AEM
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yu, H. B.
Right arrow Articles by Leung, K. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yu, H. B.
Right arrow Articles by Leung, K. Y.
Agricola
Right arrow Articles by Yu, H. B.
Right arrow Articles by Leung, K. Y.
Applied and Environmental Microbiology, August 2005, p. 4469-4477, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4469-4477.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of Putative Virulence Genes and Gene Clusters in Aeromonas hydrophila PPD134/91

H. B. Yu,1 Y. L. Zhang,2 Y. L. Lau,1 F. Yao,1 S. Vilches,3 S. Merino,3 J. M. Tomas,3 S. P. Howard,4 and K. Y. Leung1*

Department of Biological Sciences, Faculty of Science, National University of Singapore, Science Drive 4, Singapore 117543, Republic of Singapore,1 Department of Immunology, University of Washington, Seattle, Washington 98195-7650,2 Department of Microbiology, University of Barcelona, Barcelona, Spain 08071,3 Department of Microbiology and Immunology, Faculty of Medicine, University of Saskatchewan, Saskatoon, Canada S7N 5E54

Received 29 November 2004/ Accepted 14 February 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Aeromonas hydrophila is a ubiquitous gram-negative bacterium of aquatic environments, which has been implicated as a causative agent of motile aeromonad septicemia in a variety of aquatic animals (especially freshwater fish species) (2, 37). It causes gastrointestinal and extraintestinal infections in humans, including septicemia, wound infections, and gastroenteritis (16). A number of virulence factors have been identified in A. hydrophila, namely, pili and adhesins (29, 30), O-antigens and capsules (23, 48, 49), S-layers (10), exotoxins such as hemolysins and enterotoxin (7, 14), and a repertoire of exoenzymes which digest cellular components such as proteases, amylases, and lipases (20, 28).

The pathogenesis of A. hydrophila is multifactorial. Most studies to date have concentrated on the characterization of a few virulence factors in different animal models by using different strains (10, 34, 42), making it very difficult to evaluate the significance of each gene in the virulence of A. hydrophila. This study aims to identify more putative virulence determinants and characterize them in an integrated manner.

Suppressive subtraction hybridization or genomic subtraction offers a genome-level approach to identifying the genetic differences between virulent and avirulent strains of bacteria (22, 47). In this study, we used genomic subtraction to identify 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames (ORFs), which are frequently present in a group of virulent strains of A. hydrophila. In addition, four genomic islands (GIs), which differ from the rest of the genome in G+C content and which carry mobility-associated genes (integrases or transposes) and putative virulence genes, were found. These are genes expressing the O-antigen and capsule (48, 49), a phage-associated gene cluster, and a type III secretion system (TTSS) gene cluster. Subsequently, these putative virulence-associated genes were located on a preliminary physical map of A. hydrophila PPD134/91 to determine their genetic organization in A. hydrophila, which will provide insight into the molecular basis of pathogenicity of this bacterium.

We further studied the individual or combined roles of these putative virulence genes using a consistent animal model and in the same strains. Knockout mutations were constructed to elucidate the contributions of a number of these virulence genes to A. hydrophila pathogenesis. Of the single-knockout mutations examined, only that in the TTSS ({Delta}ascN) significantly affected the 50% lethal doses (LD50s) in a blue gourami infection model. In addition, a triple deletion mutant of S-layer, serine protease and metalloprotease ({Delta}ahsA {Delta}serA {Delta}mepA) increased the LD50 to the same level as the ascN mutant.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Bacterial strains and plasmids.
The bacterial strains and plasmids used in this study are listed in Table 1. A. hydrophila strains were maintained on tryptic soy agar or in tryptic soy broth (Difco/Becton Dickinson) at 25°C. Escherichia coli strains were maintained on L agar or in Luria broth (LB) (Difco/Becton Dickinson) at 37°C. When required, media were supplemented with ampicillin (100 µg/ml) or chloramphenicol (30 µg/ml). The conjugative transfer of plasmids between A. hydrophila and E. coli strains was carried out by plate mating at 30°C for 24 h.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Bacterial strains and vectors used in this study

 
DNA manipulations and Southern hybridization.
Bacterial genomic DNA was extracted using Genomic DNA isolation/purification kits (QIAGEN). Plasmid DNA was extracted using QIAprep spin miniprep kits. Restriction-digested products of PPD134/91 genomic DNA with PacI and EcoRI were cloned into pNEB193 vector (New England Biolabs), which was also cut by the same two enzymes, transformed into E. coli JM109-competent cells, and sequenced. Southern blotting was performed to characterize the distribution of the putative virulence genes of A. hydrophila PPD134/91 in other motile aeromonads with the BluGene Non-Radioactive Nucleic Acid Detection system (Gibco-BRL). Transfer of the DNA to a nylon membrane (GeneScreen, NEM Research Products), hybridization conditions, and visualization with streptaviridin-alkaline phosphate conjugates were carried out as recommended by the manufacturer.

