Role of type III secretion in Edwardsiella tarda virulence

Y. P. Tan1, J. Zheng1, S. L. Tung1, I. Rosenshine2 and K. Y. Leung1

1 Department of Biological Sciences, Faculty of Science, National University of Singapore, Science Drive 4, Singapore 117543
2 Department of Molecular Genetics and Biotechnology, Faculty of Medicine, The Hebrew University, PO Box 12272, Jerusalem 91120, Israel

Correspondence
K. Y. Leung
dbslky{at}nus.edu.sg


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Edwardsiella tarda is a Gram-negative enteric bacterium affecting both animals and humans. Recently, a type III secretion system (TTSS) was found in Ed. tarda. Such systems are generally used by bacterial pathogens to deliver virulence factors into host cells to subvert normal cell functions. Genome-walking was performed from the eseB and esrB genes (homologues of Salmonella sseB and ssrB, respectively) identified in previous studies, to determine the sequences of the TTSS. Thirty-five ORFs were identified which encode the TTSS apparatus, chaperones, effectors and regulators. Mutants affected in genes representing each category were generated and found to have decreased survival and growth in fish phagocytes. LD50 values of the mutants were increased by at least 10-fold in comparison to those of the wild-type strain. The adherence and invasion rates of the esrA and esrB mutants were enhanced while those of the other mutants remained similar to the wild-type. The eseC and eseD mutants showed slight autoaggregation in Dulbecco's Modified Eagle Medium, whereas the rest of the mutants failed to autoaggregate. Regulation of the TTSS was found to involve the two-component regulatory system esrA–esrB. This study showed that the TTSS is important for Ed. tarda pathogenesis. An understanding of this system will provide greater insight into the virulence mechanisms of this bacterial pathogen.


Abbreviations: 2-D, two-dimensional; DMEM, Dulbecco's Modified Eagle's Medium; ECP, extracellular protein; EPC, epithelioma papillosum of carp; EPEC, enteropathogenic Escherichia coli; PAI, pathogenicity island; SPI2, Salmonella pathogenicity island 2; TSA, tryptic soy agar; TTSS, type III secretion system

The GenBank accession number for the complete TTSS including the flanking sequences on the left and right sides of the cluster is AY643478.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Edwardsiella tarda is a wide-host-range Gram-negative bacterium, infecting lower and higher vertebrates such as fish (Sae-Oui et al., 1984), amphibians (Kourany et al., 1977), reptiles (Goldstein et al., 1981), birds (Cook & Tappe, 1985) and mammals, including humans (Janda & Abbott, 1993a). Its virulence factors are largely unknown. One of the established factors is its ability to invade non-phagocytic cells (Janda et al., 1991; Ling et al., 2000). When viewed by electron microscopy, the bacterium was observed to induce extensive plasma membrane projections or ruffles in HEp-2 cells (Phillips et al., 1998). It is also capable of surviving in phagocytes (Srinivasa Rao et al., 2001, 2003b), and produces haemolysins (Janda & Abbott, 1993b; Chen et al., 1996; Hirono et al., 1997) and catalases (Srinivasa Rao et al., 2003b).

More recently, two genes, eseB and esrB, homologous to the sseB and ssrB respectively of the type III secretion system (TTSS) encoded by Salmonella pathogenicity island 2 (SPI2) (Hensel et al., 1998), were found in Ed. tarda (Tan et al., 2002; Srinivasa Rao et al., 2003a). At the same time, three TTSS proteins, EseB, EseC and EseD, homologous to SseB, SseC and SseD respectively of SPI2, were identified by MS in the extracellular proteome of Ed. tarda (Srinivasa Rao et al., 2004). These findings led to speculation of the presence of a SPI2-like pathogenicity island (PAI) in Ed. tarda.

TTSSs are used by many bacterial pathogens for the delivery of virulence factors into host cells (Hueck, 1998). They encode various subsets of proteins involved in different aspects of pathogenesis. Some of them are secreted effector molecules, whereas others are involved in regulating TTSS expression, form the translocation apparatus (Knutton et al., 1998; Roine et al., 1997), or cause pore formation in the host cell membrane (Tardy et al., 1999; Wachter et al., 1999) to facilitate translocation of effector proteins into host cells.

In addition to SPI2, a SPI1-encoded TTSS is also present in Salmonella (Ochman et al., 1996). SPI1 is involved in the interaction of the bacterium with the cells of the gastrointestinal mucosa and facilitates invasion of epithelial cells (Galan & Curtiss, 1989; Galan, 1996), whereas SPI2 is primarily associated with bacterial survival and proliferation within phagocytes (Hensel, 2000).

