The Lipid A from Vibrio fischeri Lipopolysaccharide AUNIQUESTRUCTUREBEARINGAPHOSPHOGLYCEROLMOIETY

Vibrio fischeri, a bioluminescent marine bacterium, exists in an exclusive symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, whose light organ it colonizes. Previously, it has been shown that the lipopolysaccharide (LPS) or free lipid A of V. fischeri can trigger morphological changes in the juvenile squid’s light organ that occur upon colonization. To investigate the structural features that might be responsible for this phenomenon, the lipid A from V. fischeri ES114 LPS was isolated and characterized by multistage mass spectrometry (MS n ). A microheterogeneous mixture of mono- and diphosphorylated diglucosamine disaccharides was observed with variable states of acylation ranging from tetra- to octaacylated forms. All lipid A species, however, contained a set of conserved primary acyl chains consisting of an N-linked C14:0(3-OH) at the 2-position, an unusual N-linked C14:1(3-OH) at the 2�-position, and two O-linked C12:0(3-OH) fatty acids at the 3- and 3�-positions. The fatty acids found in secondary acylation were considerably more variable, with either a C12:0 or C16:1 at the 2-position, C14:0 or C14:0(3-OH) at the 2�-position, and C12:0 or no substituent at the 3�-position. Most surprising was the presence of an unusual set of modifications at the secondary acylation site of the 3-position consisting of phosphoglycerol (GroP), lysophosphatidic acid (GroP bearing C12:0, C16:0, or C16:1), or phosphatidic acid (GroP bearing either C16:0 C12:0 or C16:0 C16:1). Given their unusual nature, it is possible that these features of the V. fischeri lipid A may underlie the ability of E. scolopes to recognize its symbiotic partner. The bioluminescent bacterium Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes. Colonization of the juvenile squid’s light organ by V. fischeri begins within hours of hatching (1). From the complex bacterial community present in seawater, V. fischeri, which represents less than 1% of the bacterial population, is exclusively recruited by E. scolopes in a multistep winnowing process (2). As the colonization of the juvenile’s light organ progresses, a series of developmental changes occur in the tissues. The most dramatic of these morphogenetic events is the loss of a superficial ciliated field of cells important for the initial recruitment of V. fischeri. This process occurs over the 96 –120 h following initial colonization by the symbiont (2) and does not occur without interactions with the symbiont in the deep crypt regions of the organ. Once the colonization is established, the squid continues to maintain a population of V. fischeri in its light organ, with cycles of daily flushing and repopulation by residual bacteria. The selection process that leads to the exclusive symbiotic relationship between V. fischeri and E. scolopes involves the interaction of bacterial surface components with host tissues. Previously, we showed that bacterial lipopolysaccharide (LPS) and lipid A can induce early stage apoptosis in the cells of the ciliated field of the juvenile squid’s light organ (3). However, the effect was not species-specific, suggesting that a conserved portion of the lipid A may be the responsible component of the LPS structure (3). In later stages of the colonization process, bacterial peptidoglycan acts synergistically with LPS to induce most, if not all, of light organ morphogenesis (4). How the developmental signals of bacterial LPS and peptidoglycan, which are presented by the symbionts in the deep crypts of the light organ, are conveyed to the superficial tissue remains unknown, but one piece of the puzzle has recently been discovered. At hatching, the light organ has high levels of nitric oxide (NO). Following symbiont colonization of the crypts, the levels of both NO and the enzyme that catalyzes its production, nitric-oxide synthase, are attenuated (5). Recent studies of the system revealed that LPS and peptidoglycan work synergistically to turn down the NO/nitric-oxide synthase of the organ. In addition, this attenuation is critical for inducing the onset of the apoptotic program (6). These data provide evidence that the LPS and peptidoglycan do not work by direct interactions with