Role of cold shock proteins B and D in Aeromonas salmonicida subsp. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus).

Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens affecting plant, insect, and mammalian hosts. However, their significance in bacterial pathogens of teleost fish remains unexplored. Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is a psychrotrophic pathogen and the causative agent of furunculosis in marine and freshwater fish. Four csp genes (cspB, cspD, cspA, and cspC) have been identified in the genome of A. salmonicida J223 (wild type). Here, we evaluated the role of DNA binding proteins, CspB and CspD, in A. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus). A. salmonicida ΔcspB, ΔcspD, and the double ΔcspBΔcspD mutants were constructed and characterized. A. salmonicida ΔcspB and ΔcspBΔcspD mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. A. salmonicida ΔcspD showed a slower growth at 28°C, biofilm formation, lower survival in low temperatures and freezing conditions (-20°C, 0°C, and 4°C), deficient in lipopolysaccharide synthesis, and low virulence in lumpfish. Additionally, ΔcspBΔcspD mutants showed less survival in the presence of bile compared to the wild type. Transcriptome analysis revealed that 200, 37, and 921 genes were differentially expressed in ΔcspB, ΔcspD, and ΔcspBΔcspD, respectively. In ΔcspB and ΔcspBΔcspD virulence genes in the chromosome and virulence plasmid were downregulated. Our analysis indicates that CspB and CspD mostly act as a transcriptional activator, influencing cell division (e.g., treB), virulence factors (e.g., aexT), and ultimately virulence.

The Sec pathway gene yidC regulates the virulence of mesophilic Aeromonassalmonicida.

Aeromonas salmonicida is a common pathogenic bacterial species found in both freshwater and marine fish, leading to significant economic losses in the aquaculture industry. YidC is an accessory to SecYEG and is essential for the SecYEG transporter to insert into the bacterial membrane. However, the roles of the yidC gene on the host immune response remain unclear. Here, we compared the pathogenicity of yidC gene-deleted (ΔyidC) strain and wild-type (SRW-OG1) strain of mesophilic A. salmonicida to Orange-spotted grouper (Epinephelus coioides), and explored the impacts of yidC gene on the immune response of E. coioides to mesophilic A. salmonicida infection by using Red/ET recombineering. In this study, the E. coioides in the Secondary infected group had a 53.9 % higher survival rate than those in the Primary infected group. In addition, the adhesion ability of ΔyidC strain decreased by about 83.36 % compared with that of the wild-type (SRW-OG1) strain. Further comparison of the biological phenotype of SRW-OG1 and ΔyidC revealed that this yidC gene could regulate the expression of genes related to iron metabolism and have no effect on bacterial growth under the limited iron concentration. In the low concentration of Fe3+ and Fe2+ environment, SRW-OG1 can obtain iron ions by regulating yidC. Based on the above results, yidC gene contributed to the pathogenicity of mesophilic A. salmonicida to E. coioides, deletion of yidC gene promoted the inflammation and immune response of E. coioides to mesophilic A. salmonicida infection.

Transcriptional Signatures of Immune, Neural, and Endocrine Functions in the Brain and Kidney of Rainbow Trout (Oncorhynchus mykiss) in Response to Aeromonas salmonicida Infection.

Rainbow trout (Oncorhynchus mykiss) serves as one of the most important commercial fish with an annual production of around 800,000 tonnes. However, infectious diseases, such as furunculosis caused by Aeromonas salmonicida infection, results in great economic loss in trout culture. The brain and kidney are two important organs associated with "sickness behaviors" and immunomodulation in response to disease. Therefore, we worked with 60 trout and investigated transcriptional responses and enrichment pathways between healthy and infected trout. We observed that furunculosis resulted in the activation of toll-like receptors with neuroinflammation and neural dysfunction in the brain, which might cause the "sickness behaviors" of infected trout including anorexia and lethargy. We also showed the salmonid-specific whole genome duplication contributed to duplicated colony stimulating factor 1 (csf-1) paralogs, which play an important role in modulating brain immunomodulation. Enrichment analyses of kidneys showed up-regulated immunomodulation and down-regulated neural functions, suggesting an immune-neural interaction between the brain and kidney. Moreover, the kidney endocrine network was activated in response to A. salmonicida infection, further convincing the communications between endocrine and immune systems in regulating internal homeostasis. Our study provided a foundation for pathophysiological responses of the brain and kidney in response to furunculosis and potentially offered a reference for generating disease-resistant trout strains.

