Correspondence: Qiyao Wang, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. Tel.: +86 21 64253306; fax: +86 21 64253306; e-mail: email@example.com
Edwardsiella tarda is a Gram-negative, facultative aerobic pathogen which infects multifarious hosts including fish, amphibians and human beings. A twin-arginine translocation (Tat) gene cluster important for high-salt tolerance in E. tarda was identified previously. Here the genetic structure and pleiotropic roles of the Tat system in physiological adaptation of the bacterium were further characterized. Functional analysis indicated that tatD was not required for Tat export process and tatE might be an allelic gene of tatA in the bacterium. The results showed that disruption in the Tat system did not affect the morphology and biofilm formation in E. tarda, but did affect motility, hemagglutination, cell aggregation and infection of eukaryotic cells (e.g. macrophage J774a). Comparative proteomics analysis of subcellular proteins using two-dimensional gel electrophoresis and a qualitative shotgun protein sequencing method were implemented to identify proteins differentially expressed in E. tarda EIB202 vs. ∆tatABCD. The results revealed a large repertoire of differentially expressed proteins (n = 61), shedding light on the Tat system associated with virulence and stress-associated processes in E. tarda.
Edwardsiella tarda is a versatile Gram-negative bacterial pathogen causing edwardsiellosis in various fish and resulting in great economic losses in aquaculture worldwide (Park et al., 2012). The bacterium also infects amphibians, birds and human beings, raising increasing concerns about this important zoonotic pathogen. In the past decades, much attention has been paid to the identification of virulence factors such as hemolysin, chondroitinase, siderophore, catalase, type III secretion system (TTSS) and type VI secretion system (T6SS) involved in the pathogenicity of E. tarda (Leung et al., 2012). Little is known about the stress adaptation processes involved in the invasion and colonization of various hosts. The genome sequence of E. tarda EIB202, evaluated as highly virulent in fish models such as turbot, zebra fish and swordtail fish, disclosed the genetic properties of its physiological fitness and virulence (Wang et al., 2009), which warranted extensive proteomics analysis to identify and characterize proteins contributing to the adaptation to various environments and hosts.
Bacterial secretion systems are essential to translocate proteins to their subcellular destination. The twin-arginine translocation (Tat) pathway is involved in transporting pre-folded proteins across bacterial cytoplasmic membrane into periplasm using ∆pH as energy (Desvaux et al., 2009). The Tat system has been reported to be involved in many bacterial physiological processes (De Buck et al., 2008). In E. tarda, a Tat gene locus was identified and the Tat disrupted mutant was characterized to be associated with H2S production, hemolytic activity, stress response to temperature, NaCl, SDS and ethanol, as well as attenuated virulence toward zebra fish and turbot (Wang et al., 2013). In this study, the genetic structure and pleiotropic roles of the Tat system in the physiological adaptation in the bacterium were further characterized. We also performed comparative proteomics between wild-type E. tarda EIB202 and ∆tatABCD to identify differentially expressed proteins affected by Tat disruption.
Materials and methods
Bacterial strains and culture conditions
Edwardsiella tarda EIB202, ∆tatABCD and tatABCD+ were used in this study (Wang et al., 2013). Additional mutants ΔtatA, ΔtatB, ΔtatC, ΔtatD, ΔtatE, ΔtatAΔtatE and their corresponding complement strains tatA+, tatB+, tatC+, tatD+, tatE+ and tatABCD+ (Supporting Information, Table S1) were constructed following the site-directed mutagenesis procedures using suicide plasmid pDMK (Xiao et al., 2009) and complement plasmid pMMB206 as described previously (Wang et al., 2013). Bacterial strains were grown in Luria–Bertani medium (LB; Oxoid, Basingstoke, UK), tryptic soy broth (TSB; Difco Laboratories, Detroit, MI) or Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Eggenstein, Germany) at 28 °C.
Co-transcriptional analysis of tat gene cluster
To determine whether the tatABCD genes were transcribed as in one operon, reverse transcribed-PCR (RT-PCR) with primers within tatA and tatB, tatB and tatC, and tatC and tatD (Table S2) were performed to analyze co-transcription of the genes as described previously (Wang et al., 2011). The positive amplification was determined with primer pairs generating targeted products using cDNA as amplification template, but yielded no product using extracted RNA sample as amplification template.