In this paper, we report the identification of a TTSS in Ed. tarda. Based on sequence homology, many of the Ed. tarda TTSS genes resemble counterparts in the SPI2-encoded TTSS. We also show that the Ed. tarda TTSS is important for virulence.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids, media and culture conditions.
The bacterial strains and plasmids used are described in Table 1. Ed. tarda cultures were routinely grown in tryptic soy broth (BD Biosciences) or on tryptic soy agar (TSA, BD Biosciences) and Escherichia coli strains were cultured on Luria–Bertani (LB, BD Biosciences) agar or in LB broth. Ed. tarda and E. coli cultures were incubated at 25 and 37 °C respectively unless otherwise stated. Stock cultures were maintained in a suspension of broth with 25 % (v/v) glycerol and stored at –80 °C. When required, the appropriate antibiotics were supplemented at the following concentrations: ampicillin (100 µg ml–1), colistin (12·5 µg ml–1), gentamicin (100 µg ml–1) and kanamycin (50 µg ml–1).


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Table 1. Bacterial strains, mutants and plasmids

 
For the study of the autoaggregation phenotype of Ed. tarda wild-type and its mutants, bacterial cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) without phenol red as described previously (Srinivasa Rao et al., 2004). The bacteria were streaked on TSA and incubated for 48 h, after which a single colony was inoculated into 5 ml DMEM and cultured at 25 °C in a 5 % CO2 incubator for 24 h. The strains or mutants which aggregated to form cell clumps covering the entire round bottom of the tube (clear medium at the top) were scored ‘+’, while those which remained suspended in the culture medium (turbid) with few cells settling at the bottom of the tube were scored ‘–’ for autoaggregation. A ‘±’ score denotes bacteria that formed small clumps at the bottom of the tube. The experiment was repeated three times in three independent trials.

Genome-walking, cloning and sequencing.
In order to obtain the sequences of the TTSS cluster, genome-walking and/or inverse PCR (Sambrook & Russell, 2000) were performed concurrently from both the eseB and esrB genes obtained from our earlier work (Tan et al., 2002; Srinivasa Rao et al., 2003a). Genome-walking libraries (EcoRV, DraI, HincII, PvuII, ScaI, StuI) were constructed and PCR amplification was carried out according to the Universal GenomeWalker kit (BD Biosciences) user manual. DNA fragments amplified were cloned into the pGEM-T Easy vector system (Promega) and transformed into E. coli TOP10F' or E. coli DH5{alpha}.

The Applied Biosystems (ABI) PRISM BigDye Terminator v3.0 or 3.1 Cycle Sequencing Kit was used for the sequencing PCR. The ABI 3100 Genetic Analyser was then used for automated DNA sequencing. Sequence assembly and editing were performed with the DNASIS sequence analysis software (Hitachi Software). BLASTP and/or PSI-BLAST sequence homology analyses were done using the national center for biotechnology BLAST network service.

Construction of Ed. tarda mutants.
Insertion mutation was carried out in various TTSS genes (esaC, escA, eseB, eseC, eseD, esrA and esrB) in Ed. tarda PPD130/91. Briefly, internal fragments of each gene were obtained by PCR using the primers given in Table 2 and cloned into the pGEM-T Easy vector (Promega). These partial gene fragments were released from the vector by EcoRI digestion and religated into the suicide plasmid pFS100 (Rubires et al., 1997). The construct obtained was transformed into E. coli MC1061. pFS100 carrying the internal fragment of each TTSS gene was isolated from E. coli MC1061 and transformed into E. coli strain SM10{lambda}pir. Subsequently plate-mating was performed to allow mobilization of the suicide vector with the internal gene fragment from E. coli SM10{lambda}pir to Ed. tarda PPD130/91. Insertional inactivation then occurred, resulting in the integration of the cloned suicide vector into the respective TTSS gene of interest in the Ed. tarda genome.