Elucidating bacterial adhesion to mucosal surface by an original AFM approach.

BACKGROUND: Fish skin represents an ancient vertebrate mucosal surface, sharing characteristics with other mucosal surfaces including those of the intestine. The skin mucosa is continuously exposed to microbes in the surrounding water and is therefore important in the first line defense against environmental pathogens by preventing bacteria from accessing the underlying surfaces. Understanding the microbe-host interactions at the fish skin mucosa is highly relevant in order to understand and control infection, commensalism, colonization, persistence, infection, and disease. Here we investigate the interactions between the pathogenic bacteria Aeromonas salmonicida (A. salmonicida) and Yersinia ruckeri (Y. ruckeri), respectively, and the skin mucosal surface of Atlantic salmon fry using AFM force spectroscopy. RESULTS: The results obtained revealed that when retracting probes functionalized with bacteria from surfaces coated with immobilized mucins, isolated from salmon mucosal surfaces, rupture events reflecting the disruption of adhesive interactions were observed, with rupture strengths centered around 200 pN. However, when retracting probes functionalized with bacteria from the intact mucosal surface of salmon fish fry no adhesive interactions could be detected. Furthermore, rheological measurements revealed a near fluid-like behavior for the fish fry skin mucus. Taken together, the experimental data indicate that the adhesion between the mucin molecules within the mucous layer may be significantly weaker than the interaction between the bacteria and the mucin molecules. The bacteria, immobilized on the AFM probe, do bind to individual mucins in the mucosal layer, but are released from the near fluid mucus with little resistance upon retraction of the AFM probe, to which they are immobilized. CONCLUSION: The data provided in the current paper reveal that A. salmonicida and Y. ruckeri do bind to the immobilized mucins. However, when retracting the bacteria from intact mucosal surfaces, no adhesive interactions are detected. These observations suggest a mechanism underlying the protective function of the mucosal surface based on the clearing of potential threats by adhering them to loosely attached mucus that is subsequently released from the fish skin.

Characterization of Aeromonas salmonicida and A. sobria isolated from cultured salmonid fish in Korea and development of a vaccine against furunculosis.

Previously, Aeromonas sobria and A. salmonicida were identified to be the most prevalent species in salmonid farms in Korea. In this study, we evaluated the biochemical characteristics, antibiotic susceptibility and pathogenicity of A. salmonicida (3 isolates) and A. sobria (8 isolates) isolated from salmonids, and further investigated efficacy of A. salmonicida vaccine. In antibiotic susceptibility test, all of A. sobria isolates were resistant to amoxicillin and ampicillin. Six A. sobria and two A. salmonicida isolates were resistant to oxytetracycline. In challenge test, A. sobria isolates exhibited low pathogenicity in rainbow trout (Oncorhynchus mykiss) while one A. salmonicida isolate showed high pathogenicity with LD50 of 6.4 × 103  CFU/fish in rainbow trout and coho salmon (Oncorhynchus kisutch). Among virulence factors, secretion apparatus (ascV and ascC) and transcription regulatory protein (exsA) of type 3 secretion system and A-layer protein genes were differentially detected in DNA or cDNA of A. salmonicida isolates, indicating their contribution to the pathogenicity. A formalin-killed vaccine of highly pathogenic A. salmonicida isolate exhibited a protective effect with relative survival rate of 81.8% and 82.9% at 8 weeks and 16 weeks post-vaccination, respectively, in challenge test.

Virulence, attachment and invasion of Caco-2 cells by multidrug-resistant bacteria isolated from wild animals.

Wild animals may be considered important reservoirs for bacterial pathogens and, consequently, possible sources of infection for humans. In this study, selected multidrug-resistant bacteria (Acinetobacter spp., Aeromonas salmonicida, Klebsiella pneumoniae, Pseudomonas fluorescens and Shewanella putrefaciens) isolated from wild animals were characterized on their ability to attach and invade/internalize human colonic carcinoma (Caco-2) cells. In addition, the viability of these bacteria to survive under simulated human gastrointestinal tract conditions as well as the production of virulence factors (homoserine lactones signal molecules, gelatinases, proteases, siderophores and biofilm formation) were studied. The results suggests that all the bacteria presented the capacity to attach and internalize into Caco-2 cells. A. salmonicida and P. fluorescens exhibited the highest ability to internalize. These bacteria were also found to be the highest proteases producers. A. salmonicida and K. pneumoniae survived under simulated human gastrointestinal conditions. These were the bacteria with the highest capacity to produce biofilms. K. pneumoniae was the only bacterium producing siderophores. Taken together, the present results reinforce the need for the "One Health" initiative, underscoring the environment and the animals as important reservoirs of infectious determinants.