DNA sequencing and sequence analysis.
DNA sequencing was carried out on an Applied Biosystems PRISM 3100 genetic analyzer with an ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems). The sequences were edited using the manufacturer's software. Sequence assembly and further editing were carried out with Vector NTI DNA analysis software (InforMax). BLASTN, BLASTP, and BLASTX sequence homology analyses and a protein conserved-domain database analysis (CDD Search) were performed using the BLAST network server of the National Center for Biotechnology Information.

Genome walking and genomic subtraction.
Advantage Polymerase 2 (Clontech) was used for genome walking. Genome Walker libraries were constructed by using five restriction enzymes (DraI, EcoRV, PvuII, ScaI, and StuI). The cycling parameters for genome walking were as follows: 7 cycles of 15 s at 94°C, 4 min at 72°C, 32 cycles of 15 s at 94°C, and 3 min at 67°C. The amplified fragments were cloned into the pGEM-T Easy Vector (Promega), transformed into E. coli JM109-competent cells, and sequenced. Bacterial genome subtraction was performed as described previously by Zhang and coworkers (47).

Construction of defined insertion mutants and deletion mutants.
To obtain defined insertion mutants, oligonucleotides containing flanking XbaI restriction sites (primers sequences available upon request) were used to amplify internal fragments from the targeted genes. The amplified fragment was ligated to pGEM-T Easy vector and transformed into E. coli JM109. The internal fragment was recovered by XbaI restriction digestion and ligated to XbaI-digested and dephosphorylated suicide vector pRE112 (11). The recombinant plasmid was transformed into E. coli MC1061({lambda}pir), selecting for Cmr to isolate the pRE-112-derived plasmids. These Plasmids were transformed into E. coli S17-1({lambda}pir) and transferred by conjugation to A. hydrophila AH-1 (Apr), selecting for Cmr and Apr colonies. The insertion of plasmids into the chromosomes of these mutants was confirmed by PCR using appropriate primers.

Nonpolar deletion mutants were constructed according to the method for suicide plasmid pRE112 (11). Briefly, the targeted gene and at least 300 bp of flanking sequences were amplified with oligonucleotides containing XbaI restriction sites and then cloned into pGEM-T easy vector, followed by inverse PCR using these constructs as templates. The inverse PCR products were purified and self ligated to get the deleted constructs of targeted genes. The deletion constructs were digested with XbaI and ligated to XbaI-digested and dephosphorylated pRE112. This construct was transformed into E. coli S17-1({lambda}pir). The single-crossover mutants were obtained by conjugal transfer into A. hydrophila AH-1. Double-crossover mutants were obtained by selecting against the presence of the sacB gene carried by the vector. Mutants resistant to sucrose were isolated by being plated onto LB-sucrose agar (1% tryptone, 0.5% yeast tract, 1.5% agar, 12% sucrose). The double-crossover mutants were confirmed by PCR.

LD50 studies of fish.
Healthy blue gourami (Trichogaster trichopterus Pallas) were obtained from a commercial fish farm, maintained in well-aerated dechlorinated water at 25 ± 2°C, and acclimatized to laboratory conditions for at least 15 days. Fish were approximately 13 g each and were about 3 months old. Three groups of 10 fish each were injected intramuscularly with 0.1 ml of phosphate-buffered saline-washed bacterial cells adjusted to the required concentrations. LD50 studies were carried out in triplicate for all of the strains. Fish were monitored for mortality for 7 days, and LD50s were calculated by the method of Reed and Muench (31).

Pulse-field gel electrophoresis. Pulse-field gel electrophoresis was carried out as previously described (46). ProMega-Markers Lambda Ladders (Promega) were used as the size markers. Band sizes were calculated from at least three independent separations of restriction fragments.

Statistical analysis.
LD50 data were analyzed using Student's t test. P values of <0.05 were considered significant.

Nucleotide sequence accession numbers.
Thirteen genes of A. hydrophila AH-1 or AH-3 for construction of knockouts were assigned the following accession numbers: aerA (AY442276), bvgA and bvgS (AY841798), f85 (AY442275), flhA1 (AY841793), flhA3 (AY841794), hlyA (AY442273), hup1 (AY442272), hup3 (AY841797), mepA (AY841796), ompAI (AY442271), opdA (AY442274), and serA (AY841795).