Functional analysis of Tat system using GFP in E. tarda
To verify the role of individual Tat system components, plasmid pUTat-ssTorA::GFP expressing the signal sequence (ss) of trimethylamine N-oxide (TMAO) reductase (TorA) of E. tarda fused to GFP was introduced into EIB202 and Tat mutants according to the protocol described previously (Wang et al., 2013). The subcellular fractions of the constructed strains were extracted and GFP localization was analyzed with Western blotting.
Growth assays, production of H2S, morphology test and mobility assay
The overnight cultured strains were adjusted to the same optical density at 600 nm (OD600 nm), and then serially diluted 10-fold with LB medium. A 5-μL aliquot of each dilution was spotted onto the LB agar plates, and incubated at 28 °C or 42 °C. Production of H2S was examined on deoxycholate hydrogen sulfide lactose agar (DHL, Nissui, Japan). The morphology of strains was detected according to the Gram-stain method using a microscope and Hitachi S-4800 scanning electron microscopy. For the swimming motility assay, the tested strains with the same OD600 nm were spotted on LB agar containing 0.3% agar and cultured at 28 °C for 20 h.
Quantitative biofilm formation, hemagglutination assay and cell aggregation assay
Biofilm formation on polystyrene plate was normalized by dividing the biofilm value by the OD600 nm value of the cells (Xiao et al., 2009). Mannose-resistance hemagglutination against rat erythrocytes was determined as previously described (Wang et al., 2011). The hemagglutination titer was defined as the reciprocal of the highest dilution of tested strains that displayed complete hemagglutination. The cell aggregation was determined by the method of Srinivasa Rao et al. (2004). The overnight cultured strains were inoculated into TSB or DMEM medium with the same OD600 nm. Then the strains were cultured for 24 h without shaking and the growth condition was photographed.
Cell adherence and intracellular survival ability assay
Epithelioma papillosum of carp (EPC) cell and Madin-Darby canine kidney (MDCK) epithelial cells were employed for the adherence assay (Xiao et al., 2009). The cells were infected with EIB202 and Tat mutant strains at a multiplicity of infection (m.o.i.) of 10 for 2 h to detect the adherence ability. After 2 h incubation, the cells were washed and lysed, and the adherence rates were recorded by dividing the viable number by the initial bacterial number from triplicate experiments. The results were calculated by the adherence rate of test strains divided by that of EIB202, which was arbitrarily defined as 1. Mouse macrophage-like cell line J774a was chosen for intracellular survival assay according to previously described methods (Wang et al., 2011). J774a was infected with test strains at an m.o.i. of 100 for 1 h. Gentamicin 100 μg mL−1 was added to kill the extracellular bacteria following 2 h incubation. The results were calculated by dividing the intracellular survival rate of test strains by that of EIB202, which was arbitrarily defined as 1.
2-DGE characterization of proteins affected by Tat mutation
To identify proteins involved in the Tat pathway, comparisons of whole cell lysated proteins (WCLP), periplasmic proteins (PP) and extracellular proteins (ECP) between EIB202 and ΔtatABCD were performed using two-dimensional gel electrophoresis (2-DGE) proteomics approaches. The WCLP and ECP were isolated by trichloroacetic acid (TCA)-acetone precipitation (Srinivasa Rao et al., 2004; Wang et al., 2013). The PP were prepared by exposing cells to chloroform as described previously (Ames et al., 1984). Proteins were stored at −80 °C until use. 2-DGE with a wide pH 3–10 range IPG strips and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass (MALDI-TOF/TOF) spectrometry were performed as previously described (Wang et al., 2013). Three separate gels of each sample were analyzed at the same time.
Shotgun HPLC-ESI-MS/MS analysis
The ECP fraction for shotgun analysis was isolated by ultracentrifugation with Biomax-5K columns (Millipore). Samples were separated by 12% SDS-PAGE and the protein lane of gel then cut into slices followed by in-gel trypsin digestion (Shevchenko et al., 1996). The digested peptides were separated by shotgun HPLC-ESI-MS/MS analysis (Liu et al., 2010).