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Table 2. Primers used for PCR amplification for the construction of insertional and deletion mutants

 
In addition to the insertion mutants, non-polar deletion mutation was also performed for the esrA and esrB genes following the procedure of Edwards et al. (1998). The target gene, including at least 500 bp flanking sequences, was amplified using primers (Table 2) containing the KpnI restriction site and cloned into the pGEM-T Easy vector. Inverse PCR was then performed using these constructs as templates. The resulting PCR product was purified and self-ligated to obtain the gene of interest with non-polar deletion, which was then excised with KpnI and cloned into the suicide vector pRE112 (Edwards et al., 1998). The construct obtained was transformed into E. coli SM10{lambda}pir and mobilized into wild-type Ed. tarda PPD130/91 by conjugal transfer. Double crossover mutation was achieved by plating the bacterial cells on TSA plates with 10 % sucrose. The deletion mutants were confirmed by PCR and sequencing.

Phagocyte isolation and intracellular replication assay.
Phagocytes were isolated from the head kidney of healthy naïve blue gourami obtained from a commercial fish farm following the method of Secombs (1990) and as described by Srinivasa Rao et al. (2001) with slight modifications. Approximately 4–5x106 phagocytic cells suspended in L-15 medium (Sigma) supplemented with 5 % fetal bovine serum (Invitrogen) were seeded into each well of a 48-well tissue culture plate (Costar) and incubated for 4 h at 25 °C in a 5 % CO2 atmosphere. Afterwards, the cells were washed once with Hanks' balanced salt solution (HBSS, Sigma) and infected with Ed. tarda at an m.o.i. of 1 : 1 for 30 min (four wells per strain/mutant). The cells were then washed twice with HBSS and extracellular bacteria were killed by gentamicin (100 µg ml–1) treatment for 1·5 h. At the end of this time period, the cells were washed twice with HBSS and those in two of the wells were lysed with 1 % Triton X-100 (Sigma) and plated to obtain the bacterial count in c.f.u. Five hundred microlitres fresh L-15 medium was added into each of the remaining wells and the cells were incubated for a further 4·5 h before being lysed with 1 % Triton X-100 (Sigma) and plated.

The ratio of the c.f.u. count at the end of the 6·5 h period and that at 2 h after gentamicin treatment was obtained for each mutant and the wild-type strain. The ratio of the two time points for each mutant was expressed as a quotient of that of the wild-type strain. A value less than one indicated reduced replication in phagocytes. The experiment was repeated three times for each mutant and the wild-type strain was kept for comparison in every trial.

Adherence and internalization assays.
These assays were performed following the procedure by Wang et al. (1998) with slight modifications. Epithelioma papillosum of carp, Cyprinus carpio (EPC), cells (Wolf & Mann, 1980) were grown for 72 h to 100 % confluence in 24-well tissue culture plates (Falcon). The cells were then washed once with HBSS and infected with Ed. tarda and its mutants in minimal essential medium (MEM, Invitrogen) at an m.o.i. of 1 : 1 (four wells per bacterial strain/mutant). The plate was centrifuged at 800 g for 5 min at 4 °C and incubated at 25 °C in a 5 % CO2 incubator for 30 min. To measure the number of bacteria adhering to the EPC monolayer, the cells were washed six times with HBSS, lysed with 1 % Triton X-100 (Sigma) and the bacterial number quantified by plate counting (Elsinghorst, 1994). For measuring the number of internalized bacteria, the monolayer was washed three times with HBSS and incubated for 2 h in MEM with gentamicin (100 µg ml–1) (Sigma) to kill any remaining extracellular bacteria. The EPC cells were washed three times again with HBSS to remove gentamicin; 1 ml 1 % Triton X-100 was added to lyse the cells and the number of viable intracellular bacteria was quantified by plate counting. The adherence and invasion rates were calculated from the mean of two wells in triplicate experiments.

LD50 determination.
The LD50 of Ed. tarda PPD130/91 wild-type and mutants was determined using healthy naïve blue gourami, Trichogaster trichopterus (Pallas), obtained from a commercial fish farm as described previously (Ling et al., 2000). Fish mortality was observed for 7 days and the LD50 values were calculated according to the method of Reed & Muench (1938).

Preparation of extracellular proteins (ECPs) and two-dimensional (2-D) PAGE analysis.
ECPs from Ed. tarda PPD130/91 and its mutants were prepared as described before (Leung & Stevenson, 1988; Tan et al., 2002) and the proteins were quantified using the Bio-Rad protein assay.

2-D PAGE was used to resolve the proteins. Eighteen-centimetre Immobiline Drystrips pH 3–10 (Amersham Biosciences) were used and passive rehydration of the strips was carried out. IEF of the proteins was done using the Ettan IPGphor Isoelectric Focusing System (Amersham) under the following conditions: 50 V for 1 h, 500 V for 2 h, 4000 V for 1 h 30 min, 8000 V for 40 000 V h or 500 V for 2 h, 4000 V for 1 h 30 min, 8 000 V for 40 000 V h. The cup loading method was employed to load the protein samples prior to IEF. Silver staining was performed following the procedure of Blum et al. (1987).