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Identification of putative virulence genes using genomic subtraction.
Genomic subtraction was our first approach to identifying common virulence genes, based on screening genetic differences between virulent and avirulent strains of A. hydrophila. In our previous study, PPD35/85 was used as a driver and PPD134/91 was used as a tester, and 16 unique DNA fragments (F fragment numbers) (Table 2) derived from 115 subtracted clones were shown to be present in most of the eight virulent strains of A. hydrophila (47). However, the first subtraction library was far from complete, considering the presence of only four pairs of identical clones. Therefore, an additional round of genomic subtraction was carried out using PPD35/85 or PPD64/90 as the driver and using PPD134/91 again as the tester. The restriction enzyme EcoRV (which cuts less frequently) rather than RsaI was used to generate longer subtracted DNA fragments. Subtracted clones using PPD64/90 or PPD35/85 as the driver were named PA or PB, respectively. Southern hybridization was then performed to survey the distribution of these subtracted fragments among 8 virulent and 6 avirulent strains of A. hydrophila (the first 14 strains listed in Table 1). The results showed that seven unique DNA fragments (derived from about 100 subtracted clones) (Table 2) from the PA and PB libraries were present in most of the eight virulent strains (≥4) but absent in most of the six avirulent strains (≤2) (Table 3). Altogether, 22 unique DNA fragments (F97 and PB45 are in the same DNA fragment) representing about 200 subtracted clones were obtained from two rounds of genomic subtraction and were commonly present in the eight virulent strains (≥4) (Table 2) (47). These 22 DNA fragments were assumed to encode potential virulence genes and were subjected to further analysis.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Summary of putative virulence genes identified in A. hydrophila PPD134/91

 

View this table:
[in this window]
[in a new window]
  		TABLE 3. Distribution of PA and PB subtracted clones among virulent and avirulent strains of A. hydrophila

 
Sequence analysis of the 22 unique DNA fragments.
Genome walking was carried out to obtain complete ORFs, as well as upstream and downstream sequences, of these 22 DNA fragments to predict their genetic organizations and functions. Fifteen of the DNA fragments encode 19 proteins with homologues in other bacteria. Eleven of these homologues showed significant homology to virulence-associated factors (Table 2). These included known virulence factors of Aeromonas species such as hemolysin (HlyA and F108) and aerolysin (AerA and PA26/PB80).

The other nine genes identified are new putative virulence factors for A. hydrophila and have high similarities to known virulence proteins of other pathogens, including the homologues of a histone-like protein (HU and F52), oligopeptidase A (OpdA and F88/F109/F61/F72), outer membrane protein (OmpAI and F2/F3), VsdC and a type III secretion protein homologue (F11), acinetobactin biosynthesis protein (F20), a two-component BvgA and BvgS system, and arylsulfotransferase.

VsdC is essential for virulence in Salmonella enterica serovar Dublin (18). The VsdC homologue in A. hydrophila contains a VIP2 domain, which belongs to a family of actin-ADP-ribosylating toxins. The presence of a type III secretion protein homologue in A. hydrophila PPD134/91 correlates well with the identification of a TTSS in this strain, as discussed further below. The acinetobactin biosynthesis protein F20 is involved in iron acquisition and may have an important role in the pathogenesis of Acinetobacter baumannii (25). In Bordetella pertussis, the bvgA and bvgS two-component system controls the expression of many virulence genes, including those encoding pili (26) and adenylate cyclase (12). These bvgA and bvgS homologues may thus also be involved in the regulation of pathogenesis in A. hydrophila PPD134/91. On the other hand, arylsulfotransferase has been suggested to be a virulence factor of the Campylobacter-Wolinella family of many oral bacteria and is widely distributed in Campylobacter species (43, 45).

The rest of the eight proteins showed high homology to proteins in other bacteria that have not yet been linked to virulence. They are topoisomerase (PB28/PB35), GGDEF family protein (PB60), a YaiI/YqxD family protein (F89), para-aminobenzoate synthase (F92), a putative exported protein (F93), a transporter transmembrane protein (F99/F106), a transmembrane protein (PB28/PB35), and a reverse transcriptase-like protein (F97). The functions of these proteins in A. hydrophila pathogenesis remain to be clarified.

For the last seven DNA fragments (F32, F34, F58, F85, F87, PA1/PA4/PB62, and PA6/PA98/PB38/PB78), the ORFs encoded had no significant matches with entries in the GenBank database and may represent novel virulence determinants. These presumptive virulence factors will be studied in future experiments.

Identification of a phage-associated genomic island.
The second approach adopted to search for putative virulence genes was to look for pathogenicity island (PAI) markers. PAIs are specific genomic islands with a large unstable chromosomal region that encodes virulence genes, and they are present in most of the pathogenic bacteria (13). Recently, ssrA, a small stable RNA molecule (tmRNA) has been reported to reside in or near the junction point of a mosaic of Salmonella-specific sequences (8) and serves as the insertion site for acquired sequences such as the cryptic phage CP4-57 in E. coli and PAIs in Vibrio cholerae (17) and Dichelobacter nodusus (3).

Primers ssrA-F (5-CAAACGACGAAAACTACGC-3) and ssrA-R (5-GGTACTACATGCTTAGTC) within the conserved ssrA region were designed, and an ssrA gene was identified in A. hydrophila PPD134/91 via a PCR using these primers. Subsequently, a series of genome walking experiments led to the identification of a 23-kb DNA region. Flanked by ssrA at the left end, this region exhibits significantly lower G+C content (49.7%) than the average genome G+C content of A. hydrophila (57 to 63%), which is a common characteristic of GIs (13). It also shows a mosaic distribution of G+C content (Table 4; Fig. 1). Furthermore, two ORFs (ORF1 and ORF2) (Table 4) of this region show high similarities to the insertion element IS1650 and are adjacent to ssrA. The G+C content of thirteen ORFs (Table 4) did not differ significantly from that of A. hydrophila, indicating that they might have been acquired from species with G+C content similar to that of A. hydrophila or that the base composition of these acquired DNA has gradually adapted to the host genome (19).


