Cold shock exoribonuclease R (VacB) is involved in Aeromonas hydrophila pathogenesis

In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC 7966(T) of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4 degrees C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37 degrees C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses (LD(50)). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same LD(50) dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.     

Virulence profiles and other biological characters in water isolated Aeromonas hydrophila

Thirty water isolates of A. hydrophila were tested for potential virulence profiles, antibiotic resistance and Bacteriocin-Like Substances (BLS) production. Cytotoxic activity was present in all strains tested, 87% were hemolytic and 70% adhesive. Lysine decarboxylase reactions (LDC) positivity was correlated with virulence factors: 100% versus cytotoxicity, 84% versus adherence, 76% versus hemolytic activity. The correlation was also present in the LDC-negative strains. Hemolytic and cytotoxic activities were frequently associated: high cytotoxicity, corresponding to high hemolytic activity and vice versa. The in vitro susceptibility of A. hydrophila to 28 antibacterial agents showed that cefotaxime was the most active beta-lactam antibiotic, and Cefuroxime inhibited 90% of the strains. Isolates were resistant to Penicillin G, Ampicillin, Carbenicillin, Amoxicillin, Cephalotin and Cefaclor. Tetracycline, Chloramphenicol, Nitrofurantoine, the quinolones and the aminoglycosides (except Streptomycin) were consistently active. BLS production never emerged against closely-related microorganisms. On the contrary A. hydrophila presented a heteroinhibitory activity against non-taxonomically related genera such as Listeria spp. (L. seeligeri NCTC 11856, L. welshimeri NCTC 11857, L. ivanovii NCTC 11846) and S. aureus ATCC 25923. Although a large number of strains showed virulence determinants together with other biological characters such as antibiotic resistance and BLS production, it was not possible to relate these factors to the observed plasmids.     

Characterization of extracellular proteins produced by Aeromonas hydrophila AH-1

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.     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.     

Characterization of extracellular metallo- and serine-proteases of Aeromonas hydrophila strain B51

The extracellular proteases of Aeromonas hydrophila B51 were stable on heating (56 degrees C) and on storage at 4 degrees C or -20 degrees C. Inhibitor studies showed that 72% of the total activity was inhibited by EDTA (a metalloprotease inhibitor) and 26% was inhibited by phenylmethanesulphonyl fluoride (a serine protease inhibitor). Analytical isoelectric focussing revealed the presence of 33 proteins in the crude extracellular products. Using a casein overlay technique three separate zones of proteolytic activity were detected: a zone with pI 6.5-6.8, formed of two closely focussed bands (possibly isomers of the same protease) and completely inhibited by EDTA; a single band with pI 7.0, which was inhibited by EDTA; and a diffuse zone with pI 8.3-8.5, which was only partially inhibited by EDTA. It is concluded that the serine protease activity focussed in this latter zone. These results indicate the presence of at least four, and possibly five proteases. Our results differ substantially from those reported by other workers using different isolates and it is suggested that significant differences in the character of extracellular products and extracellular proteases exist between different isolates of A. hydrophila.     

A note on nutritional influence on extracellular protease synthesis in Aeromonas hydrophila

Several individual amino acids served as the sole nitrogen source for growth and extracellular protease synthesis by cells of Aeromonas hydrophila. In the presence of urea, ammonium compounds and glycine, enzyme production was severely repressed although cell growth was only moderately affected.     

Molecular cloning and characterization of an extracellular protease gene from Aeromonas hydrophila.

A structural gene which codes for an extracellular protease in Aeromonas hydrophilia SO2/2 and D13 was cloned in Escherichia coli C600-1 by using pBR322 as a vector. The gene codes for a temperature-stable protease with a molecular mass of approximately 38,000 daltons. The protein was secreted to the periplasm of E. coli C600-1 and purified by osmotic shock. Cloned protease (P3) was identical in molecular mass and properties to the one purified from A. hydrophila SO2/2 culture supernatant as an extracellular product.     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

Proteomics analysis reveals that CirA in Aeromonas hydrophila is involved in nutrient uptake

