Vibrios, which are gram-negative halophilic bacteria that include more than 60
species, comprise the major culturable bacteria in marine and estuarine environments.
They commonly possess two chromosomes and that the genome structure consisting of a maintained relatively flexible and smaller chromosome and a large chromosome retaining most of the essential genes is stably among species of the family
Vibrionaceae (Okada et al., 2005)
The sequenced genome of V. vulnificus YJ016 also includes two chromosomes of estimated 3377 kbp (large chromosome) and 1857 kbp (small chromosome) in size, and a plasmid of 48,508 bp. Five groups of putative pathogenicity genes encoding respectively the proteins for type IV pilus, capsular polysaccharide biosynthesis, iron acquisition, extracellular enzyme and toxin, and RTX [repeats in toxin] toxin were annotated (Chen et al., 2003).
The sequence analysis revealed an RtxA gene cluster (VVA1030, VVA1032, VVA1034, VVA1035 and VVA1036) contained in a 22-k bp region on the small chromosome. Interestingly, an additional RTX like gene (RtxL), VVA0331, was annotated also on the small chromosome. A typical type I secretion system encoding gene cluster (VVA0332-VVA0334) and a signal transduction system encoding gene cluster (VVA0325-VVA0329) containing an originally named VieSAB like three component system were found to be located respectively upstream and downstream flanking the RtxL gene (Fig. 1A). We speculate that the three gene clusters function coordinately as a virulence-associated genomic island and roles of the genomic island in the bacterial virulence were hence investigated.
2-1. RTX toxins
RTX toxins play an important role in the virulence of a variety of human and animal gram-negative bacterial pathogens. There are two categories: the hemolysins, which affect a variety of cell types, and the leukotoxins, which are cell-type and species-specific (Lally et al., 1999). It is one of a close family of membrane-targeted toxins, which have been shown to be influential not only in urinary tract infections but also hemorrhagic intestinal disease, juvenile periodontitis, pneumonia, whooping cough, and wound infections; these toxins include enterohemorrhagic O157 E. coli hemolysin (Schmidt et al., 1996), the leukotoxin of Pasteurella haemolytica (Strathdee and Lo, 1987), the hemolysins and leukotoxins of Actinobacillus spp (Burrows and Lo, 1992; Frey et al., 1993; Kraig et al., 1990), the bifunctional adenylate cyclase-hemolysin of Bordetella pertussis (Glaser et al., 1988), and the hemolysins of Proteus vulgaris (Welch, 1987), Morganella morganii (Koronakis et al., 1987), and Moraxella bovis (Gray et al., 1995).
They commonly share a posttranslational maturation and a C-terminal
calcium-binding domain of acidic glycine-rich nonapeptide repeats that has led to the RTX family nomenclature. In addition, they are exported out of the cell by type I secretion system (Welch, 2001). The posttranslational modification is unique to this toxin family, but Ca2+ binding and type I secretion are both common to other bacterial proteins (Stanley et al., 1998; Welch, 2001). In E. coli HlyA, there are 11 to 17 glycine-rich repeats. When the protein bound with Ca2+ (one calcium ion per repeat), it forms short β-strands organized in an unusual “spring-like” structure called a parallel β-barrel or β-superhelix (Baumann et al., 1993). Calcium binding is an absolute requirement for its cytotoxic activity (Boehm et al., 1990; Ludwig et al., 1988) and the binding occurs following its export action.
2-1-1.VcRtxA
In V. cholerae, RtxA encoding gene was identified as a 13,635-bp-long ORF located adjacent to ctx (cholera toxin) genes on the large chromosome (Lin et al., 1999). The deduced V. Cholerae RtxA (VcRtxA) protein with 4,545 aa in length is the second largest single-polypeptide toxin known, which caused cell rounding and
depolymerization of the actin cytoskeleton in a broad range of cell types (Boardman and Satchell, 2004; Fullner and Mekalanos, 2000; Lin et al., 1999). Like other RTX toxins, VcRtxA shares the common features of posttranslational maturation depending on RtxC activator, a C-terminal calcium-binding domain of acidic glycine-rich
nonapeptide repeats, and their exportation out of the cell by type I secretion system, RtxB and RtxD (Boardman and Satchell, 2004; Lin et al., 1999; Sheahan et al., 2004;
Welch, 2001).
