Date of Award

10-4-2016

Document Type

Thesis and Dissertation

Degree Name

Master of Science (MS)

Department

School of Biological Sciences

First Advisor

Brian J. Wilkinson

Second Advisor

Craig Gatto

Abstract

The membrane homeostasis of the psychrotolerant foodborne pathogen Listeria monocytogenes involves maintaining fluidity in a range of different conditions by incorporation of specific branched-chain fatty acids (BCFAs). BCFAs are synthesized through a well-characterized pathway from branched-chain amino acids into short branched-chain acyl-CoAs followed by elongation by the FAS II system. Branched-chain alpha-keto acid dehydrogenase (Bkd) is a major player in this pathway. MOR401 is a transposon insertion mutant deficient in Bkd and has decreased membrane BFCAs. Low levels of BCFAs in L. monocytogenes are linked to diminished growth, less resistance to antimicrobials, and a severe reduction in virulence. Rescued growth and survivability of MOR401 can be achieved by media supplementation with BCFA precursors. Feeding the mutant various branched-chain carboxylic acids induced the production of BCFAs and restored growth. After culturing in the presence of novel BCFA C6 precursors 2-ethylbutyrate and 2-methylpentanoate, MOR401 possessed restored survivability after one hour of incubation with protamine, a cationic antimicrobial peptide.

Production of BCFAs in MOR401 suggests the existence of a bypass pathway of BCFA biosynthesis. Exogenous fatty acid precursors are proposed to be utilized by the activity of the enzymes Ptb and Buk, products of genes located just upstream of the bkd gene cluster, which work to prime exogenous short chain carboxylic acids for BCFA biosynthesis. To further study the role of the proposed pathway, a buk knockout mutant was constructed and grown in BHI medium supplemented with natural and unnatural BCFA precursors to verify its ability to utilize these substrates in Buk’s absence. Lack of novel fatty acid incorporation confirms the bypass pathway enzymes’ importance in the conversion of carboxylic acids into activated of acyl CoAs for elongation and addition into the membrane.

BCFAs dominate the membrane of L. monocytogenes, which is unlike other organisms which utilize unsaturated fatty acids to maintain membrane fluidity. How then does L. monocytogenes modulate its membrane fluidity during infection of a host? Serum fatty acids are straight chain fatty acids and straight chain unsaturated fatty acids. To investigate L. monocytogenes’ likely in vivo membrane fatty acid composition and growth, wild type strain and MOR401 were cultured in fetal bovine serum. Following the ex vivo growth studies, fatty acid analysis of the cell membranes was carried out. The results show that even though L. monocytogenes does not possess the machinery needed to synthesize SCUFAs, it is indeed able to utilize and benefit from SCUFA incorporation into the membrane.

Comments

Imported from ProQuest Kuczek_ilstu_0092N_10848.pdf

Page Count

58

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