing a 0.65-kb fragment that was inserted into pMMB67HE.cam to generate pCP1194. To construct a His-Tag-tapA fusion plasmid, a BamHI site was introduced into pCP1065 by inserting a BamHI linker (10-mer) into the NruI site of tapA, generating pCP1178. Next, the 0.7-kb BamHI fragment from pCP1178, containing a truncated tapA gene (corresponding to amino acids 11 to 136 of the mature pilin), was inserted in frame into the His-Tag cloning vector, pET15b (Novagen), resulting in pCP1183.

Purification of His-Tag-TapA fusion proteins and production of anti-TapA antisera. To prepare antisera against TapA from A. hydrophila Ah65 and A. veronii bv. sobria strain BC88, overnight cultures of E. coli BL21(DE3) harboring pCP1183 or pTB028 were inoculated (1:100) into 50 ml of LB broth containing carbenicillin and grown at 37°C. When the cultures reached an optical density at 600 nm (OD₆₀₀) of ~0.6, IPTG was added, and the cells were grown for 5 h at 37°C with vigorous shaking. Preparation of lysates and purification of the ~15-kDa His-Tag-TapA fusion proteins were carried out according to the manufacturer's protocol (Xpress System; Invitrogen). Due to the insolubility of the A. veronii bv. sobria His-Tag-TapA fusion, it was necessary to purify this protein by successive solubilization in urea (44). The TapA protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel slice containing it was emulsified in 1 ml of complete Freund's adjuvant. Antisera were prepared either in New Zealand White rabbits by R & R Rabbitry Research Development (for Ah65 TapA) or in New Zealand White rabbits held at the University of Tasmania Animal Facility (for BC88 TapA) by comparable methods. Briefly, rabbits (n = 3) were administered ~100 µg of His-Tag-TapA protein subcutaneously into five sites. Booster injections of the SDS-PAGE His-Tag-TapA protein band in incomplete Freund's adjuvant were given at days 21, 35, and 49. Trial bleeds were collected on days 28, 42, and 56, and a volume bleed was done at day 63. Serum from the volume bleed of the rabbit showing the highest reactivity was used in this study. In both cases, antisera were rendered specific for TapA by absorption against an acetone powder of Ah65-A18 or BC88tapA.

Cell fractionation and Western blotting. Whole-cell lysate samples were prepared by mixing 10 µl of an overnight TSB culture with 2.5 µl of 5× sample buffer (0.3125 M Tris-HCl, pH 6.8; 50% [vol/vol] glycerol; 10% [wt/vol] SDS; 0.25% [vol/vol] 2-mercaptoethanol; 0.5% [wt/vol] bromophenol blue). For cell fractionation experiments, periplasmic contents were extracted by osmotic shock (52). Briefly, overnight cultures grown under the appropriate conditions were diluted 1:20 in TSB and incubated until the cultures reached an OD₆₀₀ of ~2. Bacteria from a 2.5-ml sample were recovered by centrifugation (15,000 × g, 5 min). They were gently resuspended in 1 ml of ice-cold 33 mM Tris-HCl (pH 8)-1 mM EDTA-0.5 M sucrose and held on ice for 10 min. The cells were pelleted at low speed (6,000 × g, 2 min) and then gently resuspended in 1 ml of ice-cold 0.5 mM MgCl₂ and held on ice for 10 min. After centrifugation (4,000 × g, 2 min), the periplasmic contents were recovered in the supernatant. The cytoplasmic contents and membrane fractions were then extracted from the pellet (35). In brief, the pellet was resuspended in 150 µl of 100 mM Tris-HCl (pH 8)-0.5 mM EDTA-0.5 M sucrose containing 15 µl of a 2-mg/ml mixture of lysozyme and 150 µl of cold distilled H₂O and held on ice for 5 min before centrifugation (15,000 × g, 3 min, 4°C). The pellet was subsequently resuspended in 1 ml of 10 mM Tris-HCl (pH 8) and subjected to three freeze-thaw cycles in liquid nitrogen, after which 33 µl of 1 M MgCl₂ containing 10 µl of a 1-mg/ml mixture of DNase I was added. After centrifugation (15,000 × g, 25 min, 4°C), the cytoplasmic contents were recovered in the supernatant, and the pellet contained the membrane fractions. For each of the fractionated samples, 20-µl aliquots were mixed with 5 µl of 5× sample buffer. SDS-PAGE was performed as described by Laemmli using discontinuous 15 or 18.5% acrylamide gels (34). The proteins were transferred to nitrocellulose (48), incubated with anti-TapA polyclonal antiserum (see above), and visualized with goat anti-rabbit alkaline phosphatase conjugate (Promega).

