Colonization State Influences the Hemocyte Proteome in a Beneficial Squid–Vibrio Symbiosis

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. Molecular & Cellular Proteomics

The Role of Hemocytes in the Hawaiian Bobtail Squid, Euprymna scolopes: A Model Organism for Studying Beneficial Host–Microbe Interactions

“…Hemocytes have also been shown to internalize OMVs (Aschtgen et al, 2016) but how these vesicles and other MAMPs are detected by hemocytes is unclear. These cells do express a number of PRRs, including peptidoglycan recognition proteins (PGRPs), a Toll-like receptor (TLR), and galectins (see below; Collins et al, 2012a; Schleicher et al, 2014). The role of hemocytes in light organ development is not completely understood, but these cells are hypothesized to be involved in restructuring of the ciliated appendages during morphogenesis and after the initiation of symbiosis (Koropatnick et al, 2007).…”

“…The role of hemocytes in light organ development is not completely understood, but these cells are hypothesized to be involved in restructuring of the ciliated appendages during morphogenesis and after the initiation of symbiosis (Koropatnick et al, 2007). A previous study implicated that gelanolytic activity via a matrix metalloproteinase (MMP) may contribute to this process (Koropatnick et al, 2014) and other work has shown an increase in MMP expression in the light organs of colonized juveniles (Chun et al, 2008) and in hemocytes from symbiotic compared to antibiotic-treated (cured) adult hosts (Collins et al, 2012a; Schleicher et al, 2014). However, regression of the ciliated appendages may not be entirely dependent upon hemocytes, as squid colonized by V. fischeri in simulated microgravity showed a decrease in hemocyte trafficking but an accelerated restructuring of the ciliated appendages (Foster et al, 2013).…”

“…These findings suggest that hemocytes can distinguish between related bacteria (all strains tested were within the Vibrionaceae ) and that colonization state influences the binding behavior of the hemocytes, specifically toward V. fischeri . These observations have led to investigations of the molecular mechanisms by which colonization alters the cellular immune response of the host (Collins et al, 2012a; Schleicher et al, 2014). …”

“…To understand how colonization state influences hemocyte gene and protein expression, two studies employed transcriptomics and proteomics to investigate circulating hemocytes in adult hosts (Collins et al, 2012a; Schleicher et al, 2014). Transcriptomics and proteomics of circulating hemocytes revealed a number of innate immune-related genes and proteins, some of which were differentially expressed between colonized and cured squid ( Figure 2 ).…”

“…Hemocytes are primarily implicated in defense against pathogens (Hillyer et al, 2003; Hillyer and Strand, 2014; Parsons and Foley, 2016), but also have metabolic functions in some species, including lipid storage and transport in mussels, oysters, snails, and insects (Allen and Conley, 1982; Pollero et al, 1985; Pollero and Garin, 1992; Adamo et al, 2008). In a number of beneficial associations, hemocytes have been found to both mediate and be influenced by the microbiota (Nyholm and McFall-Ngai, 1998; Silver et al, 2007; Nyholm et al, 2009; Rodrigues et al, 2010; Schmitz et al, 2012; Weiss et al, 2012; Schleicher et al, 2014). For example, in tse-tse flies, proper immune development, including immune gene expression and hemocyte proliferation, is dependent upon colonization by Wigglesworthia (Weiss et al, 2012).…”