Complementation of mutants.
The eseB gene and the esrA–esrB regulatory genes, including more than 1 kb of flanking sequences upstream of each gene, were amplified by PCR and cloned into pKK177-3 (Brosius & Holy, 1984), adjacent to the Ptac promoter, and pACYC184 (Fermentas Life Sciences) respectively. Each of the constructs was first transformed into E. coli DH5{alpha} and plasmid DNA was isolated. The eseB and esrA–esrB genes carried on the vector were then transformed into the respective eseB and esrA and esrB mutants. Expression of the eseB gene in these constructs is inducible by 0·1 mM IPTG.

The complemented mutants harbouring a wild-type copy of the eseB gene carried on the pKK177-3 vector were then inoculated into DMEM with and without IPTG, cultured for 24 h and compared with the wild-type and the eseB mutant. The ability of the esrA and esrB mutant complemented with the wild-type esrA–esrB genes provided in trans on the pACYC184 plasmid to autoaggregate in DMEM were also checked and their adherence and invasion rates were measured.

Statistical analysis.
The data obtained for adherence and internalization and phagocyte intracellular replication were expressed as mean±SEM. Data analysis was performed using the paired t-test (MINITAB Release 14.0 Statistical Software, Minitab) for comparison between the wild-type and each mutant. P<0·05 was considered statistically significant.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Identification of a TTSS in Ed. tarda
Using the comparative proteomics (Tan et al., 2002) and transposon tagging approaches (Srinivasa Rao et al., 2003a), we found two genes, eseB and esrB, in Ed. tarda that are homologous to members of the SPI2-encoded TTSS. Furthermore, the EseB protein and two other major proteins, homologous to SPI2 SseC and SseD, were found in the Ed. tarda secretions (Srinivasa Rao et al., 2004). These findings suggested the presence of a SPI2-like PAI. Subsequent PCR and genome-walking confirmed our hypothesis and 35 ORFs encoding the TTSS from the virulent Ed. tarda PPD130/91 were identified.

The TTSS encoding region is 30 756 bp and its organization is shown in Fig. 1(a). The cluster has a mean G+C content of 64·3 mol%, which is approximately 6–9 percentage points higher than that of the Ed. tarda chromosome (55–58 mol%) (Farmer & McWhorter, 1984). This observation is in contrast to SPI2, in which the G+C content is generally lower than the rest of the Salmonella genome (Hensel et al., 1997, 1999). Such variation in the G+C content is a distinctive feature of PAIs observed in many bacteria (Hacker et al., 1997). It suggests that the group of genes could have been acquired from an external source. The presence of this TTSS (as surveyed using eseB and esrB) in most virulent but not avirulent strains (Tan et al., 2002; Srinivasa Rao et al., 2003a) also suggests the possibility of horizontal gene transfer.



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Fig. 1. Genetic organization of TTSSs in (a) Ed. tarda PPD130/91, (b) S. enterica serovar Typhimurium (SPI2), (c) EPEC (LEE) and (d) C. violaceum ATCC 12472 (CPI2). The secretion systems essentially consist of four categories of genes, namely apparatus, chaperones, effectors and regulators, but the arrangement of the genes varies. The direction of transcription of each gene is indicated by the arrow. In Ed. tarda, the cluster is flanked by orfA–E and orfF–H on both sides. OrfA is homologous to the catalase–peroxidase. OrfB is unknown and OrfC–E are homologous to members of the tdc operon, tdcA, tdcB and tdcC. OrfF is homolgous to a hypothetical protein in E. coli and the sulfite oxidase in Burkholderia fungorum. OrfH is homologous to the putative Photobacterium profundum guanine aminohydrolase and E. coli guanine deaminase. OrfG has no homologue.

 
The predicted ORFs of the TTSS cluster are listed in Table 3 and their putative functions are given as deduced from those of the homologues in Salmonella enterica serovar Typhimurium, enteropathogenic E. coli (EPEC) and Yersinia enterocolitica. As in the case of SPI2, the genes of the Ed. tarda TTSS cluster are likewise grouped into four categories, namely Ed. tarda secretion system apparatus (esa), chaperones (esc), effectors (ese) and regulators (esr). The designation of each gene is based on its sequence homologue.


