The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins

Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain

Comparative Analysis of Membrane Vesicles from Three Piscirickettsia salmonis Isolates Reveals Differences in Vesicle Characteristics.

Membrane vesicles (MVs) are spherical particles naturally released from the membrane of Gram-negative bacteria. Bacterial MV production is associated with a range of phenotypes including biofilm formation, horizontal gene transfer, toxin delivery, modulation of host immune responses and virulence. This study reports comparative profiling of MVs from bacterial strains isolated from three widely disperse geographical areas. Mass spectrometry identified 119, 159 and 142 proteins in MVs from three different strains of Piscirickettsia salmonis isolated from salmonids in Chile (LF-89), Norway (NVI 5692) and Canada (NVI 5892), respectively. MV comparison revealed several strain-specific differences related to higher virulence capability for LF-89 MVs, both in vivo and in vitro, and stronger similarities between the NVI 5692 and NVI 5892 MV proteome. The MVs were similar in size and appearance as analyzed by electron microscopy and dynamic light scattering. The MVs from all three strains were internalized by both commercial and primary immune cell cultures, which suggest a potential role of the MVs in the bacterium's utilization of leukocytes. When MVs were injected into an adult zebrafish infection model, an upregulation of several pro-inflammatory genes were observed in spleen and kidney, indicating a modulating effect on the immune system. The present study is the first comparative analysis of P. salmonis derived MVs, highlighting strain-specific vesicle characteristics. The results further illustrate that the MV proteome from one bacterial strain is not representative of all bacterial strains within one species

Adult zebrafish challenged with membrane vesicles isolated from <i>Piscirickettsia salmonis</i>.

<p>Cumulative survival of adult zebrafish injected with 40 μg of MVs isolated from the three different strains of <i>P</i>. <i>salmonis</i> (LF-89, NVI 5692 and NVI 5892) or PBS (n = 20).</p

Size distribution and imaging analysis of <i>Piscirickettsia salmonis</i> membrane vesicles.

<p>Vesicle size and range analyzed by dynamic light scattering (left panels) (n = 3) and electron transmission microscopy imaging (right panels) of MVs isolated from LF-89, NVI 5692 and NVI 5892. Bar size, 200 nm.</p

Immune gene transcription of adult zebrafish challenged with <i>P</i>. <i>salmonis</i> and isolated membrane vesicles analyzed by RT-qPCR.

<p>Immune gene expression of kidney and spleen, isolated 14 days post injection with either 40 μg MVs isolated from three different strains of <i>P</i>. <i>salmonis</i> or 1x10⁷ CFU of the same bacteria strains (LF-89, NVI 5692 and NVI 5892). Results are presented as mean +/- SD. Asterisk indicate significantly upregulated genes compared to the PBS control p<0.05, two tailed unpaired Student’s t-test (n = 3).</p

Internalization and effect of membrane vesicles isolated from <i>Piscirickettsia salmonis</i> in fish cells.

<p>(A) Cytopathic effect of 20 μg/mL MVs in SHK-1 cells. The cytopathic effect is characterized by the production of rounded vacuoles (arrow). Bar size, 100 μm. (B) The effect of three different MV concentrations (10, 20 and 40 μg/mL) on internalization in SHK-1 cells and kidney and spleen primary leukocytes isolated from adult zebrafish assessed by flow cytometry (n = 3). Results are presented as mean ± SD. Asterisks indicate statistical significances between the different concentrations of MVs within each cell type (Two-way ANOVA, Tukey`s multiple comparison test). P value: **** < 0.0001; *** < 0.001; ** < 0.01; * < 0.1.</p