Amy J. Horneman

Genome Sequence of Aeromonas hydrophila ATCC 7966T: Jack of All Trades

The complete genome of Aeromonas hydrophila ATCC 7966(T) was sequenced. Aeromonas, a ubiquitous waterborne bacterium, has been placed by the Environmental Protection Agency on the Contaminant Candidate List because of its potential to cause human disease. The 4.7-Mb genome of this emerging pathogen shows a physiologically adroit organism with broad metabolic capabilities and considerable virulence potential. A large array of virulence genes, including some identified in clinical isolates of Aeromonas spp. or Vibrio spp., may confer upon this organism the ability to infect a wide range of hosts. However, two recognized virulence markers, a type III secretion system and a lateral flagellum, that are reported in other A. hydrophila strains are not identified in the sequenced isolate, ATCC 7966(T). Given the ubiquity and free-living lifestyle of this organism, there is relatively little evidence of fluidity in terms of mobile elements in the genome of this particular strain. Notable aspects of the metabolic repertoire of A. hydrophila include dissimilatory sulfate reduction and resistance mechanisms (such as thiopurine reductase, arsenate reductase, and phosphonate degradation enzymes) against toxic compounds encountered in polluted waters. These enzymes may have bioremediative as well as industrial potential. Thus, the A. hydrophila genome sequence provides valuable insights into its ability to flourish in both aquatic and host environments.

A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin

We recently delineated the importance of a type VI secretion system (T6SS) gene cluster in the virulence of diarrheal isolate SSU of Aeromonas hydrophila and showed that VasH, a sigma(54) activator and T6SS component, was involved in the production of its associated effectors, e.g., hemolysin-coregulated protein. To identify additional T6SS effectors and/or secreted proteins, we subjected culture supernatants from deletion mutants of A. hydrophila, namely, a Delta act mutant (a T2SS-associated cytotoxic enterotoxin-encoding gene) and a Delta act Delta vasH mutant, to 2-dimensional gel electrophoresis and mass spectrometric analysis. Based on these approaches, we identified a member of the VgrG protein family, VgrG1, that contained a vegetative insecticidal protein (VIP-2) domain at its carboxyl-terminal end. Consequently, the vgrG1 gene was cloned in pBI-EGFP and pET-30a vectors to be expressed in HeLa Tet-Off cells and Escherichia coli, respectively. We assessed the ADP-ribosyltransferase (ADPRT) activity of various domains of purified recombinant VgrG1 (rVgrG1) and provided evidence that only the full-length VgrG1, as well as its carboxyl-terminal domain encoding the VIP-2 domain, showed ADPRT activity. Importantly, bacterium-host cell interaction was needed for the T6SS to induce cytotoxicity in eukaryotic cells, and we demonstrated translocation of VgrG1. Furthermore, our data indicated that expression of the genes encoding the full-length VgrG1 and its carboxyl-terminal domain in HeLa Tet-Off cells disrupted the actin cytoskeleton, which was followed by a decrease in cell viability and an increase in apoptosis. Taken together, these findings demonstrated for the first time that VgrG1 of A. hydrophila possessed actin ADPRT activity associated with its VIP-2 domain and that this domain alone was able to induce a rounded phenotype in HeLa Tet-Off cells, followed by apoptosis mediated by caspase 9 activation.

Distribution of Virulence Factors and Molecular Fingerprinting of Aeromonas Species Isolates from Water and Clinical Samples: Suggestive Evidence of Water-to-Human Transmission†

ABSTRACT A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.