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Table 3. ORFs of Ed. tarda TTSS and the sequence homologues in S. enterica serovar Typhimurium, EPEC, C. violaceum and Y. enterocolitica

 
Previously, the ORFs were presented in two separate DNA regions (Zheng et al., 2005). Five additional ORFs have since been identified which join the two regions together and they have been designated ORF1A–ORF1E. The DNA region 1 was reversed in this case so as to join DNA region 2 to obtain a complete, contiguous TTSS cluster. For ease of reference, we have kept to the ORF numbering used earlier (Table 3).

The Ed. tarda TTSS and its counterparts SPI2, LEE and CPI2
The Ed. tarda TTSS identified in the present study showed both similarities and differences in organization to the TTSSs encoded by SPI2 (Hensel, 2000), LEE (locus of enterocyte effacement; Elliott et al., 1998) and CPI2 (Chromobacterium pathogenicity island 2; Brazilian National Genome Project Consortium, 2003; Betts et al., 2004) encoded in S. enterica serovar Typhimurium, EPEC and Chromobacterium violaceum respectively (Fig. 1). It also has homologues in the Y. enterocolitica TTSS (Table 3). The functions of several SPI2 (Ruiz-Albert et al., 2003; Zurawski & Stein, 2003) and LEE (Elliott et al., 2001; Creasey et al., 2003; Jepson et al., 2003; Tu et al., 2003; Deng et al., 2004; Kanack et al., 2004; Nougayrede & Donnenberg, 2004; O'Connell et al., 2004; Pallen et al., 2005) proteins (Fig. 1b, c) were updated based on more recent studies. In contrast to the other four bacteria, C. violaceum is generally free-living, but occasional fatal human infections have been reported (Ti et al., 1993). Recently, TTSS genes which are likely to be involved in pathogenesis were found in the genome of this bacterium (Brazilian National Genome Project Consortium, 2003; Alves de Brito et al., 2004; Betts et al., 2004).

The gene members of these TTSSs are essentially categorized into the four groups mentioned above and those involved in similar or related functions (apparatus, effectors and the corresponding chaperone, regulators) are located in close proximity (Fig. 1). In the four bacteria compared, the apparatus genes essentially exist in two regions in the cluster, and those encoding the apparatus proteins R, S, T and U are present and arranged in tandem in the exact same order and transcribed in the same direction. As for the other apparatus genes, their positions, though similar, are not identical in the four bacteria.

The gene arrangement of the Ed. tarda system (Fig. 1a) most closely resembles that in S. enterica serovar Typhimurium (Fig. 1b). The putative effector genes (eseB, eseC, eseD, eseE) and the interspersed putative chaperone genes (escA and escB) are arranged in the same order in the two bacteria. However, although both of them possess a two-component regulatory system, their locations differ. The Ed. tarda system is situated further away from the group of effector and chaperone genes unlike in SPI2, in which it is positioned immediately next to the ssaB gene and only four ORFs away from sseA. Moreover, the Ed. tarda regulatory gene members are transcribed in opposite directions, in contrast to the Salmonella ssrA–ssrB, which form one transcriptional unit (Shea et al., 1996). Therefore, with respect to the location and direction of transcription of the Ed. tarda regulatory system, there is greater similarity to the C. violaceum system (Fig. 1d).

Besides the putative esrA–esrB two-component system, an additional transcriptional regulator, which we named esrC, is also present in the Ed. tarda TTSS cluster (Zheng et al., 2005). This protein has homology to proteins of the AraC/XylS family of positive transcriptional regulators (Gallegos et al., 1997) such as VirF in Yersinia species (Wattiau & Cornelis, 1994). CV2584 of C. violaceum is also a transcriptional regulator homologous to VirF (Betts et al., 2004).

Orf10 shows weak homology to SpiC of S. enterica subsp. enterica Paratypi, SsaB of S. enterica serovar Typhimurium and a hypothetical protein (CV2598) of C. violaceum (Table 3), but it is unknown at this stage if it is an apparatus or effector protein. Orf19 has homology to a hypothetical protein (CV2589) in C. violaceum (NCBI accession no. AAQ60259 and Y. pseudotuberculosis (NCBI accession no. YP_068866), and a putative PAI protein of S. enterica subsp. enterica serovar Typhi (NCBI accession no. NP_805081) and Paratypi (NCBI accession no. YP_150694).