Electron microscopy. Bacterial cells were negatively stained with either 2% phosphotungstic acid (pH 7.2) on parlodion-coated grids or 1% uranyl acetate on Formvar-coated copper grids (28, 30). They were examined with a JEOL 100-B transmission electron microscope operated at 60 kV or a Philips 410 electron microscope at 80 kV. For immune electron microscopy (IEM), bacteria on Formvar-coated grids were washed briefly in a drop of TTB buffer (20 mM Tris-HCl, 25 mM NaCl, 0.1% [wt/vol] bovine serum albumin, 0.05% [vol/vol] Tween 20; pH 8.2) and floated on a drop of 5% bovine serum albumin in TTB for 15 min. The grids were then washed three times in TTB and reacted with 10-fold dilutions of TapA antiserum (1:10 to 1:1,000) for 60 min. Grids were again washed (three times in TTB). They were then exposed (60 min) to goat anti-rabbit immunoglobulin G conjugated with 10-nm gold particles (BioCell, Cardiff, United Kingdom) diluted 1:50 in TTB. The grids were subsequently washed and negatively stained. A 1:100 dilution of Bfp antiserum served as a positive control for the IEM (30).

Purification of pili. A. hydrophila strains containing plasmids of interest were streaked from 80°C glycerol stocks onto TSA containing appropriate antibiotics and grown at 22°C overnight. For each strain, a single colony was inoculated into 10 ml of TSB containing antibiotics and incubated overnight at 22°C without shaking. Eight 1-liter flasks each containing 500 ml of TSB plus antibiotics were inoculated with 1 ml from the 10-ml overnight culture and then incubated at 22°C for 72 h (static). After the bacteria were harvested, the pili were sheared and purified according to a procedure described elsewhere (30). For Western blotting of pilus preparations, samples containing 1 µg of total protein in 10 µl of 0.5 M Tris-HCl (pH 7.5) were mixed with 2.5 µl of 5× sample buffer.

Adhesion assays. Adhesion of bacteria to HEp-2 epithelial cells, Henle 407 intestinal cells, and fresh human intestinal tissue was assessed by bright-field microscopy (8, 27, 29). In brief, 1-ml aliquots of 5 × 10⁶ CFU were inoculated onto the semiconfluent coverslip cultures of the cell lines grown in Eagle minimal essential medium containing 5 to 10% fetal calf serum (MEM-FCS) or onto fresh samples of intestinal tissue in MEM-FCS in 24-well tissue culture plates. After incubation (60 min, 37°C, 5% CO₂), nonadherent bacteria were removed by washing (four times in phosphate-buffered saline [PBS]). The cell monolayers were fixed with 3:1 methanolacetic acid (1 ml, 5 min), stained with May-Grünwald and Giemsa stains (BDH, Poole, United Kingdom), and mounted for counting. At least three coverslip cultures were assayed for each strain in each experiment. Intestinal specimens were fixed in formalin after washing and then embedded in paraffin and sectioned. Sections (~10 µm) on glass slides were deparaffinized, hydrated, and stained with hematoxylin-eosin for light microscopic examination (29).

