Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association

Abstract

The light-organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution.

Fig. 1.

Features of early host development in the squid-vibrio symbiosis. The light-organ tissues undergo a series of symbiont-induced developmental events (see text for details), here visualized by scanning electron microscopy (Upper) and confocal microscopy (Lower). After aggregating in host mucus, at 4 h, GFP-labeled V. fischeri cells (green) enter host tissues (red). At 12 h, the symbionts induce the loss of the superficial ciliated epithelium that facilitates colonization, a process that is complete by 96 h. By 18 h, the time when host transcriptional responses were characterized, bacteria fill the epithelium-lined crypts (red) and are highly luminous. By 48 h, symbionts induce crypt-cell swelling, a phenotype that is not observed in colonizations by luxA and luxI mutants, and that correlates with their inability to persist.

Fig. 2.

Transcriptional profiles of light organs exposed to different colonization conditions. Juvenile squid were either left uninoculated (apo) or inoculated with wild-type, luxA, or luxI strains of V. fischeri, in the presence or absence of added AI. The relative similarities of patterns of gene expression under the different conditions were mapped by using change correlation average linkage. Numbers at the nodes represent percent confidence from bootstrapping analysis (n = 1,000). The relative levels of luminescence produced in the light organs under each condition are also indicated.

Fig. 3.

Localization of EsLBP in juvenile light-organ crypts using confocal immunocytochemistry (ICC) (36). (A) In aposymbiotic animals, FITC-labeled secondary antibodies localized EsLBP (green) to the apical surfaces of the crypt epithelia. (B) In symbiotic animals, the epithelia remained labeled; however, a large amount of labeling had appeared in the crypt spaces as well. The staining was not the result of V. fischeri cells directly binding the EsLBP antibody. (Inset) When these bacteria (≈1 μm), counterstained with propidium iodide (red; Left of Inset), were treated with the EsLBP antibody, there was no binding (Right). (C) Preimmune controls of both aposymbiotic and symbiotic crypts showed no nonspecific staining. In all images, counterstaining of animal tissues included TOTO3 (blue), which labels nucleic acids; and rhodamine phalloidin (red), which labels filamentous actin. Numbering indicates the two largest of the three crypts in each image; “is” indicates the ink sac. See details in SI Text.