Localization of the bacterial agent of juvenile oyster disease (Roseovarius crassostreae) within affected eastern oysters (Crassostrea virginica).

The bacterium Roseovarius crassostreae causes seasonal mortalities among commercially produced eastern oysters (Crassostrea virginica) grown in the Northeastern United States. Phylogenetically, the species belongs to a major lineage of marine bacteria (the Roseobacter clade), within which Roseovarius crassostreae is the only known pathogen to be isolated in laboratory culture. The objective of the current study was to determine the location and nature of R. crassostreae interactions with oysters affected by juvenile oyster disease (JOD). Scanning electron microscopy of diseased individuals revealed abundant colonization of the inner shell surfaces by bacteria which were morphologically similar to R. crassostreae. The same types of cells were also observed on and within layers of host-derived conchiolin on the inner valves. Most bacterial cells were alive as determined by the use of a fluorescent viability stain. Further, most were clearly attached at the cell poles, which is consistent with the ability of R. crassostreae to express polar fimbriae. When material from the pallial fluid, soft tissue and inner valve surfaces was cultured, the highest numbers of R. crassostreae were recovered from the inner valves. These samples also contained the greatest abundance of R. crassostreae as a percentage of total colonies. Cloning and sequencing of 16S rRNA genes provided culture-independent evidence of the numerical dominance of R. crassostreae among the bacterial consortia associated with the inner shell surfaces of JOD-affected animals. The ability of R. crassostreae to colonize shell and conchiolin is consistent with the described JOD-pathology and may aid the bacteria in avoiding hemocyte-mediated killing.

Domain-dependent modulation of PDGFRbeta by ganglioside GM1.

The regulation of receptor tyrosine kinases (RTKs) is important in several cellular events, including proliferation, differentiation, and apoptosis. Gangliosides are sialic acid-containing glycosphingolipids that can regulate RTK activity. The addition of ganglioside GM1 to the medium of Swiss 3T3 fibroblasts inhibits both platelet-derived growth factor (PDGF)-mediated tyrosine phosphorylation of PDGF receptor beta (PDGFRbeta) and receptor-mediated endocytosis. However, GM1 did not affect PDGF-mediated receptor phosphorylation, neuritogenesis, or endocytosis in PC12 cells stably transfected with the gene for PDGFRbeta. The ability of GM1 to modulate PDGFRbeta in 3T3 cells but not in transfected PC12 cells indicates a cell context-dependent response. We hypothesized that this inhibition of PDGFRbeta by GM1 must map to one or more domains of the receptor. Thus, a chimeric receptor was created that possessed the extracellular and transmembrane domains of the nerve growth factor (NGF) receptor TrkA and the cytoplasmic domain of PDGFRbeta (TTbeta). In 3T3 cells transfected with the TTbeta construct, GM1 did not inhibit NGF-induced tyrosine phosphorylation of the chimeric receptor or of Erk1/2 in this cell line. GM1 still inhibited PDGF-mediated tyrosine phosphorylation of endogenous PDGFRbeta and of Erk1/2 in Swiss TTbeta cells. Thus, the cytoplasmic domain of PDGFRbeta is not required for GM1-dependent inhibition of PDGFRbeta in 3T3 cells. This suggests that the inhibition of PDGFRbeta by GM1 in Swiss 3T3 fibroblasts maps to either the extracellular and/or transmembrane domain of PDGFRbeta.