2-1-2.VvRtxA
In V. vulnificus YJ016, VvRtxA protein with 5206 aa in length is the largest single-polypeptide toxin known. Comparison of the amino acid sequences of VcRtxA and VvRtxA revealed 80–90% identity throughout most regions of the toxins. VvRtxA also exerts the common features of all RTX toxins, including the GD-rich calcium binding repeats at the C-terminus and N-terminal hydrophobic repeats. Although RtxL was annotated as a second RTX protein and an entire type I secretion gene cluster is also present downstream of the RtxL encoding gene, no typical feature of RTX toxin could be identified in the 4655 aa sequences yet.
2-2. Signal transduction- two component systems (2CSs)
To subsist in nature, bacteria possess regulatory systems that permit them to recognize and adapt to a highly changing environment. The capacities are brought about largely through varying in gene expressions that are coordinated by global
regulatory networks. Pathogenic bacteria often use two-component systems (2CSs) to control expression of the genes encoding bacterial toxins, adhesions, and other virulence-associated molecules that promote their survival in the host (Locht, 1999).
Bacterial 2CS consisting of a sensor histidine kinase and a response regulator, acts to recognize specific signals and to convert this information into transcriptional or behavioral responses in order to confront the highly changing circumstances (Miller et al., 1989; Soncini and Groisman, 1996; Stock et al., 1989). After sensing the input signals, the sensor protein catalyzes an autophosphorylation reaction, which transfers a phosphate from ATP to a conserved histidine residue. The phosphate group is subsequently transferred from the histidine residue to a specific aspartate residue on the receiver domain of the cognate response regulator. Phosphorylation of the
response regulator leads to activation of the transcription-regulating activity through an appropriate conformational change (Mizuno, 1998; West and Stock, 2001).
2-2-1. VieSAB three component system
VieSAB of V. cholerae differs from the conventional 2CS in that two putative response regulators, VieA and VieB, are contained in the system and hence named three component system. Different from VieB which has a typical N-terminal phosphoreceiver domain of response regulator but lacking any recognizable DNA binding motif, the vieA gene encodes a response regulator with a LuxR-family type of DNA binding motif and an EAL domain (Galperin et al., 2001). Evidences from two different in vivo screenings have suggested that VieSAB contributes to regulate the gene expressions during infection, such as CT (cholera toxin) production (Camilli and Mekalanos, 1995; Lee et al., 2001; Tischler et al., 2002). The vieSAB genes adjacent to each other appeared to be transcribed differentially. Although both vieS and vieA expressed during in vitro growth, the expression of vieS appeared to be constitutive, while vieA expression was VieA dependent (Lee et al., 1998). VieA has been shown to control the intracellular concentration of cyclic diguanylate (cyclic di-GMP) through its EAL domain carrying a cyclic di-GMP phosphodiesterase activity. The
level of cyclic di-GMP, which is likely a secondary messenger in the cells, led to an optimal expression of vps (Vibrio exopolysaccharide synthesis) (Tischler and Camilli, 2004)
2-3. Cyclic diguanylate as a secondary messenger
In addition to 2CS, bacteria also use receptor mediated signaling system, so-called secondary messenger as a linker between extracellular signals and the downstream events (Galperin, 2004). Cyclic nucleotides (cAMP and cGMP) are among the most widely studied of this class of molecules (Botsford, 1981; Botsford and Harman, 1992). Recently, a new signaling molecule, cyclic-di(3’→5’)-guanylic acid (cyclic di-GMP), was identified as an allosteric activator in cellulose biosynthesis pathway in Gluconacetobacter xylinus (Ross et al., 1990). In addition, the
involvement of the GGDEF- and EAL- containing proteins in cyclic di-GMP metabolism has been established, whereby the GGDEF domain represents the dinucleotide cyclase, while EAL, most probably, represents the cyclic dinucleotide phosphodiesterase (Ausmees et al., 2001; Simm et al., 2004; Tal et al., 1998).