Statistical analysis was performed using spss (version 14.0). One-way anova with least-significant difference (LSD) post hoc multiple comparisons was used to compare the biofilm, adherence and intracellular survival data. An independent-sample t-test was used to analyze the protein expression between wild-type strain and ∆tatABCD. A value of P < 0.05 was considered significant.
Transcriptional analysis of the gene cluster encoding a Tat system in E. tarda
Based on the E. tarda EIB202 genome information (Wang et al., 2009), tatABCD locus was flanked by putative ubiquinone/menaquinone biosynthesis methyltransferase gene ubiB (ETAE_0149) and delta-aminolevulinic acid dehydratase gene hemB (ETAE_0154). Gene rfaH (ETAE_0155) encoding a transcriptional activator was located downstream of hemB (Fig. S1a). tatE (ETAE_2651) was located elsewhere on the chromosome. There was a 3-bp gap between tatA and tatB, an 11-bp overlap between tatB and tatC, and a 173-bp gap between tatC and tatD. In addition, there was a stable stem-loop structure (Fig. S1b) between the intergenic region of tatC and tatD.
Bioinformatics analysis with hmmtop (http://www.enzim.hu/hmmtop/index.html) was used to predict the structure of the Tat pathway component. TatA, TatB and TatE were predicted to be single-span followed by an amphipathic α-helix at the cytoplasmic side of the membrane. TatC was a polytopic membrane protein with six predicted transmembrane helices. TatD was predicted to be cytoplasmically located. Gln8, Phe20, Gly21 and Phe39 in TatA, and Glu8, Gly21, Pro22, Leu25, Pro26, Glu53 and Glu58 in TatB were highly conserved (data not shown), similar to observations in E. coli (Berks et al., 2003).
Moreover, a predicted σ70-dependent promoter was located upstream of tatA (data not shown). To determine whether the tatABCD were transcribed as in one operon, primers pairs targeting the regions covering tatA and tatB, tatB and tatC, and tatC and tatD were designed for RT-PCR. The results showed that three primer pairs generated targeted amplification products when genomic DNA or cDNA were used as the amplification template (Fig. S1c). The results confirmed a single polycistronic mRNA for the tatA, tatB, tatC, and tatD in E. tarda EIB202.
Functional analysis of the individual components in the Tat system
Tat pathway component mutants ΔtatA, ΔtatB, ΔtatC, ΔtatD and ΔtatE and their corresponding complement strains following the previously described methods (Table S1) were constructed (Wang et al., 2013). In addition, a double mutant ΔtatAΔtatE and ΔtatABCD were also constructed (Table S1). To evaluate the function of Tat pathway components in E. tarda, the plasmid pUTat-ssTorA::GFP encoding GFP protein fused to E. tarda Tat signal peptide TorA was used for subcellular location of GFP detection (Wang et al., 2013). Western blotting analysis indicated that the majority of expressed GFP was translocated into periplasm in EIB202, ΔtatD and ΔtatE as mature GFP. In contrast, GFP was detected in cytoplasm in ΔtatA, ΔtatB, ΔtatC, ΔtatAΔtatE and ΔtatABCD as precursor, demonstrating that the Tat system was deficient in these stains (Fig. 1a).
All the Tat mutants and their corresponding complement strains exhibited similar growth to that of EIB202 on LB agar at 28 °C (data not shown). When cultured at 42 °C, ΔtatAΔtatE and ΔtatABCD showed a dramatically deficient growth, and the growth of complement strain tatABCD+ restored to that of EIB202 (Fig. 1b). ΔtatA, ΔtatB and ΔtatC mutants also displayed growth deficiency, whereas their corresponding complement strains showed restored growth (Fig. 1b). ΔtatAΔtatE displayed severe growth deficiency at upshifted temperature compared with ΔtatA or ΔtatE mutants (Fig. 1b). When cultured on DHL agar, typical colonies with black center indicative of H2S-production appeared in ΔtatD and ΔtatE as that of EIB202, whereas none of the other mutants showed H2S-production (Fig. 1c).
