Abstract
The squid Euprymna scolopes and the luminescent bacterium Vibrio fischeri form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Host macrophage-like hemocytes are believed to play a role in mediating the symbiosis with V. fischeri. Previous studies have shown that the colonization state of the light organ influences the host's hemocyte response to the symbiont. To further understand the molecular mechanisms behind this process, we used two quantitative mass-spectrometry-based proteomic techniques, isobaric tags for relative and absolute quantification (iTRAQ) and label-free spectral counting, to compare and quantify the adult hemocyte proteomes from colonized (sym) and uncolonized (antibiotic-treated/cured) squid. Overall, iTRAQ allowed for the quantification of 1,024 proteins with two or more peptides. Thirty-seven unique proteins were determined to be significantly different between sym and cured hemocytes (p value < 0.05), with 20 more abundant proteins and 17 less abundant in sym hemocytes. The label-free approach resulted in 1,241 proteins that were identified in all replicates. Of 185 unique proteins present at significantly different amounts in sym hemocytes (as determined by spectral counting), 92 were more abundant and 93 were less abundant. Comparisons between iTRAQ and spectral counting revealed that 30 of the 37 proteins quantified via iTRAQ exhibited trends similar to those identified by the label-free method. Both proteomic techniques mutually identified 16 proteins that were significantly different between the two groups of hemocytes (p value < 0.05). The presence of V. fischeri in the host light organ influenced the abundance of proteins associated with the cytoskeleton, adhesion, lysosomes, proteolysis, and the innate immune response. These data provide evidence that colonization by V. fischeri alters the hemocyte proteome and reveals proteins that may be important for maintaining host-symbiont specificity.
Highlights
The mechanisms by which hemocytes of E. scolopes differentiate between the light organ symbiont V. fischeri and non-symbiotic bacteria are still under investigation, but it is clear that colonization influences the cellular response, gene expression, and proteome of this cell type
Previous proteomic analyses of the squid–vibrio symbiosis have been focused on two-dimensional gel electrophoresis or individual protein identification and characterization [10, 13, 69]
This study represents the first application of quantitative proteomics to the squid–vibrio symbiosis
Summary
Animal Collection and Maintenance—Adult E. scolopes were caught within the shallow sand flats off of Oahu, HI, by dip net. One hundred microliters of 8 M urea solution was added to the filters (no DTT, no SDS), and samples were centrifuged at 14,000 ϫ g for 15 min This step was repeated twice and was followed by the addition of 100 l of a 50 mM ammonium bicarbonate solution (provided with the FASP kit) and centrifugation at 14,000 ϫ g for 15 min. Two-dimensional LC-MS/MS Analyses—Tryptic digests of sym and cured unlabeled protein were analyzed in technical duplicates via a two-dimensional nano-LC-MS/MS system with a split-phase nano column (RP-SCX-RP [23]) on a QExactive mass spectrometer (Thermo Scientific) with 22-h runs per sample (LC as described earlier (24 –26)). For each significantly different protein, the ratio of the average NSAF for sym relative to the average NSAF for cured was Log transformed and filtered for proteins meeting a cutoff of a 1.5-fold change
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