View this table:
[in this window]
[in a new window]
  		TABLE 4. Homology and G+C content for open reading frames of phage-associated island (accession no. AY442269)

 


View larger version (10K):
[in this window]
[in a new window]
  		FIG. 1. G+C content of each region for the phage-associated island and the location of probes used for southern blot. {zam0080556370003} indicates the location of primer pairs for the probes.

 
Many putative virulence factors are bacteriophage encoded (4), and like insertion elements, bacteriophages are often associated with GIs and are involved in the transfer of virulence determinants between bacterial species (15). This island carries several homologues to bacteriophage-encoded proteins (Table 4), suggesting they are good virulence factor candidates. Furthermore, a conserved protein domain database search indicated that ORF6 contains an AAA domain which belongs to an AAA superfamily of ATPases associated with a wide variety of cellular activities, including membrane fusion, proteolysis, and DNA replication. More interestingly, both ORF16 and ORF19 contain a helix-turn-helix XRE domain, suggesting they may function as transcriptional regulators. Sixteen out of the 24 ORFs did not show any good homology in the GenBank database, suggesting that they may encode novel proteins. This putative GI thus consists of a mosaic of segments similar to those found in various bacteriophages and is therefore referred to as a phage-associated island.

The distribution of this putative GI among 14 A. hydrophila strains was surveyed by Southern analysis using probes from four regions of the DNA (Fig. 1; Table 4). The four probes hybridized with most of the eight virulent strains but only hybridized with one out of the six avirulent strains tested (Table 5). The hybridization signals were strong in virulent strains ATCC 7966, PPD134/91, and Xs91-4-1 and the avirulent strain L36 but weak in the other five virulent strains, suggesting some genetic variations among the virulent strains. The results therefore indicate that this putative GI is PPD134/91 specific and/or virulent strain specific. Identification and sequencing of this GI in other virulent strains will help to clarify this issue.


View this table:
[in this window]
[in a new window]
  		TABLE 5. Distribution of the ORFs from phage-associated island in different A. hydrophila strains

 
Phage-mediated integration events may be involved in the acquisition of this phage-associated island. Phages contribute to the evolution of bacterial pathogens through gene transfer at the time of infection (39). It has been reported that removal of a portion of a Salmonella-specific region, which is near ssrA and contains a number of mobile elements such as insertion elements and bacteriophages, conferred the greatest defect in virulence (8). This phage-associated island may also contribute to differences in host specificity or disease manifestation.

Identification of a TTSS gene cluster.
TTSSs have been reported in many gram-negative animal and plant pathogens, including A. hydrophila (6, 9, 46). As reported previously (46), an ascV homologue is present in A. hydrophila PPD134/91. Furthermore, an ascU homologue, near one end of the TTSS in A. hydrophila AH-1, was also identified in A. hydrophila PPD134/91 by PCR (ascU-F, 5-TGGTGATCGCCATCGCCGA-3; ascU-R, 5-GACGGCGCTTGCTCTTGAT-3). The identification of these two TTSS homologues strongly indicated that a TTSS cluster is also present in A. hydrophila PPD134/91. This TTSS cluster was located on the chromosome of A. hydrophila PPD134/91 (Fig. 2). The complete TTSS sequence of A. hydrophila PPD134/91 is not yet available. However, analysis of the TTSS gene cluster of A. hydrophila strain AH-1 revealed an ORF found near ascU showing high homology to the P4-family integrase of a variety of bacteria. Hueck (15) reported that the TTSS may be acquired as intact genetic blocks by horizontal gene transfer during evolution. It is thus reasonable to speculate that the integrase may have been involved in the original mobilization of the TTSS into the chromosome of A. hydrophila. Downstream sequencing of ascU in A. hydrophila PPD134/91 will facilitate the understanding of the evolutionary history of this TTSS. Moreover, complete TTSS gene clusters have been recently identified in the two A. hydrophila strains, AH-3 and SSU (accession no. AY763611) (38), which further suggests that a complete TTSS gene cluster may be also present in PPD134/91.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 2. Physical map of A. hydrophila PPD134/91. The locations of virulence genes and clusters were determined with respect to PacI fragments.