Tat system mutation affects mobility, hemagglutination activity and cell aggregation but not biofilm formation
ΔtatABCD displayed no chain-forming or remarkable colony morphological change compared with EIB202 using electron microscopy or SEM observation (data not shown). There was no statistically significant difference among EIB202, ∆tatABCD and tatABCD+ in quantitative biofilm test (data not shown). However, the tatABCD mutant was obviously defective in swimming on 0.3% agar plate, in contrast to EIB202 and tatABCD+, in which the swimming ability was partially restored (Fig. 1d). Furthermore, deletion in tatABCD resulted in dramatically impaired hemagglutination activity, whereas the complement of tatABCD rescued hemagglutination similar to EIB202 (Table 1). Interestingly, EIB202 aggregated and settled to the bottom of the tube in TSB or DMEM medium after culture for 24 h without shaking, whereas ΔtatABCD was dispersed in these two culture media (Fig. 1e).
Table 1. Effect of Edwardsiella tarda Tat system on adherence (EPC and MDCK) or intracellular survival (J774a), and hemagglutination activity
Data regarding adherence (EPC and MDCK) and intracellular survival (J774a) are representative of three replicated experiments yielding similar results. The results were presented as the mean ± SD (n = 3). The adherence and intracellular survival rate of EIB202 was defined as 1.
Values followed by different superscript letters are significantly different (P <0.05).
See Material and methods for a detailed description of the experiments.
ND, Not determined.
1 ± 0.00a
1 ± 0.00a
1 ± 0.00a
0.26 ± 0.11b
0.01 ± 0.019b
0.11 ± 0.05b
0.63 ± 0.08c
0.54 ± 0.14c
0.62 ± 0.08c
Escherichia coli Top10F'
0.50 ± 0.05c
0.15 ± 0.08d
The Tat system is involved in cell adherence and intracellular survival
Compared with EIB202, ΔtatABCD displayed a significantly lower adherence rate to EPC and MDCK cells, respectively. ΔtatABCD also presented a 90% decrease in the intracellular survival capacity in J774a compared with that of EIB202 (Table 1). tatABCD+ displayed partially restored adherence and intracellular colonization capacities.
2-DGE characterization of WCLP affected by Tat abrogation
Comparative proteomics was used to elucidate the underlying mechanisms of the Tat pathway involved in physiological adaptation in E. tarda. Overall, the WCLP proteome generating approximately 1000 reproducible spots obtained under normal growth conditions was analyzed on each 2-DGE gel (representative gels are shown in Fig. 2a and b). Of the 51 differentially expressed WCLP spots on the 2-DGE map, 29 spots (P < 0.05) displaying change greater than 1.5-fold were successfully identified with the MALDI-TOF/TOF method (Table 2). Of these, three proteins were absent and 23 proteins present in a notably reduced amount in the whole cell lysate of ∆tatABCD. Meanwhile, the majority (29) of the differentially expressed proteins were enzymes related to metabolic processes including glycolysis, citrate cycle, pentose phosphate pathway, and oxidative phosphorylation, which confirmed that the Tat pathway was involved in the redox process and was essential for energy metabolism (Palmer et al., 2005). The affected 29 WCLP by Tat inactivation were categorized to be mainly correlated with metabolism processes (Table 2).
Table 2. Identification of altered whole cell lysated protein by 2-DGE between Edwardsiella tarda EIB202 and ΔtatABCD
Spot no. was consistent with the number in Fig. 2a and b.
Fold change was given as protein expression in ∆tatABCD divided by that in EIB202.
COG – clusters of orthologous groups; C – energy production and conversion; G – carbohydrate transport and metabolism; F – nucleotide transport and metabolism; H – coenzyme transport and metabolism; E – amino acid transport and metabolism; L – replication, recombination, and repair; J – translation, ribosomal structure and biogenesis; D – a cell cycle control, cell division, chromosome partitioning; O – posttranslational modification, protein turnover, chaperones; R – general function prediction only; S – function unknown.
2-DGE characterization of periplasmic proteins affected by Tat mutant
Close to 450 separate PP spots with high reproducibility could be identified on 2-DGE gels (representative gels are exhibited in Fig. 2c and d). Of the 18 significantly (P < 0.05) altered spots with change > 1.90-fold, 10 spots were identified by MALDI-TOF/TOF. Of these, one spot was up-regulated, whereas the other nine were down-regulated when tatABCD was disrupted in E. tarda (Table 3). The putative functions of the altered proteins were mainly classified into protein groups of metabolism and posttranslational modification, protein turnover and chaperones.
