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Background: The putative needle complex subunit AscF forms a ternary complex with the chaperones AscE and AscG in the type III secretion system of Aeromonas hydrophila so as to avoid premature assembly. Previously, we demonstrated that the C-terminal region of AscG (residues 62-116) in the hetero-molecular chaperone, AscE-AscG, is disordered and susceptible to limited protease digestion., Methodology/principal Findings: Here, we report the crystal structure of the ordered AscG(1-61) region in complex with AscE at 2.4 Å resolution. Helices α2 and α3 of AscE in the AscE-AscG(1-61) complex assumes a helix-turn-helix conformation in an anti-parallel fashion similar to that in apo AscE. However, in the presence of AscG, an additional N-terminal helix α1 in AscE (residues 4-12) is observed. PscG or YscG in the crystal structures of PscE-PscF-PscG or YscE-YscF-YscG, respectively, assumes a typical tetratricopeptide repeat (TPR) fold with three TPR repeats and one C-terminal capping helix. By comparison, AscG in AscE-AscG(1-61) comprises three anti-parallel helices that resembles the N-terminal TPR repeats in the corresponding region of PscG or YscG in PscE-PscF-PscG or YscE-YscF-YscG. Thermal denaturation of AscE-AscG and AscE-AscG(1-61) complexes demonstrates that the C-terminal disordered region does not contribute to the thermal stability of the overall complex., Conclusion/significance: The N-terminal region of the AscG in the AscE-AscG complex is ordered and assumes a structure similar to those in the corresponding regions of PscE-PscG-PscF or YscE-YscF-YscG complexes. While the C-terminal region of AscG in the AscE-AscG complex is disordered and will assume its structure only in the presence of the substrate AscF. We hypothesize that AscE act as a chaperone of the chaperone to keep AscG in a stable but partially disordered state for interaction with AscF.

In the type III secretion system (T3SS) of Aeromonas hydrophila, AcrH acts as a chaperone for translocators AopB and AopD. AcrH forms a stable 1:1 monomeric complex with AopD, whereas the 1:1 AcrH-AopB complex exists mainly as a metastable oligomeric form and only in minor amounts as a stable monomeric form. Limited protease digestion shows that these complexes contain highly exposed regions, thus allowing mapping of intact functional chaperone binding regions of AopB and AopD. AopD uses the transmembrane domain (DF1, residues 16-147) and the C-terminal amphipathic helical domain (DF2, residues 242-296) whereas AopB uses a discrete region containing the transmembrane domain and the putative N-terminal coiled coil domain (BF1, residues 33-264). Oligomerization of the AcrH-AopB complex is mainly through the C-terminal coiled coil domain of AopB, which is dispensable for chaperone binding. The three proteins, AcrH, AopB, and AopD, can be coexpressed to form an oligomeric and metastable complex. These three proteins are also oligomerized mainly through the C-terminal domain of AopB. Formation of such an oligomeric and metastable complex may be important for the proper formation of translocon of correct topology and stoichiometry on the host membrane.

In the type III secretion system (T3SS) of Aeromonas hydrophila, the putative needle complex subunit AscF requires both putative chaperones AscE and AscG for formation of a ternary complex to avoid premature assembly. Here we report the crystal structure of AscE at 2.7 A resolution and the mapping of buried regions of AscE, AscG, and AscF in the AscEG and AscEFG complexes using limited protease digestion. The dimeric AscE is comprised of two helix-turn-helix monomers packed in an antiparallel fashion. The N-terminal 13 residues of AscE are buried only upon binding with AscG, but this region is found to be nonessential for the interaction. AscE functions as a monomer and can be coexpressed with AscG or with both AscG and AscF to form soluble complexes. The AscE binding region of AscG in the AscEG complex is identified to be within the N-terminal 61 residues of AscG. The exposed C-terminal substrate-binding region of AscG in the AscEG complex is induced to be buried only upon binding to AscF. However, the N-terminal 52 residues of AscF remain exposed even in the ternary AscEFG complex. On the other hand, the 35-residue C-terminal region of AscF in the complex is resistant to protease digestion in the AscEFG complex. Site-directed mutagenesis showed that two C-terminal hydrophobic residues, Ile83 and Leu84, of AscF are essential for chaperone binding.