There are six novel genes (orf1B, orf2, orf13, orf19, orf29 and orf30) with unknown function. Although orf19 has homology to putative PAI genes, the function of the latter is not known. orf29 and orf30 located next to esrB were found to belong to the TTSS cluster (Zheng et al., 2005).

TTSS boundary
Several ORFs (orfA–E and orfF–H) flanking the Ed. tarda TTSS were obtained (Fig. 1a). At one end, orfA encodes a catalase–peroxidase-like protein, orfB is unknown and orfCorfE are homologous to the tdc operon genes. The catalase–peroxidase enzyme mainly functions in protecting bacteria under oxidative stress against the deleterious effects of reactive oxygen species (Schellhorn & Hassan, 1988; Schellhorn, 1995), although it has also been implicated in virulence in some bacteria (Pym et al., 2002; Ng et al., 2004). The E. coli tdc operon is involved in amino acid transport and metabolism during anaerobic growth to provide a source of metabolic energy (Datta et al., 1987; Goss et al., 1988; Sumantran et al., 1990; Ganduri et al., 1993). At the other end, orfF has homology to a hypothetical protein in E. coli (Dobrindt et al., 2002) and sulfite oxidase enzymes in Burkholderia fungorum (NCBI accession no. ZP 00280377). orfG has no sequence homologue and orfH is homologous to the putative guanine aminohydrolase (NCBI accession no. YP 130136) and guanine deaminase in Photobacterium profundum and E. coli (Maynes et al., 2000) respectively. The guanine aminohydrolase or deaminase is involved in the conversion of guanine to xanthine and ammonia during the first step of utilization of guanine as a nitrogen source (Maynes et al., 2000). The homologues in Ed. tarda may have similar functions. These two groups of genes appear to play roles in metabolism and housekeeping for the general well-being of the bacteria. The mean G+C content of the two flanking regions (54·8 mol% for orfA–orfE and 53·5 mol% for orfF–orfH) is 54·2 mol%, which is very close to the Ed. tarda genome range (55–58 %). Therefore, they could be the flanking genes lying outside the cluster defining the TTSS boundary.

Construction and characterization of mutants
Since TTSSs are known to play important roles in virulence in many pathogenic bacteria (Hueck, 1998), there is a need to establish if this secretion system is also crucial for Ed. tarda pathogenesis. In order to address this question and to assign a more defined role to this newly discovered system in Ed. tarda, various TTSS genes, including those homologous to the regulators (esrA and esrB), apparatus (esaC), chaperone (escA) and effectors (eseB, eseC and eseD), were disrupted by insertional inactivation. For the regulator genes, non-polar deletion was also carried out. The mutants' ability to survive and replicate within phagocytes, to adhere to and internalize within non-phagocytic EPC cells, and to kill fish (LD50 determination) were examined and compared to the properties of the wild-type strain. These three phenotypes are the ones primarily associated with the SPI2 analogue (Hensel, 2000). The ability of the mutants to autoaggregate in DMEM was also checked. This phenotype was previously found to correlate with Ed. tarda virulence (Srinivasa Rao et al., 2004); hence it may be used as a rapid preliminary screening tool to identify potential attenuated mutants.

Reduced survival and growth of TTSS mutants in fish phagocytes
The results of the mutant characterization are as shown in Table 4. Both the insertion and deletion esrA and esrB mutants showed essentially similar phenotypes. All the mutants tested had decreased ability to survive and replicate within phagocytes when compared to the wild-type strain, thus showing that the TTSS may be important for Ed. tarda to resist phagocytic killing and to replicate within the immune cells. This observation was similar to that in Salmonella. Disruption of SPI2 genes such as spiR (ssrA), spiA (ssaC) (Ochman et al., 1996), sseB and sseC (Hensel et al., 1998) affected intracellular survival and replication of the mutants in murine macrophages.


