Chapter 4: Supplemental file 4.3 ITRAQ.xls

The squid, Euprymna scolopes, and the bacterium, Vibrio fischeri, form a highly specific relationship that is used to study how beneficial bacteria interact with animal hosts. Even though the light organ of E. scolopes is exposed to the outside marine environment, it can only be colonized by V. fischeri. For this reason E. scolopes must carefully control the light organ microenvironment. One mechanism that the squid utilizes to regulate this symbiosis is a daily expulsion of V. fischeri from the light organ. The squid expels the symbiont, in addition to some host tissue, as a thick exudate that was collected and analyzed by high-throughput mass spectrometry (MS)-based proteomics. As expected, many of the V. fischeri proteins identified exemplify the lifestyle of the symbiont in the light organ including quorum sensing and the management of oxidative stress. The function of one of the V. fischeri proteins identified, a bacterial immunoglobulin-like surface protein, was further characterized by creating a mutant deficient in this protein. Host proteins within the light organ also highlighted the oxidative microenvironment, but proteins associated with innate immunity were prominent as well. The innate immune response, including macrophage-like hemocytes, of E. scolopes is considered to provide an additional regulatory component to this host-microbe relationship. In the absence of adaptive immunity, the hemocytes still have the ability to differentiate V. fischeri from non-symbiotic bacteria. To get a better understanding of the molecular mechanisms involved in determining host-symbiont specificity, two quantitative proteomic techniques (iTRAQ and label-free spectral counting) were used to characterize the influence of light organ colonization on the squid hemocytes. These studies demonstrated that the presence of V. fischeri in the light organ can alter the composition of the hemocyte proteome by affecting proteins related to the cytoskeleton, adhesion, and innate immunity. Further characterization of symbiont recognition by the host involved the analysis of the transcript distribution and protein localization of two carbohydrate-binding galectins. These studies represent the first application of a variety of MS-based proteomic techniques to the squid-vibrio symbiosis. In addition, the data generated from these analyses have laid the foundation for the characterization of many new target proteins, which may have important functions in regulating and shaping the outcome of host-microbe relationships in general.