N-Acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila

In this study, we delineated the role of N-acylhomoserine lactone(s) (AHLs)-mediated quorum sensing (QS) in the virulence of diarrhoeal isolate SSU of Aeromonas hydrophila by generating a double knockout Delta ahyRI mutant. Protease production was substantially reduced in the Delta ahyRI mutant when compared with that in the wild-type (WT) strain. Importantly, based on Western blot analysis, the Delta ahyRI mutant was unable to secrete type VI secretion system (T6SS)-associated effectors, namely haemolysin coregulated protein and the valine-glycine repeat family of proteins, while significant levels of these effectors were detected in the culture supernatant of the WT A. hydrophila. In contrast, the production and translocation of the type III secretion system (T3SS) effector AexU in human colonic epithelial cells were not affected when the ahyRI genes were deleted. Solid surface-associated biofilm formation was significantly reduced in the Delta ahyRI mutant when compared with that in the WT strain, as determined by a crystal violet staining assay. Scanning electron microscopic observations revealed that the Delta ahyRI mutant was also defective in the formation of structured biofilm, as it was less filamentous and produced a distinct exopolysaccharide on its surface when compared with the structured biofilm produced by the WT strain. These effects of AhyRI could be complemented either by expressing the ahyRI genes in trans or by the exogeneous addition of AHLs to the Delta ahyRI/ahyR(+) complemented strain. In a mouse lethality experiment, 50 % attenuation was observed when we deleted the ahyRI genes from the parental strain of A. hydrophila. Together, our data suggest that AHL-mediated QS modulates the virulence of A. hydrophila SSU by regulating the T6SS, metalloprotease production and biofilm formation.

Characterization of Aeromonas hydrophila Wound Pathotypes by Comparative Genomic and Functional Analyses of Virulence Genes

ABSTRACT Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases. In a previously published case report, we described a subject with a polymicrobial wound infection that included a persistent and aggressive strain of A. hydrophila (E1), as well as a more antibiotic-resistant strain of A. hydrophila (E2). To better understand the differences between pathogenic and environmental strains of A. hydrophila, we conducted comparative genomic and functional analyses of virulence-associated genes of these two wound isolates (E1 and E2), the environmental type strain A. hydrophila ATCC 7966T, and four other isolates belonging to A. aquariorum, A. veronii, A. salmonicida, and A. caviae. Full-genome sequencing of strains E1 and E2 revealed extensive differences between the two and strain ATCC 7966T. The more persistent wound infection strain, E1, harbored coding sequences for a cytotoxic enterotoxin (Act), a type 3 secretion system (T3SS), flagella, hemolysins, and a homolog of exotoxin A found in Pseudomonas aeruginosa. Corresponding phenotypic analyses with A. hydrophila ATCC 7966T and SSU as reference strains demonstrated the functionality of these virulence genes, with strain E1 displaying enhanced swimming and swarming motility, lateral flagella on electron microscopy, the presence of T3SS effector AexU, and enhanced lethality in a mouse model of Aeromonas infection. By combining sequence-based analysis and functional assays, we characterized an A. hydrophila pathotype, exemplified by strain E1, that exhibited increased virulence in a mouse model of infection, likely because of encapsulation, enhanced motility, toxin secretion, and cellular toxicity. IMPORTANCE Aeromonas hydrophila is a common aquatic bacterium that has increasingly been implicated in serious human infections. While many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. This paper demonstrates, by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. Our results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms. Aeromonas hydrophila is a common aquatic bacterium that has increasingly been implicated in serious human infections. While many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. This paper demonstrates, by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. Our results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms.

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.

Complex Evolutionary History of the Aeromonas veronii Group Revealed by Host Interaction and DNA Sequence Data