Removable intestinal tie adult rabbit diarrhea (RITARD) model. Wild-type and tapA mutant Aeromonas strains were tested in New Zealand White rabbits (1,250 to 1,650 g; 7 to 9 weeks of age; 3 to 4 weeks postweaning) according to the protocol of Pazzaglia et al. (41). Each strain was tested in nine rabbits. Five control rabbits received E. coli EC101. Each rabbit received 10¹⁰ CFU in 10 ml of BHIB injected into the jejunum close to the ligament of Treitz. Animals were monitored for 7 days for diarrheal symptoms and shedding of Aeromonas organisms in feces. The animals were sacrificed on day 8 postchallenge.

Infant mouse colonization. BALB/c mice, obtained from a breeding colony held at the University of Tasmania, were inoculated orally with bacteria under test conditions according to the protocol of Attridge et al. (3). Log-phase cultures of the wild-type and mutant Aeromonas strains were diluted to obtain a culture containing 2 × 10⁸ CFU per ml of each strain, and 5 µl of blue food coloring was added to facilitate the monitoring of the inoculation procedure. Three- to five-day-old infant mice were taken from their mothers 4 h prior to oral infection. They were inoculated by gastric lavage with 50 µl of bacterial suspension (~10⁷ CFU per mouse) and held at 25°C for 24 h, after which time they were sacrificed and their intestines were removed. The intestines were homogenized in 5 ml of PBS, and Aeromonas bacteria were quantitated by plate counts of serial dilutions of these homogenates on TSAY.

Statistical analysis. The differences in adherence to cell lines by wild-type and tapA mutant strains and between groups of mice inoculated with these strains were analyzed by the Student's t test using Microsoft Excel software.

	RESULTS

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Construction of mutant strains. Initially, tapA and tapD mutants (Ah65N-A18 and Ah65N-D33.2, respectively) of A. hydrophila Ah65N were constructed as described in Materials and Methods. Strain Ah65 was originally isolated from rainbow trout (Salmo gairdneri) and called "A. hydrophila" (36). Its 16S ribosomal DNA sequences, however, were identical to those of the HG2 definition strain (A. M. Carnahan, personal communication). Ribotyping was unable to identify it definitively but putatively classified it as belonging to HG3 (M. Altwegg, personal communication). The strain was relatively poorly adherent to epithelial and intestinal cell lines and thus proved a poor choice for in vivo experiments designed to investigate the role of Tap in intestinal colonization and virulence. Hence, a tapA mutant strain of a dysenteric isolate of A. veronii bv. sobria (strain BC88) was subsequently constructed for such functional investigations. Strain BC88 was originally isolated (in 1983) at the Princess Margaret Hospital, Perth, Western Australia, from the stool of a child with bloody diarrhea. The virulence-associated factors of this strain included the ability to produce enterotoxin (positive suckling mouse assay), cytotoxin (Vero cell assay), and hemolysin (titer of >512 versus rabbit erythrocytes). It was also able to invade HEp-2 cells and was highly adhesive to epithelial and intestinal cell lines and intestinal tissue. In addition, it was the strain from which we had purified and characterized the type IV bundle-forming pilus colonization factor (29, 30).

Exotoxic activities of A. veronii bv. sobria strain BC88 and A. hydrophila tapA mutant strains. Mutation in tapA did not affect the ability of strain BC88 to produce exotoxic activities. The hemolytic titer of the mutant was 1,024, as was the titer of the wild-type strain (200 µl of broth supernatant in the first well, doubling dilutions in PBS; 37°C, 1 h; 4°C, 1 h). Cytotoxic titers of both strains for Vero cells were identical at 128 (50 µl of broth supernatant in 150 µl of MEM in the first well; doubling dilutions in MEM; 40 min, 37°C, 5% CO₂). Supernatants (100 µl) from both strains also gave positive results (intestinal-weight/remaining-body-weight ratios of 0.087 and 0.085 for the wild-type and mutant strains, respectively) in the suckling mouse enterotoxin assay (eight mice per group). The hemolytic titers in the tapA mutant of A. hydrophila Ah65 were not significantly different from those of the wild-type strain. However, mutation in tapD decreased the hemolytic activity titer in broth supernatants from 128 in the wild-type to 0 (42).

