In vitro genomic and proteomic evidence of a type IV pili-like structure in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is an intracellular γ-proteobacteria and the etiological agent of piscirickettsiosis, which causes massive economic losses in the Chilean salmon industry. The type IV pili (T4P) play an important role in adherence to host cell surfaces and bacterial pathogenicity. T4P contains a variable number of components, as predicted in P. salmonis genomes. However, no studies have determined if P. salmonis possesses T4P. The aims of this investigation were to identify T4P components in the P. salmonis type strain LF-89T, evaluate respective transcript expressions, and analyze the main putative T4P proteins using bioinformatics and proteomic approaches. Two main clusters of P. salmonis T4P genes were found. Expression of the pilA gene was upregulated at 4 h post-infection (hpi), while pilQ was upregulated 4 days post-infection. At 16 hpi, pilB and pilD were strongly upregulated. The PilA amino acid sequence analysis showed a conserved N-terminal domain and sequence motifs critical for T4P biosynthesis. MudPIT analysis revealed PilA in the P. salmonis LF-89T proteome, and TEM showed pili-like filamentous structures on the P. salmonis surface. These results strongly suggest the presence of a T4P-like structure in P. salmonis.

Combined effects of high stocking density and Piscirickettsia salmonis treatment on the immune system, metabolism and osmoregulatory responses of the Sub-Antarctic Notothenioid fish Eleginops maclovinus.

The aim of this study was to evaluate immunological, metabolic and osmoregulatory secondary stress responses in Eleginops maclovinus specimens submitted to three different stocking densities: i) low (3.1 kg m(-3)), medium (15 kg m(-3)) and high (60 kg m(-3)) during 10 days, alone or in combination with a previous treatment of a protein extract of the pathogen Piscirickettsia salmonis (0.5 µg g weight body(-1)). Plasma, liver, gill and kidney samples were obtained at the end of both experiments. Plasma cortisol and amino acid levels increased, while plasma glucose, triglyceride and lactate levels decreased at higher stocking densities. However, no effects were observed on serum Immunoglobulin type M (IgM anti P. salmonis level) values. Gill Na(+), K(+)-ATPase activity enhanced under these experimental conditions, suggesting an osmotic imbalance. Energy metabolism changes, assessed by metabolite concentrations and enzyme activities, indicated a reallocation of energetic substrates at higher stocking densities. Specimens inoculated with a protein extract of P. salmonis and maintained at different stocking densities showed primary stress response, as all groups enhanced plasma cortisol concentrations. Serum IgM levels increased after treatment with P. salmonis extract but a negative influence of high stocking density on IgM production was observed when immune system was activated. Furthermore, treatment with P. salmonis protein extract evoked deep changes in the metabolite stores in all tissues tested, indicating a mobilization of energy substrates in response to infection. The results show that stocking density induced immunological, metabolic and osmoregulatory secondary stress responses in E. maclovinus specimens and that previous treatment with P. salmonis compromise these changes.

Chemical structure of the carbohydrate backbone of the lipopolysaccharide from Piscirickettsia salmonis.

Elucidation of the carbohydrate backbone structure of the lipopolysaccharide (LPS) from Piscirickettsia salmonis, the etiological agent of the salmonid rickettsial septicemia, is described. Structural information was established by a combination of monosaccharide and methylation analyses of LPS, and by NMR and mass spectrometries of oligosaccharides obtained through the use of various chemical degradations of the native polymer. The following structure of the backbone sugars was determined on the basis of the combined data from these experiments: [formula see text] The presence of two consecutive residues of diacetylated pseudaminic acid (Pse5,7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid) in the LPS appears to be unique among polysaccharides containing this acidic sugar. Similarly, the presence of 4-aminoarabinose (Ara4N, 4-amino-4-deoxy-l-arabinopyranose) on O-4 of the α-GlcN1P of the lipid A moiety is a unique feature of this LPS.

ISPsa2, the first mobile genetic element to be described and characterized in the bacterial facultative intracellular pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is a novel, aggressive, facultative Gram-negative bacterium that drastically affects salmon production at different latitudes, with particular impact in southern Chile. Initially, P. salmonis was described as a Rickettsia-like, obligate, intracellular Alphaproteobacteria, but it was reclassified recently as a facultative intracellular Gammaproteobacteria. This designation has prompted the independent growth of the bacterium to a pure state for detailed study of its biology, genetics and epidemiology, properties that are still relatively poorly characterized. The preliminary sequence analysis of a 992-bp fragment of pure P. salmonis DNA allowed us to characterize a novel and complete 863-bp insertion sequence in the bacterial genome (named ISPsa2), which has a novel 16/16bp perfectly inverted terminal repeat flanking a 726-bp ORF that encodes a putative transposase (Tnp-Psa). The coding sequence of the enzyme shares similarities to that described in some Bacillus species and particularly to those of the IS6 family. ISPsa2 carries its own promoter with standard -10 and -35 sequences, suggesting an interesting potential for plasticity in this pathogenic bacterium. Additionally, the presence of ISPsa2 was confirmed from three isolates of P. salmonis collected from different epizootics in Chile in 2010.

Structural features of lipid A of Piscirickettsia salmonis, the etiological agent of the salmonid rickettsial septicemia.

The composition and structure of lipid A isolated from the lipopolysaccharide (LPS) of Piscirickettsia salmonis were investigated by chemical analyses, gas chromatography/mass spectrometry (GCMS), and electrospray ionization (ESI) combined with the tandem mass spectrometry (MS/MS). Our study revealed moderate compositional and structural heterogeneity of lipid A with respect to the content of phosphate groups and 4-amino-4-deoxy-L-arabinopyranose (Ara4N) residues and with regard to the degree of acylation. It appeared that at least two molecular species were present in lipid A. The major species represented the hexaacyl lipid A consisting of the ss-(1--> 6)-linked D-glucosamine (GlcN) disaccharide backbone carrying two phosphate groups. The first one at the glycosidic hydroxyl group of the reducing GlcN I and the second one at the O-4' position of the non-reducing GlcN II. The primary fatty acids consisted of three 3-hydroxytetradecanoic [C14:0(3-OH)] and one 3-hydroxyhexadecanoic [C16:0(3-OH)] acids. The latter was amide-linked to GlcN I and one C14:0(3-OH) was amide-linked to GlcN II. Two secondary fatty acids were represented by C14:0(3-OH) and were equally distributed between the O-2' and O-3' positions. The phosphate group at O-4' carried a non-stoichiometric substituent Ara4N. The minor lipid A species contained exclusively C14:0(3-OH) with an asymmetric distribution (4+2) at GlcN II and GlcN I, respectively. The P. salmonis lipid A resembles structurally strongly endotoxic enterobacterial lipid A. This could be one of the reasons for the observed high endotoxicity of P. salmonis.