Systemic infection in European perch with thermoadapted virulent Aeromonas salmonicida (Perca fluviatilis).

In non-salmonid fish, Aeromonas salmonicidacan cause local infections with severe skin ulcerations, known as atypical furunculosis. In this study, we present a systemic infection by a virulent A. salmonicidain European perch (Perca fluviatilis).This infection was diagnosed in a Swiss warm water recirculation aquaculture system. The isolate of A.  salmonicida encodes a type three secretion system (TTSS) most likely located on a plasmid similar to pAsa5/pASvirA, which is known to specify one of the main virulence attributes of the species A. salmonicida. However, the genes specifying the TTSS of the perch isolate show a higher temperature tolerance than strains isolated from cold-water fish. The function of the TTSS in virulence was verified in a cytotoxicity test using bluegill fry and epithelioma papulosum cyprinid cells.

The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains.

BACKGROUND: Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity. RESULTS: Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel. CONCLUSION: Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain's genetic profile from pathogenic to environmental.

Comparative pathogenicity of Vibrio spp., Photobacterium damselae ssp. damselae and five isolates of Aeromonas salmonicida ssp. achromogenes in juvenile Atlantic halibut (Hippoglossus hippoglossus).

Juvenile Atlantic halibut (~100 mg, Hippoglossus hippoglossus) were exposed to Vibrio proteolyticus, a Vibrio spp. isolate, Photobacterium damselae ssp. damselae and five different isolates of Aeromonas salmonicida ssp. achromogenes via an hour-long bath immersion to ascertain their variation in pathogenicity to this fish species. Results were analysed using Kaplan-Meier survival analysis. Analysis of the data from challenges using A. salmonicida ssp. achromogenes revealed three survival values of zero and a spread of values from 0 to 28.43. Challenges using a Vibrio spp isolate, V. proteolyticus and P. damselae resulted in Kaplan-Meier survival estimates of 31.21, 50.41 and 57.21, respectively. As all bacterial species tested could induce juvenile halibut mortalities, they must all be considered as potential pathogens. However, the degree of pathogenicity of A. salmonicida is isolate dependent.

Aeromonas salmonicida Growth in Response to Atlantic Salmon Mucins Differs between Epithelial Sites, Is Governed by Sialylated and N-Acetylhexosamine-Containing O-Glycans, and Is Affected by Ca2.

Aeromonas salmonicida causes furunculosis in salmonids and is a threat to Atlantic salmon aquaculture. The epithelial surfaces that the pathogen colonizes are covered by a mucus layer predominantly comprised of secreted mucins. By using mass spectrometry to identify mucin glycan structures with and without enzymatic removal of glycan residues, coupled to measurements of bacterial growth, we show here that the complex Atlantic salmon intestinal mucin glycans enhance A. salmonicida growth, whereas the more simple skin mucin glycans do not. Of the glycan residues present terminally on the salmon mucins, only N-acetylglucosamine (GlcNAc) enhances growth. Sialic acids, which have an abundance of 75% among terminal glycans from skin and of <50% among intestinal glycans, cannot be removed or used by A. salmonicida for growth-enhancing purposes, and they shield internal GlcNAc from utilization. A Ca2+ concentration above 0.1 mM is needed for A. salmonicida to be able to utilize mucins for growth-promoting purposes, and 10 mM further enhances both A. salmonicida growth in response to mucins and binding of the bacterium to mucins. In conclusion, GlcNAc and sialic acids are important determinants of the A. salmonicida interaction with its host at the mucosal surface. Furthermore, since the mucin glycan repertoire affects pathogen growth, the glycan repertoire may be a factor to take into account during breeding and selection of strains for aquaculture.