2-3-1. Biological function of GGDEF- and EAL- containing proteins
The GGDEF domain dubbed based on its conserved sequence motif (170 aa defined in HMM profile) was firstly discovered in the response regulator PleD that controls cell differentiation in the swarmer-to-stalked cell transition in Caulobacter crescentus (Hecht and Newton, 1995) and the EAL domain dubbed based on its
conserved sequence motif (245 aa defined in HMM profile) was originally described in a study of virulence related to the 2CS BvgR in Bordetella pertussis (Merkel et al., 1998). Up to now, the mass sequencing of bacterial genomes detected an abundance of GGDEF and EAL containing proteins (Galperin et al., 2001). In the sequenced prokaryotic genomes, 1601 and 1016 copies of GGDEF and EAL domain respectively,
have been found and 147 and 88 representative architectures of GGDEF and EAL domain were defined according to Pfam database
(http://www.sanger.ac.uk/Software/Pfam/). More and more experimental data of GGDEF and EAL proteins have been reported with time, and the biological functions of them were marshaled as below:
Aggregative behavior and biofilm formation
Several experimental evidences have shown that GGDEF and EAL containing proteins influence the bacterial morphotypes, which have been characterized by an increased production of extracellular matrix components, such as fimbriae and exopolysaccharide, by aggregative behavior and by enhanced biofilm formation.
Some GGDEF or EAL protein homologs have been identified as regulators of this adhesive behavior. In Salmonella, AdrA, a GGDEF protein, is required for cellulose synthesis but not for the formation of aggregative fimbriae (Romling et al., 2000;
Zogaj et al., 2001). In Pseudomonas, WspR, a CheY-GGDEF protein, controls the expression of an acetylated cellulose polymer and a fimbrial component of the biofilm matrix (Spiers et al., 2002; Spiers et al., 2003) and PvrR, a CheY-EAL protein, has been described as a regulator of biofilm formation and aggregation (Drenkard and Ausubel, 2002). Besides, RocS and MbaA, GGDEF-EAL proteins, were both identified in genetic screens for V. cholerae mutants defective in biofilm formation and maturation. The rocS mutant seemed to possess a general defect in switching to the rugose phenotype (Rashid et al., 2003). MbaA was shown to be also required at a later stage of biofilm formation (Bomchil et al., 2003).
Bacterial motility
There are experiments suggesting that cyclic di-GMP acts as a messenger to direct the transition from sessility to motility (Simm et al., 2004) and some
GGDEF-EAL protein homologs have been identified. In V. parahaemolyticus, SrcC
controls swarmer cell differentiation, including the production of lateral flagella and synthesis of capsular polysaccharide in order to grow on surfaces (Boles and
McCarter, 2002; Guvener and McCarter, 2003). MorA is a regulator of GGDEF-EAL protein affecting flagellar development and biofilm formation in diverse
Pseudomonas species (Choy et al., 2004). FimX, another GGDEF-EAL protein, is
required for the bacterial type IV pilus-mediated twitching motility (Huang et al., 2003). Additionally, in Caulobacter crescentus, cells that lack functional PleD, a CheY-GGDEF protein, are hypermotile, unable to eject the flagellum during the swarmer-to-stalked cell transition, and failing to fully synthesize a stalk structure (Aldridge and Jenal, 1999; Aldridge et al., 2003).
3. Specific aims
In this study, we aimed to investigate the likely roles of the GGDEF-containing protein VVA0326, the EAL-containing protein VVA0328 and RtxL. The specific experiments performed are as following:
1.1. Several Vibrio vulnificus YJ016 derived mutants carrying respectively VVA0326, VVA0328 and RtxL gene deletion were constructed. Phenotypic analysis of the mutants including growth rate analysis, biofilm formation assay, swimming motility, flagellar and exopolysaccharide synthesis were carried out.
1.2. VVA0326-GFP and VVA0328-GFP (Green Florescence Protein) fusion protein constructs were obtained to demonstrate the localized distribution of the
overexpressed proteins in E. coli JM109.
2. We have also employed phylogeny analysis to study the evolutionary relationship of GGDEF- and EAL- containing proteins in the bacteria.
Materials and methods