Aeromonas salmonicida Type I pilus system contributes to host colonization but not invasion.

The host-adherence strategies employed by Aeromonas salmonicida subsp, salmonicida, the etiological agent of an infectious bacteremia of salmonids, are poorly understood. In addition to the outer protein coat or S-layer, A. salmonicida has both Type I and Type IV pili loci. The A. salmonicida Type I or Fim pilus is encoded by an operon with genes for a chaperone, an usher, and 3 pilus subunits and is predicted to be similar to the Pap fimbriae of uropathogenic Escherichia coli, which are considered significant virulence factors. A Fim-deficient strain of A. salmonicida strain A449, delta fim, was created by deleting this operon. Virulence of delta fim was unchanged in direct live challenges of Atlantic salmon Salmo salar L., a natural host for A. salmonicida. A measure of clinically inapparent (covert) infections suggested Fim was required to establish or maintain a covert infection. This was confirmed by an ex vivo adherence and invasion assay using freshly excised salmon gastrointestinal (GI) tract, which showed that, compared to the parental strain, the ability of the isogenic delta fim mutant strain to adhere to the salmon GI tract was reduced but, once adhered, its ability to invade was unchanged. Thus the Fim pilus functions as an adhesin in A. salmonicida and the presence of a functional Fim improved the efficiency of A. salmonicida infection of Atlantic salmon.

Contribution of type IV pili to the virulence of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon (Salmo salar L.).

Aeromonas salmonicida subsp. salmonicida, a bacterial pathogen of Atlantic salmon, has no visible pili, yet its genome contains genes for three type IV pilus systems. One system, Tap, is similar to the Pseudomonas aeruginosa Pil system, and a second, Flp, resembles the Actinobacillus actinomycetemcomitans Flp pilus, while the third has homology to the mannose-sensitive hemagglutinin pilus of Vibrio cholerae. The latter system is likely nonfunctional since eight genes, including the gene encoding the main pilin subunit, are deleted compared with the orthologous V. cholerae locus. The first two systems were characterized to investigate their expression and role in pathogenesis. The pili of A. salmonicida subsp. salmonicida were imaged using atomic force microscopy and Tap- and Flp-overexpressing strains. The Tap pili appeared to be polar, while the Flp pili appeared to be peritrichous. Strains deficient in tap and/or flp were used in live bacterial challenges of Atlantic salmon, which showed that the Tap pilus made a moderate contribution to virulence, while the Flp pilus made little or no contribution. Delivery of the tap mutant by immersion resulted in reduced cumulative morbidity compared with the cumulative morbidity observed with the wild-type strain; however, delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild type. Unlike the pili of other piliated bacterial pathogens, A. salmonicida subsp. salmonicida type IV pili are not absolutely required for virulence in Atlantic salmon. Significant differences in the behavior of the two mutant strains indicated that the two pilus systems are not redundant.

Comprehensive EST analysis of Atlantic halibut (Hippoglossus hippoglossus), a commercially relevant aquaculture species.

BACKGROUND: An essential first step in the genomic characterisation of a new species, in this case Atlantic halibut (Hippoglossus hippoglossus), is the generation of EST information. This forms the basis for subsequent microarray design, SNP detection and the placement of novel markers on genetic linkage maps. RESULTS: Normalised directional cDNA libraries were constructed from five different larval stages (hatching, mouth-opening, midway to metamorphosis, premetamorphosis, and post-metamorphosis) and eight different adult tissues (testis, ovary, liver, head kidney, spleen, skin, gill, and intestine). Recombination efficiency of the libraries ranged from 91-98% and insert size averaged 1.4 kb. Approximately 1000 clones were sequenced from the 5'-end of each library and after trimming, 12675 good sequences were obtained. Redundancy within each library was very low and assembly of the entire EST collection into contigs resulted in 7738 unique sequences of which 6722 (87%) had matches in Genbank. Removal of ESTs and contigs that originated from bacteria or food organisms resulted in a total of 7710 unique halibut sequences. CONCLUSION: A Unigene collection of 7710 functionally annotated ESTs has been assembled from Atlantic halibut. These have been incorporated into a publicly available, searchable database and form the basis for an oligonucleotide microarray that can be used as a tool to study gene expression in this economically important aquacultured fish.

Expression of and secretion through the Aeromonas salmonicida type III secretion system.

Aeromonas salmonicida subsp. salmonicida is the aetiological agent of furunculosis, a disease of farmed and wild salmonids. The type III secretion system (TTSS) is one of the primary virulence factors in A. salmonicida. Using a combination of differential proteomic analysis and reverse transcriptase (RT)-PCR, it is shown that A. salmonicida A449 induces the expression of TTSS proteins at 28 degrees C, but not at its more natural growth temperature of 17 degrees C. More modest increases in expression occur at 24 degrees C. This temperature-induced up-regulation of the TTSS in A. salmonicida A449 occurs within 30 min of a growth temperature increase from 16 to 28 degrees C. Growth conditions such as low-iron, low pH, low calcium, growth within the peritoneal cavity of salmon and growth to high cell densities do not induce the expression of the TTSS in A. salmonicida A449. The only other known growth condition that induces expression of the TTSS is growth of the bacterium at 16 degrees C in salt concentrations ranging from 0.19 to 0.38 M NaCl. It is also shown that growth at 28 degrees C followed by exposure to low calcium results in the secretion of one of the TTSS effector proteins. This study presents a simple in vitro model for the expression of TTSS proteins in A. salmonicida.