Table 3. Identification of altered periplasmic proteins by 2-DGE between Edwardsiella tarda EIB202 and ΔtatABCD
HPLC-ESI-MS analysis of ECP affected by Tat disruption
Edwardsiella tarda ECP were difficult to analyze by 2-DGE because of their intrinsic tendency to self-aggregate during isoelectric focusing (data not shown). Therefore, a shotgun HPLC-ESI-MS/MS proteomics approach was chosen to identify ECP in E. tarda. Results demonstrated that 29 and 51 proteins were identified in EIB202 and ΔtatABCD, respectively (Table 4). The ECP of ΔtatABCD shared the same 29 proteins identified in the ECP of EIB202. The additional 22 different ECP of ΔtatABCD are listed in Table 4. TTSS- and T6SS-related secretion proteins, putative hemolysin precursor, GapA, and zinc metalloproteinase aureolysin were identified in both EIB202 and ΔtatABCD. Among the annotated ECP using shotgun HPLC-ESI-MS/MS in EIB202, 17.2% were assigned as COGs category O (posttranslational modification, protein turnover, chaperones). Another three dominant portions were carbohydrate transport and metabolism (G, 13.8%), cell wall/membrane/envelope biogenesis (M, 10.3%), and amino acid transport and metabolism (E, 10.3%). For the annotated ΔtatABCD ECP, 17.6% of the identified ECP belonged to G, followed by O (13.7%).
Table 4. Extracellular proteins of Edwardsiella tarda EIB202 and ∆tatABCD identified by shotgun HPLC-ESI-MS/MSa
ECP with the unique peptide counts ≥ 2 identified in E. tarda EIB202 and ∆tatABCD.
For details of the COG catalog, see Tables 2 and 3.
M – cell wall/membrane/envelope biogenesis; P – inorganic ion transport and metabolism; A – RNA processing and modification; ECP identified in ∆tatABCD but was absent in EPC of EIB202; K – transcription.
Wang et al. (2013) characterized the E. tarda Tat system as it related to physiological response, virulence and high salt-stress (4% NaCl). Here we further analyzed the transcriptional structure of the Tat gene cluster, characterized the function of each of the Tat genes, and compared the proteomes of E. tarda EIB202 and ΔtatABCD under normal culture conditions.
The results (Fig. 2a–c) indicated that TatA, TatB and TatC were essential for Tat transportation, whereas TatD and TatE were not essential for Tat function in E. tarda, similar to Escherichia coli (Wexler et al., 2000; Berks et al., 2003). The Tat pathway may be responsible for swimming in E. tarda as in Vibrio cholerae (Zhang et al., 2009), Agrobacterium tumefaciens (Ding & Christie, 2003) and Pseudomonas aeruginosa (Ochsner et al., 2002). However, the predicted Tat pathway substrates did not include flagella-related genes (Wang et al., 2009). The transcriptional level of flagella synthesis and assembly genes flhD (ETAE_1336), fliA (ETAE_2126), and motA (ETAE_1338) were analyzed in the Tat mutants and no significant change was observed in the transcription of these genes compared with that of the wild type (data not shown). Therefore, the defective motility of tat mutant is probably an indirect effect similar to the findings in P. aeruginosa (Ochsner et al., 2002).
The ability to adhere to host cells is essential for bacterial infection (Xiao et al., 2009). The Tat system was observed to be involved in cell adherence and intracellular survival in various eukaryotic cell lines in E. tarda (Table 1). Tat mutants in other pathogenic bacteria such as V. cholerae (Zhang et al., 2009), Legionella pneumophila (De Buck et al., 2005) and Salmonella enterica (Reynolds et al., 2011) also associate or replicate poorly in eukaryotic cells. In S. enterica, the reduced infection of cultured macrophages by the Tat mutants may represent a difficulty phagocytosing which is caused by the chain-forming phenotype of the Tat mutants that were not easily internalized by macrophages (Reynolds et al., 2011). However, E. tarda Tat mutant showed no chain-forming phenotype in various growth phases (data not shown), indicating that other factors are associated with the defects in cell adherence and intracellular replication. The inability of the Tat mutants to adhere or replicate in macrophages probably contributes to the attenuated phenotype in fish models (Wang et al., 2013). These results suggest the Tat system has pleiotropic roles in physiological adaptation and virulence in the bacterium.