Protection and antibody reactivity following vaccination of lumpfish (Cyclopterus lumpus L.) against atypical Aeromonas salmonicida.

Atypical Aeromonas salmonicida is frequently associated with disease and mortality in farmed lumpfish (Cyclopterus lumpus L). Challenge experiments using different modes of exposure identified both high and low pathogenic isolates. Intraperitoneal vaccination induced production of high levels of specific antibodies particularly in fish given multiple injections. The immune sera contained antibodies cross reactive with both high and low pathogenic isolates. SDS-PAGE and LC/MSMS analyses showed that the highly virulent isolate expressed the virulence array protein (A-layer) while the less virulent isolate did not. Vaccines, containing the highly virulent isolate, formulated as a monovalent or as a trivalent vaccine, provided 73 and 60 relative percent survival (RPS) respectively, following intraperitoneal challenge. The detection of high levels of specific antibodies in immune sera and the protection provided by the test vaccines strongly indicate that it is possible to vaccinate lumpfish against atypical A. salmonicida and most probably also against other infectious bacterial diseases.

Involvement of LuxS in Aeromonas salmonicida metabolism, virulence and infection in Atlantic salmon (Salmo salar L).

Quorum sensing is a bacterial density dependent communication system, which regarded to regulate co-operative behaviors of community and mediated by extracellular signal molecules named autoinducers (AI). Among various signals, autoinducer-2 (AI-2) is believed to be the messengers inter species and produced by LuxS. For Aeromonas salmonicida (A. salmonicida), an opportunistic pathogen to many cold-water teleost, little information has been known about the function of AI-2 and LuxS. Therefore, our aim was to preliminarily clarify the function of LuxS in A. salmonicida. The consequences demonstrated that wild type A. salmonicida exhibited AI-2 activity and luxS defective mutant strain fail to produce AI-2 signals. Furthermore, it was suggested that luxS deficiency could impact bacterial morphology, surface properties and virulence dramatically. Challenge experiment showed a tendency that immune factors expressed earlier when Atlantic salmon was infected with ΔluxS strain. Overall, we hypothesis that AI-2 quorum sensing could regulate the expression of A-layer protein coding gene vapA, and then influence bacterial survival ability when suffered from attack of the host immune system. Though additional studies are warranted, our study will supply a new thinking to control the damage caused by A. salmonicida.

The biofilteration ability of oysters (Crassostrea gigas) to reduce Aeromonas salmonicida in salmon culture.

Pathogen contamination in the environment is inevitable with the rapid development of intensive aquaculture. Therefore, alternative ecofriendly biological strategies to control pathogenic bacteria are required. However, our aim was to investigate the ability of oysters (Crassostrea gigas) to filter the important opportunistic pathogen, Aeromonas salmonicida (strain C4), using a green fluorescent protein tag (GFP) in the Atlantic salmon (Salmo salar) farming wastewater. Hence, A. salmonicida removal efficiency and ingestion rate were detected in two different oyster stages (larvae and adults). To evaluate the practical performance of oysters as A. salmonicida biofilter, adult oysters were applied to an integrated constructed wetlands system (ICWS) and their long-term C4-GFP removal efficiency was recorded for 60 days. Overall, our results clearly indicated that oysters had substantial A. salmonicida removal ability via their ingestion process when observed under a fluorescent microscope. Approximately 88-95% of C4-GFP was removed by oyster larvae at an ingestion rate of 6.4 × 103-6.2 × 105 CFU/h·ind, while 79-92% of C4-GFP was removed by adult oysters at an ingestion rate of 2.1 × 104-3.1 × 106 CFU/h·ind. Furthermore, 57.9 ± 17.2% of C4-GFP removal efficiency was achieved when oysters were applied to ICWS. We, therefore, concluded that using oysters as a biofilter represents an effective alternative for removing A. salmonicida from aquaculture wastewater. However, the fate of oysters after ingesting the pathogenic bacteria, acting as a potential reservoir or vector for pathogens, is still debatable. This research provides the basis for the application of oysters as a biofilter to remove pathogens from aquaculture wastewater in industrialized production.

Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real-time bioluminescence imaging.

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual-labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild-type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24-h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.

Phenotype of Aeromonas salmonicida sp. salmonicida cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants and its virulence in rainbow trout (Oncorhynchus mykiss).