Aeromonas veronii biovar sobria, Aeromonas veronii biovar veronii, and Aeromonas allosaccharophila are a closely related group of organisms, the Aeromonas veronii Group, that inhabit a wide range of host animals as a symbiont or pathogen. In this study, the ability of various strains to colonize the medicinal leech as a model for beneficial symbiosis and to kill wax worm larvae as a model for virulence was determined. Isolates cultured from the leech out-competed other strains in the leech model, while most strains were virulent in the wax worms. Three housekeeping genes, recA, dnaJ and gyrB, the gene encoding chitinase, chiA, and four loci associated with the type three secretion system, ascV, ascFG, aexT, and aexU were sequenced. The phylogenetic reconstruction failed to produce one consensus tree that was compatible with most of the individual genes. The Approximately Unbiased test and the Genetic Algorithm for Recombination Detection both provided further support for differing evolutionary histories among this group of genes. Two contrasting tests detected recombination within aexU, ascFG, ascV, dnaJ, and gyrB but not in aexT or chiA. Quartet decomposition analysis indicated a complex recent evolutionary history for these strains with a high frequency of horizontal gene transfer between several but not among all strains. In this study we demonstrate that at least for some strains, horizontal gene transfer occurs at a sufficient frequency to blur the signal from vertically inherited genes, despite strains being adapted to distinct niches. Simply increasing the number of genes included in the analysis is unlikely to overcome this challenge in organisms that occupy multiple niches and can exchange DNA between strains specialized to different niches. Instead, the detection of genes critical in the adaptation to specific niches may help to reveal the physiological specialization of these strains.

Mutation in the S-ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila.

A diarrheal isolate SSU of Aeromonas hydrophila produces a cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. Our laboratory has characterized from the above Aeromonas strain, in addition to Act, the type 3- and T6-secretion systems and their effectors, as well as the genes shown to modulate the production of AI-1-like autoinducers, N-acylhomoserine lactones (AHLs) involved in quorum sensing (QS). In this study, we demonstrated the presence of an S-ribosylhomocysteinase (LuxS)-based autoinducer (AI)-2 QS system in A. hydrophila SSU and its contribution to bacterial virulence. The luxS isogenic mutant of A. hydrophila, which we prepared by marker exchange mutagenesis, showed an alteration in the dynamics and architecture of the biofilm formation, a decrease in the motility of the bacterium, and an enhanced virulence in the septicemic mouse model. Moreover, these effects of the mutation could be complemented. Enhanced production of the biofilm exopolysaccharide and filaments in the mutant strain were presumably the major causes of the observed phenotype. Our earlier studies indicated that the wild-type A. hydrophila with overproduction of DNA adenine methyltransferase (Dam) had significantly reduced motility, greater hemolytic activity associated with Act, and an enhanced ability to produce AI-1 lactones. Furthermore, such a Dam-overproducing strain was not lethal to mice. On the contrary, the luxS mutant with Dam overproduction showed an increased motility and had no effect on lactone production. In addition, the Dam-overproducing luxS mutant strain was not altered in its ability to induce lethality in a mouse model of infection when compared to the parental strain which overproduced Dam. We suggested that an altered gene expression in the luxS mutant of A. hydrophila SSU, as it related to biofilm formation and virulence, might be linked with the interruption of the bacterial metabolic pathway, specifically of methionine synthesis.

Functional genomic characterization of virulence factors from necrotizing fasciitis-causing strains of Aeromonas hydrophila.

ABSTRACT The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF.

Virulence factor–activity relationships (VFAR) with specific emphasis on Aeromonas species (spp.)

The human population most commonly inflicted with Aeromonas infection includes young children, the elderly and immunocompromised individuals. Importantly, the isolation rate of Aeromonas species from children suffering from diarrhea is similar in developing and developed countries. It is becoming clear that only a small subset of Aeromonas species belonging to a particular hybridization group causes disease in humans. Human infections with this pathogen occur by consuming contaminated food and water. Aeromonas species were isolated from wounds of patients during the tsunami in southern Thailand. Further, increased numbers of this pathogen were recovered from floodwater samples during Hurricane Katrina in New Orleans. Among various species of Aeromonas, A. hydrophila, A. caviae and A. veronii biovar sobria are mainly responsible for causing disease in humans. Our laboratory has isolated various virulence factors from a diarrheal isolate SSU of A. hydrophila and molecularly characterized them. In addition to various virulence factors produced by Aeromonas species, the status of the immune system plays an important role in inducing disease by this pathogen in the host. Taken together, we have made significant advances in better understanding the pathogenesis of Aeromonas infections, which will help in differentiating pathogenic from non-pathogenic aeromonads. This review covers virulence aspects of a clinical isolate of A. hydrophila.