Date of Award

12-1-2018

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Rader, Bethany

Abstract

The interactions between eukaryotic host organisms and their beneficial microbial symbionts are currently the subject of intense interest for their effects on the physiology, immunobiology and development of both the host and the symbiont. The Hawaiian bobtail squid Euprymna scolopes is host to two known beneficial symbioses; the light organ (LO) symbiosis which exclusively hosts Vibrio fischeri, and a bacterial community within the female reproductive organ, the accessory nidamental gland (ANG), and one as-of-yet uncharacterized beneficial symbiosis in the intestine. In order to establish this partnership, the host must be able to differentiate between beneficial symbionts and potentially harmful pathogenic symbionts. The establishment of the LO symbiosis, for which we understand the most, is mediated in part by the interaction between host recognition of microbe-associated molecular patterns (MAMPs), such as the endotoxin, lipopolysaccharide (LPS). Although LPS acts as a signal to promote this symbiosis, it is also a potent immunostimulant and requires modification. Alkaline phosphatase (AP) has been shown in vertebrates aid in maintaining persistent relationships between host and symbiont as it has a significant role in intestinal homeostasis and health by “detoxifying” the immunostimulatory lipid A moiety of LPS. In these studies, it has been shown that, host intestinal AP (IAP) activity is upregulated by the intestinal microbiota, thus reducing the host inflammatory responses to microbial LPS. In recent research, it has been suggested that E. scolopes alkaline phosphatases (EsAPs) are present in the LO and are modified by the host to be inactive in a circadian manner, promoting daily symbiont expulsion. In this study, we used E. scolopes as a model to understand the role of host AP in modulation of the symbiont’s LPS that enable initial colonization and long-term persistence of the V. fischeri symbiosis in the host, and to characterize EsAPs in the intestinal and ANG beneficial symbioses. Therefore, our aims were to; (1) identify the expression pattern of esap transcripts in the LO, ANG and intestine of E. scolopes, 2) localize the EsAP protein and activity in the LO, ANG and the intestine, (3) assures the essentiality of AP activity on the squid-vibrio symbiosis, through the use of AP inhibitors. We found that both esap1 and esap2 transcripts were differentially expressed in response to symbiont levels in the juvenile squid LO over the colonization process, to the day/ night cycle of the adult squid and were upregulated in cured relative to symbiotic ANGs and intestines. EsAP protein is localized on the apical side of the epithelial lining, and in the lumen of the symbiotic juvenile LO and adult central core (CC), the apical side of the epithelia in the epithelia-lined tubules of the ANG and in the apical brush border of the intestinal epithelia. Moreover, EsAP activity was low in the aposymbiotic LO, in times corresponding to low bacterial load during the venting time (12, 36, 60 hours of hatching) in symbiotic animals, and in LO of adult animals at 7 am, just prior to venting, relative to all other time points. Juvenile animals in which AP was inhibited, displayed higher expression of LO genes encoding for immune proteins; estlrs, esgalectin, espgrps, esc3 and esnos and a lower bacterial load relative to uninhibited animals at 48 hours post-incubation with AP inhibitor. Moreover, these animals displayed higher expression of genes encoding for cytoskeleton proteins (actin, spectrin, Hsp 90, myosin, troponin, tubulin and arginine kinase) expression compared to the uninhibited ones. These data from inhibited juvenile animals, correlates to the 7 am time point of adult animals which showed similar expression of genes that encode for immune and cytoskeleton proteins. Based on these results, we suggest that EsAPs are required for persistent colonization of the LO and modulating the “toxicity” of V. fischeri LPS, and that AP activity is a conserved mechanism for immune tolerance to beneficial symbionts across multiple functionally distinct organs.

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