The symbiosis between the squid, Euprymna scolopes, and the bacterium, Vibrio fischeri, serves as a model for understanding interactions between beneficial bacteria and animal hosts. The establishment and maintenance of the association is highly specific and depends on the selection of V. fischeri and exclusion of non-symbiotic bacteria from the environment. Current evidence suggests that the host’s cellular innate immune system, in the form of macrophage-like hemocytes, helps to mediate host tolerance of V. fischeri. To begin to understand the role of hemocytes in this association, we analyzed these cells by high-throughput 454 transcriptomic and liquid chromatography/tandem mass spectrometry (LC-MS/MS) proteomic analyses. 454 high-throughput sequencing produced 650, 686 reads totaling 279.9 Mb while LC-MS/MS analyses of circulating hemocytes putatively identified 702 unique proteins. Several receptors involved with the recognition of microbial-associated molecular patterns were identified. Among these was a complete open reading frame to a putative peptidoglycan recognition protein (EsPGRP5) with conserved residues for amidase activity. Assembly of the hemocyte transcriptome showed EsPGRP5 had high coverage, suggesting it is among the 5% most abundant transcripts in circulating hemocytes. Other transcripts and proteins identified included members of the conserved NF-κB signaling pathway, putative members of the complement pathway, the carbohydrate binding protein galectin, and cephalotoxin. Quantitative Real-Time PCR of complement-like genes, cephalotoxin, EsPGRP5, and a nitric oxide synthase showed differential expression in circulating hemocytes from adult squid with colonized light organs compared to those isolated from hosts where the symbionts were removed. These data suggest that the presence of the symbiont influences gene expression of the cellular innate immune system of E. scolopes.

SUMMARY Plasmodium infection begins with the bite of an anopheline mosquito, when sporozoites along with saliva are injected into a vertebrate host. The role of the host responses to mosquito saliva components in malaria remains unclear. We observed that antisera against Anopheles gambiae salivary glands partially protected mice from mosquito-borne Plasmodium infection. Specifically, antibodies to A. gambiae TRIO (AgTRIO), a mosquito salivary gland antigen, contributed to the protection. Mice administered AgTRIO antiserum showed lower Plasmodium liver burden and decreased parasitemia when exposed to infected mosquitoes. Active immunization with AgTRIO was also partially protective against Plasmodium berghei infection. A combination of AgTRIO antiserum and antibodies against Plasmodium circumsporozoite protein, a vaccine candidate, further decreased P. berghei infection. In humanized mice, AgTRIO antiserum afforded some protection against mosquito-transmitted Plasmodium falciparum. AgTRIO antiserum reduced the movement of sporozoites in the murine dermis. AgTRIO may serve as an arthropod-based target against Plasmodium to combat malaria.

The beneficial symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium, Vibrio fischeri, provides a unique opportunity to study host/microbe interactions within a natural microenvironment. Colonization of the squid light organ by V. fischeri begins a lifelong association with a regulated daily rhythm. Each morning the host expels an exudate from the light organ consisting of 95% of the symbiont population in addition to host hemocytes and shed epithelial cells. We analyzed the host and symbiont proteomes of adult squid exudate and surrounding light organ epithelial tissue using 1D- and 2D-polyacrylamide gel electrophoresis and multidimensional protein identification technology (MudPIT) in an effort to understand the contribution of both partners to the maintenance of this association. These proteomic analyses putatively identified 1581 unique proteins, 870 proteins originating from the symbiont and 711 from the host. Identified host proteins indicate a role of the innate immune system and reactive oxygen species (ROS) in regulating the symbiosis. Symbiont proteins detected enhance our understanding of the role of quorum sensing, two-component signaling, motility, and detoxification of ROS and reactive nitrogen species (RNS) inside the light organ. This study offers the first proteomic analysis of the symbiotic microenvironment of the adult light organ and provides the identification of proteins important to the regulation of this beneficial association.

The key step during the initiation of malaria is for motile Plasmodium parasites to exit the host dermis and infect the liver. During transmission, the parasites in the form of sporozoites, are injected together with mosquito saliva into the skin. However, the contribution of vector saliva to sporozoite activity during the establishment of the initial infection of the liver is poorly understood. Here we identify a vector protein by mass spectrometry, with similarity to the human gamma interferon inducible thiol reductase (GILT), that is associated with saliva sporozoites of infected Anopheles mosquitoes and has a negative impact on the speed and cell traversal activity of Plasmodium. This protein, referred to as mosquito GILT (mosGILT) represents an example of a protein found in mosquito saliva that may negatively influence sporozoite movement in the host and could lead to new approaches to prevent malaria.

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