Precise deletion of genes related to virulence can be used as a strategy to produce attenuated bacterial vaccines. Here, we study the deletion of the cyclic-3',5'-adenosine monophosphate (cAMP) receptor protein (Crp) in Aeromonas salmonicida, the aetiologic agent of furunculosis in marine and freshwater fish. The Crp protein is a conserved global regulator, controlling physiology processes, like sugar utilization. Deletion of the crp gene has been utilized in live attenuated vaccines for mammals, birds and warm water fish. Here, we characterized the crp gene and reported the effect of a crp deletion in A. salmonicida virulent and non-virulent isolates. We found that A. salmonicida Δcrp was not able to utilize maltose and other sugars, and its generation time was similar to the wild type. A. salmonicida ∆crp showed a higher ability of cell invasion compared to the wild type. Fish challenges showed that A. salmonicida ∆crp is ~6 times attenuated in Oncorhynchus mykiss and conferred protective immunity against the intraperitoneal challenge with A. salmonicida wild type. We concluded that deletion of A. salmonicida crp influences sugar utilization, cell invasion and virulence. Deletion of crp in A. salmonicida could be considered as part of an effective strategy to develop immersion live attenuated vaccines against furunculosis.

Transcriptional changes in innate immunity genes in head kidneys from Aeromonas salmonicida-challenged rainbow trout fed a mixture of polycyclic aromatic hydrocarbons.

We previously observed that exposure to a complex mixture of high molecular weight polycyclic aromatic hydrocarbons (PAHs) increased sensitivity of rainbow trout (Oncorhynchus mykiss) to subsequent challenge with Aeromonas salmonicida, the causative agent of furunculosis. In this study, we evaluate potential mechanisms associated with disease susceptibility from combined environmental factors of dietary PAH exposure and pathogen challenge. Rainbow trout were fed a mixture of ten high molecular weight PAHs at an environmentally relevant concentration (7.82µg PAH mixture/g fish/day) or control diet for 50 days. After 50 days of PAH exposure, fish were challenged with either Aeromonas salmonicida at a lethal concentration 30 (LC30) or growth media without the pathogen (mock challenge). Head kidneys were collected 2, 4, 10 and 20 days after challenge and gene expression (q<0.05) was evaluated among treatments. In animals fed the PAH contaminated diet, we observed down-regulation of expression for innate immune system genes in pathways (p<0.05) for the terminal steps of the complement cascade (complement component C6) and other bacteriolytic processes (lysozyme type II) potentially underlying increased disease susceptibility after pathogen challenge. Increased expression of genes associated with hemorrhage/tissue remodeling/inflammation pathways (p<0.05) was likely related to more severe head kidney damage due to infection in PAH-fed compared to control-fed fish. This study is the first to evaluate transcriptional signatures associated with the impact of chronic exposure to an environmentally relevant mixture of PAHs in disease susceptibility and immunity.

Differential effects of exposure to parasites and bacteria on stress response in turbot Scophthalmus maximus simultaneously stressed by low water depth.

The stress response of turbot Scophthalmus maximus was evaluated in fish maintained 8 days under different water depths, normal (NWD, 30 cm depth, total water volume 40 l) or low (LWD, 5 cm depth, total water volume 10 l), in the additional presence of infection-infestation of two pathogens of this species. This was caused by intraperitoneal injection of sublethal doses of the bacterium Aeromonas salmonicida subsp. salmonicida or the parasite Philasterides dicentrarchi (Ciliophora:Scuticociliatida). The LWD conditions were stressful for fish, causing increased levels of cortisol in plasma, decreased levels of glycogen in liver and nicotinamide adenine dinucleotide phosphate (NADP) and increased activities of G6Pase and GSase. The presence of bacteria or parasites in fish under NWD resulted in increased cortisol levels in plasma whereas in liver, changes were of minor importance including decreased levels of lactate and GSase activity. The simultaneous presence of bacteria and parasites in fish under NWD resulted a sharp increase in the levels of cortisol in plasma and decreased levels of glucose. Decreased levels of glycogen and lactate and activities of GSase and glutathione reductase (GR), as well as increased activities of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and levels of nicotinamide adenine dinucleotide phosphate (NADPH) occurred in the same fish in liver. Finally, the presence of pathogens in S. maximus under stressful conditions elicited by LWD resulted in synergistic actions of both type of stressors in cortisol levels. In liver, the presence of bacteria or parasites induced a synergistic action on several variables such as decreased activities of G6Pase and GSase as well as increased levels of NADP and NADPH and increased activities of GPase, G6PDH and 6PGDH.