Effect of tapA mutation on epithelial and intestinal cell adhesion. A. hydrophila Ah65 was relatively poorly adherent to HEp-2 cells (<8 bacteria per cell). Studies of quantitative counts of bacterial adherence to HEp-2 cells, however, showed no difference between numbers of wild-type (Ah65N) or tapA mutant (Ah65N-A18) bacteria recovered from cells. The percentages (means ± standard deviations) of cell-associated bacteria were 17.6 ± 2.4 and 19.4 ± 1.8 for the wild-type and tapA mutant strains, respectively.

Virulence of the tapA mutant of A. veronii biovar sobria BC88 in the rabbit (RITARD) model. Wild-type and tapA mutant strains of A. veronii bv. sobria BC88 were compared for virulence in the RITARD model. The results are summarized in Table 2.

Effect of tapA mutation on the intestinal colonization of infant mice by A. veronii bv. sobria BC88. Wild-type and tapA mutant strains were also compared for their ability to colonize the intestines of infant mice. Strains were administered alone or in competition experiments in which both strains were administered to the same animal (Fig. 2). In the former case, the colonization of infant mice by A. veronii bv. sobria strain BC88 wild-type and tapA mutant strains yielded the following recovery results: wild-type strain (inoculum of 1.4 × 10⁷), 3.1 × 10⁷ ± 4.4 × 10⁷ CFU; and tapA mutant strain (inoculum of 2.1 × 10⁷), 2.6 × 10⁷ ± 2.0 × 10⁷ CFU. The inoculum in each case was determined retrospectively by use of plate counts. The recovery was calculated as the total number of bacteria (CFU ± the standard deviation) recovered from intestinal tissue after 24 h (six mice per group).

View larger version (10K): [in this window] [in a new window]   FIG. 2.   Comparative ability of A. veronii bv. sobria strain BC88 wild-type and tapA mutant strains to colonize infant mice. Mice were fed mixed suspensions of wild-type and mutant bacteria. Input ratios are indicated below each test group (two experiments). Each point represents the wild-type/mutant output ratio of bacteria recovered from the intestines of individual mice (seven and six mice for experiments 1 and 2, respectively).

Examination of TapA expression. To evaluate the significance of these functional investigations, it was important to determine whether TapA was expressed and able to be assembled on the bacterial cell surface. To this end, antiserum to TapA of A. hydrophila Ah65N and A. veronii bv. sobria strain BC88 were prepared using His-Tag-TapA fusion proteins as described in Materials and Methods. The TapA proteins of these two strains are antigenically distinct. Hence, it was necessary to prepare antiserum to TapA of each strain. For A. hydrophila strain Ah65N, anti-TapA serum was used to examine the wild type and the tapA and tapD mutants and their respective complemented mutant strains for Tap pilin expression by using Western blotting. More limited experiments were done with A. veronii bv. sobria strain BC88. A tapA complemented strain of this organism was not examined, nor was a TapD mutant. However, bacterial shearing experiments and IEM were performed with both strains to investigate whether Tap pili were assembled on the cell surface.

View larger version (32K): [in this window] [in a new window]   FIG. 3.   Western immunoblot of whole-cell extracts from A. hydrophila Ah65N probed with Ah65 TapA polyclonal antiserum. Lane 1, Ah65N (wild-type) plus pMMB67HE.cam (vector); lane 2, Ah65N-A18 (tapA mutant) plus pMMB67HE.cam; lane 3, Ah65N-A18 (pCP1194) (tapA complemented strain); lane 4, Ah65N-D33.2 (tapD mutant) plus pMMB67HE.cam; lane 5, Ah65N-D33.2 (pCP1140) (tapD complemented strain). Numbers at the left represent protein molecular masses in kilodaltons.