We cloned and sequenced an ADP-ribosylating toxin (AexT) from a mesophilic Aeromonas hydrophila strain AH-3 with a type III secretion system (T3SS). This toxin only showed homology, in genes and proteins, with the first half of A. salmonicida AexT. The A. hydrophila AexT showed ADP-ribosyltransferase activity, translocation through the T3SS system, and this A. hydrophila T3SS system is inducible under calcium-depleted conditions. The A. hydrophila aexT mutant showed a slight reduction in their virulence assayed by several methods when compared to the wild-type strain, while an A. hydrophila T3SS mutant is highly reduced in virulence on the same assays. The A. hydrophila AexT is the first described and the smallest T3SS effector toxin found in mesophilic Aeromonas with a functional T3SS.

Aeromonas hydrophila is a ubiquitous Gram-negative bacterium which can cause motile aeromonad septicemia in both fish and humans. A. hydrophila secretes many extracellular proteins associated with pathogenicity and environmental adaptability. In this study, an extracellular proteome map of A. hydrophila AH-1 was constructed. The major extracellular virulence factors were characterized by comparing the proteomes of various deletion mutants with that of the wild type. The results suggested that serine protease was involved in the processing of a toxin and secreted enzymes such as hemolysin, glycerophospholipid-cholesterol acyltransferase and metalloprotease. We also showed that expressions of polar and lateral flagellins were under the control of temperature, FlhA, LafK, and RpoN. In addition, three novel proteins (potential effector proteins including one ExoT-like protein) were revealed to be secreted via the type III secretion system (TTSS) of A. hydrophila AH-1. Another novel finding was the demonstration of a crosstalk between the lateral flagellar system and the TTSS in A. hydrophila. These results showed that proteomics is a powerful tool for characterizing virulence factors. The construction of proteome maps will provide a valuable means of finding potential candidates for developing suitable diagnostics and therapeutics for this emerging pathogen.

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.

Aeromonas hydrophila is a gram-negative opportunistic pathogen in fish and humans. Many bacterial pathogens of animals and plants have been shown to inject anti-host virulence determinants into the hosts via a type III secretion system (TTSS). Degenerate primers based on lcrD family genes that are present in every known TTSS allowed us to locate the TTSS gene cluster in A. hydrophila AH-1. A series of genome walking steps helped in the identification of 25 open reading frames that encode proteins homologous to those in TTSSs in other bacteria. PCR-based analysis showed the presence of lcrD homologs (ascV) in all of the 33 strains of A. hydrophila isolated from various sources. Insertional inactivation of two of the TTSS genes (aopB and aopD) led to decreased cytotoxicity in carp epithelial cells, increased phagocytosis, and reduced virulence in blue gourami. These results show that a TTSS is required for A. hydrophila pathogenesis. This is the first report of sequencing and characterization of TTSS gene clusters from A. hydrophila. The TTSS identified here may help in developing suitable vaccines as well as in further understanding of the pathogenesis of A. hydrophila.

The genetic organization and sequences of the group II capsule gene cluster of Aeromonas hydrophila PPD134/91 have been determined previously. The purified capsular polysaccharides can increase the ability of avirulent strain PPD35/85 to survive in naive tilapia serum but have no inhibitory effect on the adhesion of PPD134/91 to carp epithelial cells. In this study, the presence of group II capsules among 33 randomly chosen A. hydrophila strains was examined by electron microscopy and genetic analysis. Ten strains were found to produce group II capsules. A PCR detection system was developed to identify two types of group II capsules (IIA and IIB) based on their genetic organization in the region II gene clusters. Group IIA capsules in the authors' collection of A. hydrophila strains are mainly found in the O : 18 and O : 34 serogroups, while group IIB capsules are found in the O : 21 and O : 27 serogroups. The presence of group II capsules in A. hydrophila strongly correlates with the serum and phagocyte survival abilities (seven out of ten strains). The results indicate that the authors' PCR detection system can constitute a reliable assay for the classification of group II capsules in A. hydrophila.