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Table 4. Characterization of wild-type Ed. tarda PPD130/91 and its TTSS mutants

For adherence and internalization and macrophage replication, values are presented as mean±SEM of triplicate trials. Values with different superscript letters are significantly different (P<0·05).

 
esrA and esrB mutants show increased adherence to and internalization within EPC cells but greatly reduced LD50
Earlier work showed that Ed. tarda is able to adhere to and invade epithelial cells (Janda et al., 1991; Ling et al., 2000). In this study, the esrA and esrB insertion and non-polar deletion mutants were found to have increased adherence and internalization, whereas the rest of the mutants showed adherence and internalization similar to the wild-type strain. The high internalization is probably a consequence of increased adherence because this is a prerequisite for bacterial entry (Neves et al., 1994; Edelman et al., 2003). Our observations are in contrast to the reduced adherence seen in S. enterica serovar Typhimurium SPI2 mutants (Deiwick et al., 1998). In Salmonella species, SPI2 mutations (ssaT and ssaV) affect the expression of SPI1 genes and hence decrease the ability of the bacteria to invade cultured epithelial cells. In Ed. tarda, it is not known if a SPI1-like cluster involved in internalization is present and, so far, only the haemolysin-associated genes, homologous to shlA and hpmA, have been shown to be related to bacterial entry into epithelial cells (Strauss et al., 1997). However, the putative esrA–esrB regulatory system does appear to affect the genes associated with adherence and invasion in a manner opposite to that observed in Salmonella species (Deiwick et al., 1998). EsrA–EsrB may thus control a system with some anti-internalization activity, which maintains only a certain level of bacterial entry into epithelial cells. Therefore, internalization increases when the putative two-component system is disrupted. Alternatively, the effect of the regulatory system on adherence and invasion may be indirect, mediated through the action of other sets of genes. Complementation of the esrA and esrB insertion mutants with a wild-type copy of the genes restored adherence and internalization to levels similar to the wild-type strain (Table 4).

Mutations in Salmonella spiA (ssaC) and effector genes like spiC do not affect bacterial invasion (Ochman et al., 1996; Uchiya et al., 1999) as seen in Ed. tarda esaC (spiA homologue) and effector (eseB, eseC and eseD) mutants in the present study.

When the virulence of these mutants was compared, all of them were found to have attenuated virulence. Virulence of mutants with disruption in esrA and esrB was highly attenuated, with LD50 values raised at least two orders of magnitude above that of the wild-type, whereas the rest of the mutants had LD50 values increased by about 10-fold. In the case of Salmonella, mutants harbouring SPI2 insertions were attenuated in mice (Hensel et al., 1998; Shea et al., 1996). Inactivation of regulator (ssrA) (Ochman et al., 1996), effector (sseA, sseB, sseC and spiC) or apparatus (spiA) genes led to strong attenuation of virulence (Hensel et al., 1998; Uchiya et al., 1999). This differs from the case of Ed. tarda infection, in which the regulator genes appear to play a more significant role in virulence, as their disruption led to more dramatic attenuation. However, mutations in other Salmonella effector genes caused only slight (sseF and sseG) or no detectable attenuation of virulence (sseE) (Hensel et al., 1998). Therefore, the extent of involvement of each gene in pathogenesis may vary in different bacteria.

EseB and autoaggregation in DMEM
All the TTSS mutants were attenuated and their ability to autoaggregate was reduced or abolished (Table 4). From the results obtained, failure of the eseB mutant to autoaggregate may indicate that secretion of EseB is required for autoaggregation. The EseB protein, homologous to EspA of EPEC, may be a component of an extracellular filamentous organelle or appendage that mediates autoaggregation. Therefore, when this gene is disrupted, the mutant fails to autoaggregate. Further evidence that EseB is likely to be involved in mediating the autoaggregation phenotype was provided by the restoration of this ability in the complemented eseB mutant upon IPTG induction of the eseB gene (data not shown). This phenotype was not observed in the complemented but uninduced mutant. In EPEC, espD mutants secrete low levels of EspA and produce shorter filaments (Knutton et al., 1998) and in enterohaemorrhagic E. coli, synthesis of the EspD protein is essential for the formation of EspA-containing filaments (Kresse et al., 1999). Mutations in eseC and eseD (homologous to espD and espB respectively) in Ed. tarda may also have affected the proper formation of putative EseB filaments which, in turn, reduced autoaggregation (Table 4). The EPEC CesD chaperone is essential for the proper secretion of EspB and EspD (Wainwright & Kaper, 1998). EscA, a homologue of CesD, may also be a putative chaperone essential for the proper secretion of EseB, EseC and/or EseD proteins. Disruption of EscA may therefore affect the proper formation of the putative EseB appendages, thus making autoaggregation impossible.

These results are clearly indicative of the participation of the putative Ed. tarda TTSS in phagocyte survival and virulence. Although this system was shown to affect the adherence and invasion of Ed. tarda, its precise involvement awaits further investigation in our ongoing experiments. For the insertion mutants, even though there could be potential polar effects on TTSS genes downstream, the characterization of mutants with disruption in genes from all the categories (apparatus, chaperone, effector and regulator), and the restoration of phenotypes in complemented mutants, should reflect the contribution of the TTSS to Ed. tarda pathogenesis, particularly in the three virulence-associated phenotypes examined.