View larger version (91K): [in this window] [in a new window]   FIG. 4.   Western immunoblot of A. veronii bv. sobria strain BC88 whole-cell extracts probed with BC88 TapA polyclonal antiserum. Lane 1, BC88 (wild-type); lane 2, BC88tapA (tapA mutant). Numbers at the left represent protein molecular masses in kilodaltons.

Effect of mutation of tapD on the production of TapA by A. hydrophila Ah65N. Whole-cell lysates from Ah65N-D33.2 (tapD mutant), containing either the vector, pMMB67HE.cam alone, or a TapD-expressing plasmid (pCP1140) were also examined by Western blotting (Fig. 3, lanes 4 and 5, respectively) to determine if, as for other type IV pilus gene homologs, the type IV peptidase encoded by tapD processes the tapA prepilin into a form that can be assembled into a pilus structure.

View larger version (43K): [in this window] [in a new window]   FIG. 5.   Western immunoblot of the cellular fractions of A. hydrophila Ah65N and A. veronii bv. sobria strain BC88. (A) wt, Ah65N (wild type); tapD, Ah65N-D5 (tapD mutant). (B) wt, BC88 (wild type); PERI, periplasm; CYTO, cytoplasm; MEM, membranes. Unprocessed pre-TapA and mature TapA pilin species are indicated on the right, with molecular mass standards on the left.

Detection of TapA on the bacterial cell surface. To determine if TapA is assembled into pili on the cell surface, electron microscopic examination of wild-type A. hydrophila Ah65N and isogenic tapA and tapD mutant strains (Ah65N-A18 and Ah65N-D5) was undertaken. Bacteria were grown on TSAY at 22°C and negatively stained. All three strains displayed numerous pili. IEM with anti-TapA serum could not establish whether any of the filamentous structures were Tap pili and, hence, whether they were missing in mutant strains (data not shown). Previous studies with A. hydrophila isolates have shown that the vast majority of pili on the surface of this species are "short-rigid," type I pili (13, 17, 28).

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Western immunoblot of pili sheared from A. hydrophila strain Ah65N wild-type and mutant strains probed with TapA polyclonal antiserum. Lane 1, Ah65N (wild-type); lane 2, Ah65N-A18 (tapA mutant); lane 3, Ah65N-A18 + pCP1194 (tapA complemented strain); lane 4, Ah65N-D33.2 (tapD mutant). The TapA pilin species is indicated on the right, molecular mass standards on the left.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

This study has examined the expression and functional significance of a second Aeromonas type IV pilus, Tap, identified following the cloning of its biogenesis gene cluster, tapABCD. This pilus gene cluster is widespread in Aeromonas species and has homologs in a number of other gram-negative bacteria (4, 10). Tap pili are distinct from the Bfp type IV pili which are expressed on diarrhea-associated Aeromonas species and are known to be important intestinal cell adhesins and colonization factors (14, 15, 20, 29, 37). In contrast to Bfp pili, Tap pili have never been isolated from Aeromonas species, and there have been no previous studies to investigate their significance for Aeromonas virulence. For P. aeruginosa, related pili are a major virulence-associated adhesin (12) and also play an important role in microcolony formation in biofilms (39). For V. cholerae, however, for which the organization of the homologous gene cluster, pilABCD, shows a striking similarity to that of the Aeromonas tap gene cluster (genes grouped together and transcribed in the same direction), this pilus type is reportedly not important for adhesion to HEp-2 cells or for the colonization of infant mice (10). Moreover, it is not required for V. cholerae adherence to some solid substrates, questioning its role in adherence in the environment, despite its 100% conservation between the classical and El Tor biotypes (51).

We prepared and used specific mutants of two Aeromonas strains (A. hydrophila Ah65, the strain from which the tap gene cluster was originally cloned, and a dysenteric, fecal isolate of A. veronii bv. sobria, strain BC88) to investigate the expression and function of Tap pili. The A. veronii bv. sobria strain was chosen for the in vivo functional studies because the poor cell adhesion and low virulence of strain Ah65 made it unsuitable for in vivo studies. It was established that mutations in tapA of this strain did not affect the production of exotoxins considered important for diarrhea induction.