 
Mapping of putative virulence genes on the physical map of A. hydrophila PPD134/91.
With a variety of novel putative virulence genes identified in this study, it was necessary to understand their genetic organization in the genome of A. hydrophila PPD134/91 to provide insight into the mechanisms of gene regulation and the molecular basis of pathogenicity of this bacterium. A physical map of A. hydrophila was therefore constructed. We chose the AT-rich recognition site endonusclease PacI to digest the PPD134/91 genomic DNA, based on the high G+C content (57 to 63%) of Aeromonas species (21). This yielded 19 fragments (named C1 to C19), with sizes of 11 to 733 kb. To link these fragments, PPD134/91 genomic DNA was digested with PacI and EcoRI and cloned into the pNEB193 vector. More than 100 clones were obtained, 65 of which contained sequences flanked by PacI sites. The clones whose sequences were homologous to the different portions of the same gene were considered joined to each other. For those clones which could not be joined together according to the above rule, genome walking was carried out to link them by searching for the shared PacI sites. The two half sites of PacI were located in two different clones ("clone pairs"). All of the clones (totaling 38 of the PacI- and EcoRI-digested fragments, i.e., 19 clone pairs) were prepared as probes and hybridized with PacI-digested fragments of genomic DNA from A. hydrophila PPD134/91. The two digested fragments which hybridized with the "clone pairs" were thus linked to each other. In this way, all of the fragments generated by PacI digestion of PPD134/91 were linked, and a physical map was formed (Fig. 2).

The 22 unique DNA fragments from the two rounds of genomic subtraction as well as the O-antigen and capsule clusters (48, 49), the phage-associated island, and the TTSS gene cluster have been positioned on the physical map by Southern hybridization (Fig. 2). The results showed O-antigen and capsule gene clusters were located at different regions of the chromosome (Fig. 2), while these two clusters were located near each other in E. coli (33). The locations of the TTSS cluster and the F11 fragment, which contains a type III secretion protein homologue, are quite far away from each other. This was not surprising, as the genes encoding a secretion apparatus are usually clustered, while the genes encoding the secreted proteins and their transcriptional regulators are often located in unlinked positions (15).

Ten subtracted clones consisting of seven unique DNA fragments were found to be located in the same PacI-digested fragment C14 (Fig. 2) of strain PPD134/91. However, there is as yet no indication that these virulence genes are located in a PAI. Most of these genes were not clustered in the same PacI-digested fragments in the genomes of seven other virulent strains by Southern analysis (data not shown).

Construction and characterization of mutants.
One strategy to examine whether the putative virulence genes identified in this study are involved in pathogenesis is to construct knockouts and test their virulence in the blue gourami fish model. Previous attempts to introduce plasmids or transposons into A. hydrophila PPD134/91 were not successful. A genetic barrier may exist in strain PPD134/91 and prevent the construction of knockouts in this strain. Therefore, other well-studied pathogenic strains of A. hydrophila such as AH-1 and AH-3 (24, 46) were used as the hosts for the construction of mutants. Fifteen genes in A. hydrophila AH-1 and/or AH-3, which are homologous to their corresponding genes in A. hydrophila PPD134/91, were successfully cloned for the construction of knockouts. In general, insertion mutants were constructed first to examine the effect of the target genes. If the LD50s of insertion mutants increased by at least 0.5 log, deletion mutants were then constructed in the same genes to confirm that the attenuation was not due to polar effects. We also included several deletion mutants such as {Delta}ompAI, {Delta}ascN, and {Delta}bvgA to confirm that there were no differences in the LD50s between insertion and deletion mutations. vsdC, the putative type III secretion protein (F11), and the phage-associated island sequences could not be successfully cloned from strains AH-1 and AH-3, and these genes were thus not characterized in this study. The construction of knockouts in these PPD134/91-specific genes is ongoing and will shed light on their roles in PPD134/91 pathogenesis.

The genes used for construction of mutants can be classified into three groups. Group I includes genes expressing hemolysin (hlyA) and aerolysin (aerA), which have been reported to be associated with the virulence of A. hydrophila (1, 42). Group II includes genes expressing histone-like protein (hup1 for AH-1 and hup3 for AH-3) (36); opdA, expressing ligopeptidase A (27); bvgA, bvgS, and ascN (46); and ompAI, expressing an outer membrane protein (40). These gene products are homologous to known virulence factors of other pathogens. Group III includes genes expressing a novel putative virulence protein (f85) and a recently identified polar flagellar assembly protein homologue, FlhA (flhA1 for AH-1 and flhA3 for AH-3), which appears to be involved in biofilm formation (H. B. Yu and K. Y. Leung, unpublished data).

Our results showed that the LD50s of all the mutants except ascN were comparable to that of the wild-type strains by using blue gourami fish as the infection model. The LD50s of the wild types AH-1 and AH-3 were 105.3 and 106.5, respectively. LD50s of both insertion and deletion mutants of ascN in AH-1 were determined to be 106.3. Wong and coworkers (42) reported that inactivation of hlyA or aerA alone showed no statistically significant attenuation in a suckling mouse model when compared to the wild type. Our virulence assay showed similar results for these genes, and extended this finding to most of the other virulence genes which we analyzed. In all the mutants tested, only ascN exhibited an LD50 1 log higher than that of the wild type-strain AH-1. AscN, a homologue of the YscN of Yersinia species (41), possibly interacts with membrane-bound components of the TTSS apparatus to energize secretion or to provide the energy for the assembly of the secretion apparatus. Disruption or deletion of ascN may thus render the TTSS nonfunctional, leading to the increase in LD50s and consistent with our previous study (46). The fact that most of the virulence genes did not attenuate the mutants suggested that the pathogenesis of A. hydrophila is multifactorial in nature. Disruption of more than one gene or a whole gene cluster as in the case of TTSS appears to be necessary to observe differences in the LD50s. On the other hand, the virulence of A. hydrophila may be also bacterial strain, infection route, and animal model dependent. It has been reported that a single mutation in cytotoxic enterotoxin of A. hydrophila SSU resulted in an ~300-fold increase in LD50s by intraperitoneal injection in Swiss-Webster mice (44); a single mutation in elastase of A. hydrophila AG2 resulted in an ~100-fold increase in LD50s by intraperitoneal challenge in rainbow trout (5).