Increasing genomic diversity and evidence of constrained lifestyle evolution due to insertion sequences in Aeromonas salmonicida.

BACKGROUND: Aeromonads make up a group of Gram-negative bacteria that includes human and fish pathogens. The Aeromonas salmonicida species has the peculiarity of including five known subspecies. However, few studies of the genomes of A. salmonicida subspecies have been reported to date. RESULTS: We sequenced the genomes of additional A. salmonicida isolates, including three from India, using next-generation sequencing in order to gain a better understanding of the genomic and phylogenetic links between A. salmonicida subspecies. Their relative phylogenetic positions were confirmed by a core genome phylogeny based on 1645 gene sequences. The Indian isolates, which formed a sub-group together with A. salmonicida subsp. pectinolytica, were able to grow at either at 18 °C and 37 °C, unlike the A. salmonicida psychrophilic isolates that did not grow at 37 °C. Amino acid frequencies, GC content, tRNA composition, loss and gain of genes during evolution, pseudogenes as well as genes under positive selection and the mobilome were studied to explain this intraspecies dichotomy. CONCLUSION: Insertion sequences appeared to be an important driving force that locked the psychrophilic strains into their particular lifestyle in order to conserve their genomic integrity. This observation, based on comparative genomics, is in agreement with previous results showing that insertion sequence mobility induced by heat in A. salmonicida subspecies causes genomic plasticity, resulting in a deleterious effect on the virulence of the bacterium. We provide a proof-of-concept that selfish DNAs play a major role in the evolution of bacterial species by modeling genomes.

Use of in vivo induced antigen technology to identify genes from Aeromonas salmonicida subsp. salmonicida that are specifically expressed during infection of the rainbow trout Oncorhynchus mykiss.

BACKGROUND: Aeromonas salmonicida is a major fish pathogen associated with mass mortalities in salmonid fish. In the present study, we applied In Vivo Induced Antigen Technology (IVIAT), a technique that relies on antibodies adsorbed against in vitro cultures of the pathogen, to a clinical isolate of A. salmonicida subsp. salmonicida. RESULTS: The results from IVIAT allowed identification of four proteins that were upregulated in the fish samples: A UDP-3-O-acyl-N-acetylglucosamine deacetylase, an RNA polymerase sigma factor D as well as TonB and a hypothetical protein. Subsequent investigations were performed using real-time PCR and cDNA synthesised from infected spleen, liver and anterior kidneys. These confirmed that the transcription level of each of these genes was significantly upregulated during the infection process compared to bacteria in vitro. CONCLUSIONS: The present studied identified four genes that were upregulated during the infectious process and are likely to play a role in the virulence of A. salmonicida. Because these are antigenic they might constitute potential targets for the development of new vaccine as well as therapeutic agents.

Aeromonas salmonicida-secreted protein AopP is a potent inducer of apoptosis in a mammalian and a Drosophila model.

Some pathogens are able to establish themselves within the host because they have evolved mechanisms to disrupt host innate immunity. For example, a number of pathogens secrete preformed effector proteins via type III secretion apparatuses that influence innate immune or apoptotic signalling pathways. One group of effector proteins that usurp innate immune signalling is the YopJ-like family of bacterial effector proteins, which includes AopP from Aeromonas salmonicida. Aeromonas species are known to cause gastrointestinal disease in humans, and are associated mainly with subcutaneous wound infections and septicaemia in other metazoans, particularly fish. AopP has been reported to have inhibitory activity against the NF-κB pathway in cultured cells, although the pathological outcomes of AopP activity have not been examined. Here, we show that AopP has potent pro-apoptotic activity when expressed in cultured mammalian macrophage or epithelial cells, or when ectopically expressed in Drosophila melanogaster haemocytes or imaginal disk epithelial cells. Furthermore, apoptosis was significantly elevated upon concurrent AopP expression and TNF-α cellular stimulation. Together, our results demonstrate how the specificity of a YopJ-like protein towards signalling pathways directly governs cellular pathological outcome in disease.