The Ed. tarda TTSS is regulated by a putative esrAesrB two-component system
Bacteria often sense environmental stimuli via two-component sensor-response regulatory systems (Garcia Vescovi et al., 1996; Pratt & Silhavy, 1995). In Salmonella species, the expression of SPI2 genes is dependent on the SsrA–SsrB system which is encoded within the cluster itself (Cirillo et al., 1998; Deiwick et al., 1999; Hensel et al., 1998; Ochman et al., 1996; Shea et al., 1996). Since Ed. tarda also possess a very similar EsrA–EsrB two-component system, this could likewise be involved in the regulation of the putative TTSS. When the ECP profiles of the wild-type and esrA and esrB mutants were analysed using 2-D PAGE (Fig. 2a, b; only the esrB mutant ECP profile is presented), the EseB, EseC and EseD proteins were missing or much reduced. This shows that the two-component system is likely to be controlling TTSS expression and/or protein secretion. For the eseD mutant (Fig. 2c), the EseB protein, but not EseC and EseD, was seen in the ECP, indicating that production and/or secretion of the latter two proteins may have been co-regulated. As described and discussed above, the esrA and esrB mutants were highly attenuated. This may be attributed to the loss of TTSS involvement in pathogenesis as a result of the disruption of its regulatory system. Without these important virulence factors, the bacteria failed to establish an infection in the fish host.



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Fig. 2. 2-D ECP profile of (a) wild-type, (b) esrB and (c) eseD insertional mutants. The TTSS-encoded proteins (circled) are lost or much diminished in the esrB mutant. The esrA mutant has the same ECP profile as the esrB mutant (not shown). In the eseD mutant, only the EseB protein is present in the ECP.

 
Conclusion
The identification of a putative TTSS in Ed. tarda certainly offers greater insight into the pathogenesis of this bacterium. The results of the characterization studies demonstrate the importance of the secretion system in phagocyte survival and bacterial replication and virulence. It also appears to affect Ed. tarda adherence to and internalization into epithelial cells. The restoration of these phenotypes in complemented mutants confirms the involvement of the TTSS in these functions. The Ed. tarda system is similar to that in Salmonella species in terms of the gene categories, the associated functions and regulation by a two-component system. However, differences do exist between the two systems, such as the position of the genes, their arrangement and organization, and regulation of the TTSS. Therefore, the two bacteria could have acquired the virulence gene cluster from different sources during evolution.

In Ed. tarda, the esrAesrB two-component system is critical for regulating not only the expression of the secretion system genes, but also genes outside the cluster, such as the evp genes (Srinivasa Rao et al., 2004; Zheng et al., 2005). Therefore, it could also be regulating the adherence and invasion genes, either directly or indirectly, as in the case of ssrA–ssrB in SPI2, which controls gene expression both within the cluster and elsewhere in the chromosome (Beuzon et al., 2000; Brumell et al., 2003; Cirillo et al., 1998).

The apparatus, chaperone and effector genes, on the other hand, may play more defined roles in the system, unlike the regulators, so their inactivation brings about more specific changes. This is clearly reflected in the high attenuation of the mutants with regulator disruption because additional virulence factors (e.g. Evp proteins) may be lost (Srinivasa Rao et al., 2004) besides those encoded by the TTSS. This contrasts with the more subtle decrease in virulence of the other mutants resulting from the loss of specific TTSS virulence factors. However, further studies are essential to understand the mechanism of the TTSS and to assign a more definitive role to each member of the cluster. These studies are currently ongoing in our laboratory.


   ACKNOWLEDGEMENTS
 
The authors are grateful to the National University of Singapore and the Lee Hiok Kee Foundation for providing the research grant for this work. Special thanks to Dr J. M. Tomas from the University of Barcelona, Spain and Dr T. T. Ngiam and Dr H. Loh from the Agri-food and Veterinary Authority (AVA) of Singapore for providing the pFS100 suicide plasmid and the Ed. tarda PPD130/91 strain respectively. We also would like to thank Dr Patrick Tang for critical comments which helped in improving the manuscript.


   REFERENCES
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ABSTRACT
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Received 3 March 2005; revised 29 March 2005; accepted 10 April 2005.



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