In partial fulfillment of the requirements for the degree of

Master of Science in Biology

in the

School of Biological Sciences

Hannah Lachmayr

Will defend her thesis

Assay Development for and Evaluation of Sphingolipid Hydrolysis Activity in Isolated Soil Bacteria and Venoms from Loxosceles reclusa and Kukulcania hibernalis

July 22nd, 2021

At 3:00 pm

https://bluejeans.com/293947171/9175

Thesis Advisor:

Alfred H. Merrill, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Committee Members:

Kostas Konstantinidis, Ph.D.

School of Civil & Environmental Engineering

School of Biological Sciences

Georgia Institute of Technology

Thomas DiChristina, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Janet Hatt, Ph.D.

School of Civil & Environmental Engineering

Georgia Institute of Technology

Abstract: Several Sicariidae spiders, such as the brown recluse spider Loxosceles reclusa, and some pathogenic bacteria contain phospholipase D (PLase D) enzymes that act on phospholipid substrates. PLases D that cleave sphingomyelin (SM) to release the headgroup choline are also called sphingomyelinases D (SMases D). Although the lipid product of the L. reclusa SMase D was initially thought to be ceramide 1-phosphate (Cer1P), a transphosphatidylation mechanism has been shown to produce ceramide 1,3-cyclic phosphate (Cer(1,3)P). Since SMase D is presumably the major agent in the necrosis and toxicity of the brown recluse spider venom, a rigorous but simple assay would aid in detecting the presence of activities that convert SM to Cer(1,3)P. After synthesizing fluorescent products that are known to be made by various types of SMases (i.e., NBD-ceramide, -Cer1P and -Cer(1,3)P), a solvent was identified that fully resolves these compounds as well as the substrate NBD-SM using silica gel plates. When the assay was applied to venom from L. reclusa, the product was the expected NBD-Cer(1,3)P. The assay was applied to venom from Kukulcania hibernalis, a spider that has been suggested to have SMase D but whose products have not yet been determined. NBD-Cer(1,3)P was found to be produced by K. hibernalis venom. Since at least the venoms of L. reclusa and K. hibernalis hydrolyze SM to Cer(1,3)P, which is toxic to humans, we isolated bacteria from the soil with enzymatic activity to degrade this cyclic Cer(1,3)P product. Single isolates of soil microorganisms were selected based on their ability to grow on the restrictive carbon source SM or Cer(1,3)P and then examined for their cleavage of NBD-SM and/or NBD-Cer(1,3)P. Genome sequencing of two isolates taxonomically matched to Klebsiella variicola, a category of gram-negative bacterium that can be a human pathogen. Therefore, this work has resulted in a new methodology for analysis of the PLase D/SMase D activities that produce Cer(1,3)P. The assay’s application has confirmed the nature of the SMase D in L. reclusa venom and established that SMase D activity is also present in the venom of K. hibernalis. Our findings have opened new lines of investigation into microorganisms that can metabolize sphingolipids and that could have potential clinical utility through cleaving Cer(1,3)P produced by necrotizing SMases D.