Evidence for the Participation of the Proteasome in Symbiont-Induced Tissue Morphogenesis

Abstract

The mediation of remote events in host tissues by a bacterial partner is a phenomenon reported in both plant and animal symbioses (1,2). Underlying these events are complex cellular and molecular dialogues orchestrating the indirect communication between the symbionts and host cells. One such example is the morphogenesis of the light organ of juveniles of the squid Euprymna scolopes, a process that is induced by symbionts residing deep within these host tissues (1). The most dramatic consequence of this process is the loss of the light organ’s superficial ciliated epithelium, resulting in a remodeling of the organ accompanied by hemocyte infiltration into this tissue and apoptosis of associated cells. The host genes regulated by the symbionts to mediate this morphogenesis remain to be determined. Here we provide evidence that the symbionts induce an increase in host mRNA coding for the C8 subunit of the proteasome, a highly regulated complex that degrades proteins. C8 subunit mRNA localized to the hemocytes infiltrating the tissue undergoing morphogenesis. Experiments with inhibitors of proteasome activity suggested that these changes in gene expression are accompanied by modifications in proteasome activity. These findings support a role for the expression and activity of the proteasome in development of the host’s light organ. In the association of the squid Euprymna scolopes Berry, 1913, with its bacterial symbiont Vibrio fischeri, a specialized host light organ is colonized anew each generation (Fig. 1A; for review, see Nyholm and McFall-Ngai, 2004 (1)). The complex architecture of the hatchling light organ facilitates inoculation by the symbiont. Each lateral surface of the bilobed organ consists of a conspicuous ciliated epithelial field with a base and two protruding appendages, which are composed of a single epithelial layer overlying a sinus. During inoculation, the cilia of these fields entrain the symbionts into the proximity of three pores at the base of each field. V. fischeri cells migrate through these pores and down ciliated ducts to colonize deep crypt spaces on either side of the organ. Throughout the life of the host, the extracellular symbionts reside in the crypts, in physical contact with two host cell types: the polarized epithelium that lines each crypt, and macrophage-like blood cells, or hemocytes, which are transient inhabitants of the crypt. Approximately 12 h after inoculation, V. fischeri cells induce a dramatic, irreversible morphogenesis of the light organ that occurs over about 96 h (Fig. 1B). Most dramatic is the complete regression of the ciliated fields that mediate the inoculation process. The symbionts induce this developmental program remotely. Specifically, the crypts where V. fischeri cells reside and signal morphogenesis are several cell layers away from the ciliated fields that regress. Cellular events that correlate with the symbiont-induced regression include hemocyte infiltration into the sinuses of the appendages and apoptosis of the cells composing the superficial ciliated epithelium (3) (Fig. 1C). We employed a subtractive hybridization method with cDNA libraries of light organs from 12-h aposymbiotic (i.e., exposed to other environmental bacteria, but with no V. fischeri cells present) and symbiotic juvenile animals to identify candidate host genes involved in the response to the symbiont-induced signal for morphogenesis (4, 5). One candidate (GenBank accession no. AY616011), which comprised 30% of the clones identified as more abundant in the symbiotic light organs, encoded a host mRNA most similar to C8 subunits of the mammalian proteasome. The proteasome is a highly regulated enzyme complex responsible for the majority of cellular protein degradation via the ubiqReceived 6 December 2005; accepted 26 May 2006. * To whom correspondence should be addressed, at Department of Medical Microbiology and Immunology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706. E-mail: mjmcfallngai@wisc.edu Reference: Biol. Bull. 211: 1–6. (August 2006) © 2006 Marine Biological Laboratory