In addition, double deletions mutations were made in strain AH-1, such as S-layer and metalloprotease ({Delta}ahsA {Delta}mepA; LD50 = 105.7), S-layer and serine protease ({Delta}ahsA {Delta}serA; LD50 = 105.7), and a triple-deletion mutant of S-layer, metalloprotease, and serine protease ({Delta}ahsA {Delta}serA {Delta}mepA; LD50 = 106.5). Different proteases have been shown to be involved in A. hydrophila virulence (5). The double-deletion mutants resulted in about half of a log increase in LD50s, but the attenuation was not statistically significant (P > 0.05). However, fish infected with these double mutants survived 1 to 2 days longer than those infected with the wild type at the same lethal dosage. Statistically significant attenuation was observed for the triple mutant, where the LD50 increased about 1 log. Our results therefore strongly support the idea that virulence factors in A. hydrophila pathogenesis work in a concerted manner and multiple factors are required to produce the observed deleterious effect.

Conclusion.
A variety of putative virulence genes in A. hydrophila have been identified by both genomic subtraction and GI analysis in this study. These include known A. hydrophila virulence genes (encoding hemolysin and aerolysin), as well as other genes showing homologies to known virulence factors, such as bvgA, bvgS, vsdC, and ompAI, which have not yet been examined with A. hydrophila. In addition, the putative virulence gene clusters, such as the presence of a phage-associated island and a TTSS in A. hydrophila PPD134/91, were established. Subsequent positioning of these putative genes and gene clusters on a physical map of strain PPD134/91 has helped us in better understanding the chromosome organization and the molecular mechanisms of pathogenicity in this bacterium.

This is the first report to present a comparative study of different virulence factors in A. hydrophila pathogenesis by constructing knockouts in the same strains and infecting the same infection host. Our virulence assay of these mutants demonstrated that, as is increasingly observed for other pathogens, virulence in A. hydrophila is complex and involves multiple virulence factors, which may work in concert. Construction of more multiple mutants, infection of different animal models and inclusion of other functional assays are under way to elucidate the mechanisms of these putative virulence factors. The putative virulence genes presented in this work will thus form the basis for further investigation of the pathogenesis of A. hydrophila and will be useful for data-mining for the development of effective vaccines, diagnostic and novel therapeutics against animal and human infection caused by motile aeromonads.


   ACKNOWLEDGMENTS
 
This work was supported in part by the Biomedical Research Council of Singapore (BMRC), support from A*STAR to K.Y.L., support by the Plan Nacional de I + D grants (Ministerio de Ciencia y Tecnología, Spain) and the Generalitat de Catalunya to J.M.T., and by a grant from the Canadian Institutes of Health Research to S.P.H.