The colicin I receptor (CirA) is a well-studied outer membrane protein that has been reported to play important roles in antibiotic resistance, virulence, and iron homeostasis, although its exact physiological roles require further investigation. In this study, differentially expressed proteins between the ΔahcirA and wild-type (WT) strains of Aeromonas hydrophila were compared using quantitative proteomics. Bioinformatics analysis revealed that the expression of peptide, histidine, and arginine ATP-binding cassette (ABC) transporter system-related proteins was significantly higher in the ΔahcirA strain. Subsequent growth assays revealed that ΔahcirA grew slower than the WT strain in nutrient-limited medium when supplemented with dipeptide, histidine, and arginine as the carbon source. Far-western blot analysis further confirmed that AhCirA can directly bind to histidine/arginine and dipeptide small-molecule substrates in addition to their periplasmic-binding proteins, AhDppA and AhHisJ, respectively. These results indicate that AhCirA may play an important role in the uptake of amino acids and peptides as a channel-forming porin while also directly interacting with ABC transporters to transport nutrient substances into the plasma membrane. Overall, this study demonstrates that AhCirA is a multifunctional protein in A. hydrophila and extends our understanding of known nutrient transport mechanisms among bacteria.     

RAPD analysis of Aeromonas salmonicida and Aeromonas hydrophila

The randomly amplified polymorphic DNA (RAPD) technique was used to analyse the genetic differentiation of 13 strains of Aeromonas salmonicida subsp. salmonicida , and seven strains of Aer. hydrophila. Reproducible profiles of genomic DNA fingerprints were generated by polymerase chain reaction (PCR) using a single randomly designed primer. The RAPD profiles of all the non-motile aeromonads, Aer. salmonicida subsp. salmonicida were identical. However, profiles of the motile aeromonads, Aer. hydrophila differed between isolates. These findings reveal genomic homogeneity in Aer. salmonicida subsp. salmonicida and genetic variety in Aer. hydrophila strains.     

Inhibition of frog antimicrobial peptides by extracellular products of the bacterial pathogen Aeromonas hydrophila

Aims:  To determine whether the extracellular products (ECPs) from Aeromonas hydrophila , a frog bacterial pathogen that is resistant to skin antimicrobial peptides of three different frog species Xenopus laevis , Litoria aurea and Litoria raniformis , can modulate the activity of these peptides.
Methods and Results:  ECPs were collected from cultures of Klebsiella pneumoniae , a pathogen susceptible to skin antimicrobial peptides of all three tested frog species, and from cultures of Aer .  hydrophila . They were tested for protease activity and for inhibition of the antimicrobial activity of natural peptide mixtures and single peptides of all three frog species against Escherichia coli ATCC 25922. ECPs from cultures of Aer .  hydrophila grown for 16, 24 and 36 h showed protease activity and inhibited the antibacterial activity of all peptides against E .  coli ATCC 25922. In contrast, the ECPs from cultures of Kl .  pneumoniae neither had protease activity nor inhibited the activity of any peptides.
Conclusion:  The proteolytic ECPs of Aer .  hydrophila have the ability to inhibit the skin antimicrobial peptides of frogs.
Significance and Impact of the Study:  The results of this study provide new information on the association of ECPs with the resistance of Aer .  hydrophila to frog antimicrobial peptides.     

Survival of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria in soil

The survival of mesophilic Aeromonas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. Two cytotoxic ( Aer. hydrophila and Aer. caviae ) and one invasive ( Aer. sobria ) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas , the number of living cells was determined over at least 5 months. For all strains the survival curves were characterized by an initial re-growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. The times necessary to achieve a 95% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer. hydrophila and Aer. sobria , while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas -associated human infections.     

Mutagenesis and isolation of Aeromonas hydrophila genes which are required for extracellular secretion.

Transposon mutagenesis was used to isolate mutants of Aeromonas hydrophila which were deficient in the production of extracellular proteins. The culture supernatants of two of the mutants were essentially devoid of the proteins normally secreted by the parent strain, despite their continued synthesis. Western immunoblot analysis of one of these proteins indicated that normal signal sequence processing occurred but that normal zymogen activation did not, and cell fractionation experiments indicated that both mutants accumulated the three different extracellular proteins assayed in a position external to the cytoplasmic membrane, presumably in the periplasm. The two mutants differed, however, in that one was lysed during the osmotic shock procedures and also contained severely reduced amounts of two of the major protein components of the outer membrane. The wild-type chromosomal regions into which the transposon had been inserted in the two mutants were cloned. In each case, transconjugants of the mutants containing the corresponding cloned fragment were complemented for the defects in secretion, and one of the mutants was complemented by the heterologous clone as well, suggesting the possibility of an interaction between these two genes or gene products. These results indicate that two separate functions which are required for extracellular secretion were interrupted in the insertion mutants and that one of these is also critically important in the biogenesis of the outer membrane.     