The sequences of the O-antigen and capsule gene clusters of the virulent Aeromonas hydrophila strain PPD134/91 were determined. The O-antigen gene cluster is 17,296 bp long and comprises 17 genes. Seven pathway genes for the synthesis of rhamnose and mannose, six transferase genes, one O unit flippase gene, and one O-antigen chain length determinant gene were identified by amino acid sequence similarity. PCR and Southern blot analysis were performed to survey the distribution of these 17 genes among 11 A. hydrophila strains of different serotypes. A. hydrophila PPD134/91 might belong to serotype O:18, as represented by JCM3980; it contained all the same O-antigen genes as JCM3980 (97 to 100% similarity at the DNA and amino acid levels). The capsule gene cluster of A. hydrophila PPD134/91 is 17,562 bp long and includes 13 genes, which were assembled into three distinct regions similar to those of the group II capsule gene cluster of Escherichia coli and other bacteria. Regions I and III contained four and two capsule transport genes, respectively. Region II had five genes which were highly similar to capsule synthesis pathway genes found in other bacteria. Both the purified O-antigen and capsular polysaccharides increased the ability of the avirulent A. hydrophila strain PPD35/85 to survive in naïve tilapia serum. However, the purified surface polysaccharides had no inhibitory effect on the adhesion of A. hydrophila PPD134/91 to carp epithelial cells.

Aeromonas hydrophila, a normal inhabitant of aquatic environments, is an opportunistic pathogen of a variety of aquatic and terrestrial animals, including humans. A. hydrophila PPD134/91 is defined as virulent whereas PPD35/85 is defined as avirulent on the basis of their different LD50 values in fish. Suppression subtractive hybridization (SSH) was used to identify genetic differences between these two strains. Sixty-nine genomic regions of differences were absent in PPD35/85, and the DNA sequences of these regions were determined. Sixteen ORFs encoded by 23 fragments showed high homology to known proteins of other bacteria. ORFs encoded by the remaining 46 fragments were identified as new proteins of A. hydrophila, showing no significant homology to any known proteins. Among these PPD134/91-specific genes, 22 DNA fragments (21 ORFs) were present in most of the eight virulent strains studied but mostly absent in the seven avirulent strains, suggesting that they are universal virulence genes in A. hydrophila. The PPD134/91-specific genes included five known virulence factors of A. hydrophila: haemolysin (hlyA), protease (oligopeptidase A), outer-membrane protein (Omp), multidrug-resistance protein and histone-like protein (HU-2). Another 47 DNA fragments (44 ORFs) were mainly present in PPD134/91, indicating the heterogeneity among motile aeromonads. Some of these fragments encoded virulence determinants. These included genes for the synthesis of O-antigen and type II restriction/modification system. The results indicated that SSH is successful in identifying genetic differences and virulence genes among different strains of A. hydrophila.

Aeromonas hydrophila is a Gram-negative bacterium that is pathogenic in fish, causing motile aeromonad septicaemia. It can enter (invade) fish cells, and survive as an intracellular parasite. The host-pathogen interaction and signal transduction pathway were studied by screening signal transduction inhibitors using carp epithelial cells and a virulent strain of the bacterium, PPD134/91. Genistein, a tyrosine kinase inhibitor, postponed internalization of A. hydrophila into host cells, suggesting that tyrosine phosphorylation plays a role in internalization. In contrast, staurosporine, a protein kinase C inhibitor, and sodium orthovanadate, a protein tyrosine phosphatase inhibitor, accelerated internalization of PPD134/91. Other virulent strains of A. hydrophila were also examined and it is likely that all strains, irrespective of serogroup, use the same signalling pathway to facilitate bacterial uptake.