We are grateful to Michael Janda from the California Department of Health Services for providing us with some of the A. hydrophila isolates.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, Faculty of Science, National University of Singapore, Science Drive 4, Singapore 117543, Republic of Singapore. Phone: (65) 6874 7835. Fax: (65) 6779 2486. E-mail: dbslky{at}nus.edu.sg. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Allan, B. J., and R. M. Stevenson. 1981. Extracellular virulence factors of Aeromonas hydrophila in fish infections. Can. J. Microbiol. 27:1114-1122.[Medline]
  2. Austin, B., and C. Adams. 1996. Fish pathogens, p. 197-243. In B. Austin, M. Altwegg, P. J. Gosling, and S. W. Joseph (ed.), The genus Aeromonas. John Wiley and Sons, New York, N.Y.
  3. Billington, S. J., J. L. Johnston, and J. I. Rood. 1996. Virulence regions and virulence factors of the ovine footrot pathogen, Dichelobacter nodosus. FEMS Microbiol. Lett. 145:147-156.[CrossRef][Medline]
  4. Boyd, E. F., and H. Brussow. 2002. Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends Microbiol. 10:521-529.[CrossRef][Medline]
  5. Cascon, A., J. Yugueros, A. Temprano, M. Sanchez, C. Hernanz, J. M. Luengo, and G. Naharro. 2000. A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila. Infect. Immun. 68:3233-3241.[Abstract/Free Full Text]
  6. Chacon, M. R., L. Soler, E. A. Groisman, J. Guarro, and M. J. Figueras. 2004. Type III secretion system genes in clinical Aeromonas isolates. J. Clin. Microbiol. 42:1285-1287.[Abstract/Free Full Text]
  7. Chakraborty, T., M. A. Montenegro, S. C. Sanyal, R. Helmuth, E. Bulling, and K. N. Timmis. 1984. Cloning of enterotoxin gene from Aeromonas hydrophila provides conclusive evidence of production of a cytotoxic enterotoxin. Infect. Immun. 46:435-441.[Abstract/Free Full Text]
  8. Conner, C. P., D. M. Heithoff, S. M. Julio, R. L. Sinsheimer, and M. J. Mahan. 1998. Differential patterns of acquired virulence genes distinguish Salmonella strains. Proc. Natl. Acad. Sci. USA 95:4641-4645.[Abstract/Free Full Text]
  9. Cornelis, G. R., and F. Van Gijsegem. 2000. Assembly and function of type III secretory systems. Annu. Rev. Microbiol. 54:735-774.[CrossRef][Medline]
  10. Dooley, J. S. G., and T. J. Trust. 1988. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein. J. Bacteriol. 170:499-506.[Abstract/Free Full Text]
  11. Edwards, R. A., L. H. Keller, and D. M. Schifferli. 1998. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207:149-157.[CrossRef][Medline]
  12. Glaser, P., H. Sakamoto, J. Bellalou, A. Ullmann, and A. Danchin. 1988. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 7:3997-4004.[Medline]
  13. Hacker, J., and J. B. Kaper. 2000. Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54:641-679.[CrossRef][Medline]
  14. Howard, S. P., S. Macintyre, and J. T. Buckley. 1996. The genus Aeromonas, p. 267-286. In B. Austin, M. Altwegg, P. J. Gosling, and S. Joseph (ed.), Toxin. John Wiley & Sons, Singapore, Singapore.
  15. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:379-433.[Abstract/Free Full Text]
  16. Janda, J. M. 2001. Aeromonas and Plesiomonas, p. 1237-1270. In M. Sussman (ed.), Molecular medical microbiology. Academic Press, San Diego, Calif.
  17. Karaolis, D. K. R., J. A. Johnson, C. C. Bailey, E. C. Boedecker, J. B. Kaper, and P. R. Reeves. 1998. A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains. Proc. Natl. Acad. Sci. USA 95:3134-3139.[Abstract/Free Full Text]
  18. Krause, M., C. Roudier, J. Fierer, J. Harwood, and D. Guiney. 1991. Molecular analysis of the virulence locus of the Salmonella dublin plasmid pSDL2. Mol. Microbiol. 5:307-316.[CrossRef][Medline]
  19. Lawrence, J. G., and H. Ochman. 1996. Amelioration of bacterial genome: rates of change and exchange. J. Mol. Evol. 44:383-397.
  20. Leung, K. Y., and R. M. W. Stevenson. 1988. Tn5-induced protease-deficient strains of Aeromonas hydrophila with reduced virulence for fish. Infect. Immun. 56:2639-2644.[Abstract/Free Full Text]
  21. MacInnes, J. I., T. J. Trust, and J. H. Crosa. 1979. Deoxyribonucleic acid relationships among members of the genus Aeromonas. Can. J. Microbiol. 25:579-586.[Medline]
  22. Mahairas, G. G., P. J. Sabo, M. J. Hickey, D. C. Singh, and C. K. Stover. 1996. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178:1274-1282.[Abstract/Free Full Text]
  23. Martinez, M. J., D. Simon-Pujol, F. Congregado, S. Merino, X. Rubires, and J. M. Tomas. 1995. The presence of capsular polysaccharide in mesophilic Aeromonas hydrophila serotypes O:11 and O:34. FEMS Microbiol. Lett. 128:69-73.[Medline]
  24. Merino, S., A. Aguilar, M. M. Nogueras, M. Regue, S. Swift, and J. M. Tomas. 1999. Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34. Infect. Immun. 67:4008-4013.[Abstract/Free Full Text]
  25. Mihara, K., T. Tanabe, Y. Yamakawa, T. Funahashi, H. Nakao, S. Narimatsu, and S. Yamamoto. 2004. Identification and transcriptional organization of a gene cluster involved in biosynthesis and transport of acinetobactin, a siderophore produced by Acinetobacter baumannii ATCC 19606T. Microbiology 150:2587-2597.[Abstract/Free Full Text]