Difference in the extracellular products of two strains of Aeromonas hydrophila virulent and weakly virulent for fish

In this study we first compared the toxigenic profile of Aeromonas hydrophila strains virulent and weakly virulent for rainbow trout. The separation of the toxic factor for fish is also described. Both strains produced hemolytic, enterotoxic, and dermonecrotic activities; the weakly virulent strain produced 20 times more hemolysin. Only the cell-free supernatant of the virulent strain produced a toxic factor for fish. After passage on Sephacryl S-200, the toxic factor for fish was separated from the hemolysin. This toxic factor is heat labile.     

Role of virulence plasmid of Aeromonas hydrophila in the pathogenesis of ulcerative disease syndrome in Clarias batrachus

Pathogenic Aeromonas hydrophila (strain VB21), a multiple-drug resistance strain contains a plasmid of about 21 kb. After curing of plasmid, the isolates became sensitive to antimicrobials, to which they were earlier resistant. The cured bacteria exhibited significant alterations in their surface structure, growth profile and virulence properties, and failed to cause ulcerative disease syndrome (UDS) when injected into the Indian catfish Clarias batrachus. Routine biochemical studies revealed that the plasmid curing did not alter the biochemical properties of the bacteria. After transformation of the plasmid into cured A. hydrophila the bacterium regained its virulence properties and induced all the characteristic symptoms of UDS when injected into fish. Thus, the plasmid plays a pivotal role in the phenotype, growth and virulence of A. hydrophila and pathogenesis of aeromonad UDS.     

Electroporation-mediated transformation of Aeromonas hydrophila

A strain of Aeromonas hydrophila producing copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate, abbreviated as PHBHHx, was successfully transformed by electroporation. The plasmid used was a broad host range plasmid pBBR1MCS. Electroporation conditions were varied systemically to develop an electroporation protocol. The optimal yield of transformant was approximately 4x10(2) CFU/microg DNA at 12.5 kV/cm and 1000 Omega, resulting in a time constant of approximately 5 ms. The A. hydrophila transformants expressed plasmid-encoded resistance to chloromphenicol. Plasmid DNA in the A. hydrophila transformant was stably maintained. This is the first report of transformation of bacteria A. hydrophila.     

Polar flagellum biogenesis in Aeromonas hydrophila

Mesophilic Aeromonas spp. constitutively express a single polar flagellum that helps the bacteria move to more favorable environments and is an important virulence and colonization factor. Certain strains can also produce multiple lateral flagella in semisolid media or over surfaces. We have previously reported 16 genes (flgN to flgL) that constitute region 1 of the Aeromonas hydrophila AH-3 polar flagellum biogenesis gene clusters. We identified 39 new polar flagellum genes distributed in four noncontiguous chromosome regions (regions 2 to 5). Region 2 contained six genes (flaA to maf-1), including a modification accessory factor gene (maf-1) that has not been previously reported and is thought to be involved in glycosylation of polar flagellum filament. Region 3 contained 29 genes (fliE to orf29), most of which are involved in flagellum basal body formation and chemotaxis. Region 4 contained a single gene involved in the motor stator formation (motX), and region 5 contained the three master regulatory genes for the A. hydrophila polar flagella (flrA to flrC). Mutations in the flaH, maf-1, fliM, flhA, fliA, and flrC genes, as well as the double mutant flaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in the pomB stator gene did not affect polar flagellum motility, in contrast to the motX mutant, which was unable to swim even though it expressed a polar flagellum. Mutations in all of these genes did not affect lateral flagellum synthesis or swarming motility, showing that both A. hydrophila flagellum systems are entirely distinct.     

Survival of genetically-marked Aeromonas hydrophila in water

Survival of a genetically-marked Aeromonas hydrophila was monitored in water microcosms. There was no apparent loss of a marker plasmid which encoded the xylE reporter gene during prolonged incubation in lake water. Survival was best in sterile lake water but in sea water, cells died rapidly during the first 9 d, recovered up to day 12 and thereafter numbers fell up to 28 d accompanied by loss of the plasmid in a proportion of the cells.