Inactivation of the pilus subunit gene, tapA, had no effect on the adherence ability of either of the above Aeromonas strains to HEp-2 cells. For A. veronii bv. sobria strain BC88, adherence to the intestinal cell line (Henle 407) and intestinal tissue was also not significantly different for the wild type and for the tapA mutant strain. Since type I and Bfp pili are the predominant pilus types seen on these bacterial strains (17, 30), this result may not be entirely unexpected. Experiments aimed at visualizing Tap pili on the bacterial cell surface by IEM were not successful, despite the growth of A. veronii bv. sobria strain BC88 under conditions previously shown to increase non-Bfp pilus expression (30). It is possible that the TapA antisera did not recognize the proteins in their native conformation. (TapA antisera were prepared against denatured recombinant proteins prepared by SDS-PAGE.) Furthermore, it is possible that the His-Tag sequence or the absence of disulfide bonds in the recombinant proteins may have altered their folding and, hence, their antigenicity compared to the native proteins. However, expression studies did establish that TapA was produced and present at the cell surface. Western blots of wild-type and tapD mutant and complemented strains established that TapA is processed by the type IV leader peptidase/N-methyltransferase, TapD and localizes in the cell membrane. Shearing experiments using Western blot comparisons of TapA from wild-type and mutant strains suggested that TapA was most likely present in the form of pili. There are other possible explanations for the detection of TapA in sheared preparations from the wild type but not the tapD mutant. TapA pilin may not be assembled into pili in Aeromonas species but may be more surface accessible in the wild type compared to a mutant carrying a lesion in the type IV peptidase. In this case, failure to cleave off the prepilin leader peptide would prevent it from crossing into the outer membrane. However, this is unlikely since other investigators have shown that processed and unprocessed pilin is distributed equally in the cytoplasmic and outer membranes in P. aeruginosa (38). Another possible explanation is that the assembly of Tap pili is regulated by, or requires the presence of, another protein with a role similar to that postulated for PilC in the assembly of Neisseria gonorrhoeae type IV pili (23). Under the conditions tested here, this protein may not be expressed in sufficient quantity to promote assembly of large numbers of Tap pili on the cell surface. In any event it is clear that if Tap pili are present on the surface of Aeromonas spp. they exist in only small numbers under standard bacterial growth conditions.

For Tap pili to play a role in vivo, conditions in the intestine should favor expression. However, in in vivo experiments, mutation of TapA did not affect intestinal colonization of rabbits or infant mice or significantly alter the ability of Aeromonas spp. to cause diarrheal symptoms in the RITARD model. Symptoms caused by A. veronii bv. sobria strain BC88 in rabbits were not severe in comparison to effects observed with other enteropathogenic bacteria such as V. cholerae (2) and Providencia alcalifaciens (1). Nevertheless, both the wild-type and mutant strains caused significant, short-lived diarrhea (55%, 10 of 18 rabbits overall inoculated with either strain) compared with the E. coli surgical controls which showed no symptoms. In two mouse models, there was also no evidence that the mutation decreased colonization ability. These latter results are in agreement with those of the V. cholerae pilA mutant studies in mice (10). Discernible, short-lived differences in colonization could have been missed in these in vivo studies, however, given the presence of the Bfp pilus intestinal colonization factor. Mutagenesis of the latter awaits the cloning of the Bfp pilin gene.

Further studies are required to identify factors that may influence the expression of Tap pili and to determine why the genes encoding them (and related pili in V. cholerae) are so widely conserved. The widespread distribution of the tap gene cluster in all Aeromonas species (including nonclinical species) and the results obtained in this study, however, suggest that Tap pili are not as significant as Bfp for intestinal colonization by diarrheagenic Aeromonas species.