Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
Interactions between beneficial microbial symbionts and their eukaryotic hosts are currently a subject of high interest for their effects on physiology, immunology, and overall development of both host and microbe. Euprymna scolopes, also known as the Hawaiian bobtail squid, is an important model system for studying host microbe interactions due to its highly specific colonization of the light organ (LO) by the bioluminescent Vibrio fischeri. To establish this highly specific symbiotic colonization, the host must be able to recognize the symbiont and differentiate from non symbionts and potentially harmful pathogens. This is mediated in part by host recognition of the lipopolysaccharide (LPS) produced by the symbiont. While LPS can act as a signal to help promote symbiosis between the two organisms, it is also a potent endotoxin and inducer of the innate immune system which can wreak havoc on the health of the host if not for remediation by both host and symbiont. Alkaline phosphatases have been previously shown to help maintain the relationship between squid and vibrio by detoxifying the endotoxin portion of LPS, Lipid A. It works by dephosphorylating Lipid A, reducing its endotoxin activity and presumably reducing host immune responses to the symbiont. Activity of E. scolopes alkaline phosphatase (EsAPs) was shown to be low just prior to dawn, at which point the squid vents out ~ 95% of the bacteria in the LO through a process called venting. We hypothesize that the lack of active EsAP in the LO induces an immune response that is part of the mechanism controlling venting. In addition to the host mechanisms of LPS detoxification, the symbiont itself may possess similar mechanisms to avoid the host’s immune response. The enzyme LpxF is a 4’-phosphatase that has been most extensively studied in Francisella tularensis subsp. novicida. It has been shown to decrease endotoxin activity by removing the 4’-phosphate group from Lipid A, allowing the bacteria to go unnoticed by the immune system during initial infection. In mutants where LpxF was knocked out, the host was able to quickly clear the infection, showing detoxification of its own Lipid A was necessary for the bacteria to remain in the host. VF1206 is a potential homologue of lpxF in V. fischeri, helping to maintain symbiosis. In this study, we make a first attempt at studying the possible immune responses associated with venting by investigating the following aims; (1) identifying genetic markers of the innate immune system expressed at different time points in the day-night cycle in adult squid tissues through RT-PCR and (2) characterizing genes identified in an operon predicted to function in membrane remodeling and (3) characterizing activity of VF1206, a predicted Lipid A phosphatase. RT-PCR of squid tissues was unsuccessful, likely due to incompatibility of the RNA extraction protocol used with inherent salt and fat concentrations in squid tissues . Continued troubleshooting methods will be utilized to optimize this protocol for use with squid tissues before further analysis of immune genes can be continued. V. fischeri strains with individual null mutations in genes VF1206 - VF1210, several of which are predicted to function in membrane dynamics, had growth advantages both without antibiotics and in the presence of sublethal doses of antibiotics when compared to the ES114 wildtype. Specifically, VF1207 and VF1209 consistently had growth advantages in in the absence and presence of antibiotics and VF1206 consistently had growth advantages in the presence of antibiotics. The mutant strains were also observed to have motility defects when compared to the wildtype. Decreased antibiotic susceptibility and motility indicate genes within this operon likely affect cell membrane dynamics. VF1206 specifically is predicted to detoxify LPS. VF1206 was cloned into an expression vector containing an MBP tag, however remained insoluble and at low concentration when expressed. Given that E. coli is also gram-negative and possesses Lipid A, VF1206::MBP was then cloned into a vector for expression in the eukaryotic yeast K. lactis. Future studies will focus on the further characterization of the VF1206 gene.
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