Proteomic comparison of subcellular protein profiles of EIB202 with that of ∆tatABCD indicated that the Tat system was essential for expression or transportation of various proteins that contribute to the stress adaptation and virulence in the bacterium. The majority of differentially expressed proteins were related to metabolic processes, which confirmed that the Tat pathway was involved in redox processes and essential for energy metabolism (Palmer et al., 2005). The differently expressed proteins contained non-classical ‘-RR-’ or no signal peptides. However, the proteomic identified cytoplasmic or periplasmic proteins, FrdA, ETAE_2959, ETAE_3142 (Table 2), ClpB, PepD, EATE_1540 (Table 3) and ETAE_3014 (Wang et al., 2013), which were up- or down-regulated in the specific subcellular proportions upon Tat mutation, contained typical Tat signal sequences. Whether these signal sequences are actually recognized by the E. tarda Tat pathway deserves further investigation. Specifically, the previously predicted Tat substrate ETAE_3142 (Wang et al., 2009) disappeared in the proteome of ΔtatABCD (Table 2), suggesting the Tat pathway is not only involved in the substrate transportation processes but is also essential in the control of the expression of the substrates. In addition, the significant up-regulation of HemB (Table 2) may a result of the destruction of the stem-loop between tatC and tatD in E. tarda ΔtatABCD strain that may function as a rho-independent transcription terminator for rfaH (downstream of tatD) and be responsible for lower levels of tatD-containing transcripts (Wexler et al., 2000).
The Tat system has been found to be involved in several important processes such as the biogenesis of the cell envelope (Berks et al., 2003). In E. tarda, ΔtatABCD displayed no apparent growth defects during in vitro growth in normal rich medium and exhibited no chain-forming cell morphology, although FtsZ was significantly down-regulated (Table 2), which creates a ring structure at septa and is crucial for cell division (Bi et al., 1991). However, the E. tarda Tat pathway appears to be directly or indirectly involved in various physiological processes and adaptation to stresses such as high temperature, high-salt tolerance, SDS and ethanol (Wang et al., 2013). The significant down-regulation of molecular chaperone ClpB and GrpE (Zolkiewski, 1999) (Table 3) as well as the previously identified oxidizing disulfide catalyst DsbA (Wang et al., 2013) strongly underlined the weakened stress-resistance of ΔtatABCD. In E. coli, the chaperone teams ClpB/DnaK and DnaK/DnaJ/GrpE are involved in suppressing and reversing protein aggregation modulated by heat and other stresses (Zolkiewski, 1999). FklB, a newly characterized peptidyl-prolyl cis-trans isomerase promoting stress tolerance and pathogenicity in several bacteria (Obi et al., 2011), displayed decreased expression by 1.69-fold in ΔtatABCD (Table 2). In addition, a virulence-related T6SS structure protein EvpF was identified with 1.60-fold down-regulated expression in ΔtatABCD (Table 2), indicating that T6SS function may be affected by the Tat pathway. Furthermore, the down-regulation of PstS under high-salt conditions (Wang et al., 2013), which makes up the ABC-type phosphate transport system and affects the uptake of Pi and expression of virulence-related genes (Srinivasa Rao et al., 2003), may also contribute to the slight virulence attenuation in E. tarda Tat mutant.
In summary, our data describe the essential roles of the Tat system in stress response and virulence associated phenotypes such as motility, hemagglutination, cell aggregation, cell adherence and intracellular survival in J774a. The extensive examination of the differential E. tarda proteomes upon Tat disruption provide deep insights into the function of the Tat system. Further analysis of these identified proteins using gene expression and bioinformatics tools will yield valuable information on E. tarda biology.
This work was supported by grants from the National High Technology Research and Development Program of China (2013AA093101), Special Fund for Agroscientific Research in the Public Interest (nyhyzx-201303047), and Shanghai Leading Academic Discipline Project (No. B505).