  26. Mooi, F. R., H. G. J. Held, A. R. T. Avest, K. G. Welinder, I. Livey, B. A. M. Zeijst, and W. Gaastra. 1987. Characterization of fimbrial subunits from Bordetella species. Microb. Pathog. 2:473-484.[CrossRef][Medline]
  27. Novak, P., P. H. Ray, and I. K. Dev. 1986. Localization and purification of two enzymes from Escherichia coli capable of hydrolyzing a signal peptide. J. Biol. Chem. 261:420-427.[Abstract/Free Full Text]
  28. Pemberton, J. M., S. P. Kidd, and R. Schmidt. 1997. Secreted enzymes of Aeromonas. FEMS Microbial. Lett. 152:1-10.[CrossRef][Medline]
  29. Pepe, C. M., M. W. Eklund, and M. S. Strom. 1996. Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion. Mol. Microbiol. 19:857-869.[CrossRef][Medline]
  30. Quinn, D. M., C. Y. Wong, H. M. Atkinson, and R. L. Flower. 1993. Isolation of carbohydrate-reactive outer membrane proteins of Aeromonas hydrophila. Infect. Immun. 61:371-377.[Abstract/Free Full Text]
  31. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27:493-497.
  32. Rubirés, X., F. Saigi, N. Piqué, N. Climent, S. Merino, S. Albertí, J. M. Tomás, and M. Regué. 1997. A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives. J. Bacteriol. 179:7581-7586.[Abstract/Free Full Text]
  33. Schnaitman, C. A., and J. D. Klena. 1993. Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol. Rev. 57:655-682.[Abstract/Free Full Text]
  34. Sha, J., E. V. Kozlova, and A. K. Chopra. 2002. Role of various enterotoxins in Aeromonas hydrophila-induced gastroenteritis: generation of enterotoxin gene-deficient mutants and evaluation of their enterotoxic activity. Infect. Immun. 70:1924-1935.[Abstract/Free Full Text]
  35. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1:784-791.[CrossRef]
  36. Stinson, M. W., R. McLaughlin, S. H. Choi, Z. E. Juarez, and J. Barnard. 1998. Streptococcal histone-like protein: primary structure of HlpA and protein binding to lipoteichoic acid and epithelial cells. Infect. Immun. 66:259-265.[Abstract/Free Full Text]
  37. Thune, R. L., L. A. Stanley, and K. Cooper. 1993. Pathogenesis of gram-negative bacterial infections in warm water fish. Annu. Rev. Fish Dis. 3:37-68.
  38. Vilches, S., C. Urgell, S. Merino, M. R. Chacon, L. Soler, G. Castro-Escarpulli, M. J. Figueras, and J. M. Tomas. 2004. Complete type III secretion system of a mesophilic Aeromonas hydrophila strain. Appl. Environ. Microbiol. 70:6914-6919.[Abstract/Free Full Text]
  39. Wagner, P. L., and M. K. Waldor. 2002. Bacteriophage control of bacterial virulence. Infect. Immun. 70:3985-3993.[Free Full Text]
  40. Weiser, J. N., and E. C. Gotschlich. 1991. Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1. Infect. Immun. 59:2252-2258.[Abstract/Free Full Text]
  41. Woestyn, S., A. Allaoui, P. Wattiau, and G. R. Cornelis. 1994. YscN, the putative energizer of the Yersinia Yop secretion machinery. J. Bacteriol. 176:1561-1569.[Abstract/Free Full Text]
  42. Wong, C. Y., M. W. Heuzenroeder, and R. L. Flower. 1998. Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model. Microbiology 144:291-298.[Abstract]
  43. Wyss, C. 1989. Campylobacter-Wolinella group organisms are the only oral bacteria that form arylsulfatase-active colonies on a synthetic indicator medium. Infect. Immun. 57:1380-1383.[Abstract/Free Full Text]
  44. Xu, X. J., M. R. Ferguson, V. L. Popov, C. W. Houston, J. W. Peterson, and A. K. Chopra. 1998. Role of a cytotoxic enterotoxin in Aeromonas-mediated infections: development of transposon and isogenic mutants. Infect. Immun. 66:3501-3509.[Abstract/Free Full Text]
  45. Yao, R., and P. Guerry. 1996. Molecular cloning and site-specific mutagenesis of a gene involved in arylsulfatase production in Campylobacter jejuni. J. Bacteriol. 178:3335-3338.[Abstract/Free Full Text]
  46. Yu, H. B., P. S. Srinivasa Rao, H. C. Lee, S. Vilches, S. Merino, J. M. Tomas, and K. Y. Leung. 2004. A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis. Infect. Immun. 72:1248-1256.[Abstract/Free Full Text]
  47. Zhang, Y. L., C. T. Ong, and K. Y. Leung. 2000. Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish. Microbiology 146:999-1009.[Abstract/Free Full Text]
  48. Zhang, Y. L., E. Arakawa, and K. Y. Leung. 2002. Novel Aeromonas hydrophila PPD134/91 genes involved in O-antigen and capsule biosynthesis. Infect. Immun. 70:2326-2335.[Abstract/Free Full Text]
  49. Zhang, Y. L., Y. L. Lau, E. Arakawa, and K. Y. Leung. 2003. Detection and genetic analysis of group II capsules in Aeromonas hydrophila. Microbiology 149:1051-1060.[Abstract/Free Full Text]

Applied and Environmental Microbiology, August 2005, p. 4469-4477, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4469-4477.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yu, H. B.
Right arrow Articles by Leung, K. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yu, H. B.
Right arrow Articles by Leung, K. Y.
Agricola
Right arrow Articles by Yu, H. B.
Right arrow Articles by Leung, K. Y.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Microbiol. Mol. Biol. Rev. Eukaryot. Cell All ASM Journals