ORCID
https://orcid.org/0000-0002-4677-0364
Date of Award
Spring 5-15-2017
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Alpha-defensins are broad-spectrum antimicrobial peptides secreted by Paneth cells of the intestinal epithelium, as a part of host innate immune defense. However, specific groups of bacteria are able to inhabit the intestinal lumen despite the antimicrobial pressure exerted by alpha-defensins. Thus, I proposed that intestinal bacteria have genes involved in alpha-defensin resistance. To this end, I screened a fecal metagenomic Escherichia coli library for mechanisms of alpha-defensin resistance. I identified a 2661 base pair nucleotide sequence (pA081 insert) predicted to originate from the intestinal bacteria Ruminococcus callidus, that when expressed in E. coli, confers resistance to the intestinal alpha-defensin Cryptdin-4 (Crp4). A 367 base pair fragment of the pA081 insert, crs81 (cryptdin-4 resistance sequence 81), was sufficient for E. coli Crp4 resistance. By RNAseq, E. coli expressing the pA081 insert or crs81 have decreased expression of the crp gene, which encodes the global metabolic transcriptional regulator, CRP (cAMP receptor protein). Findings of decreased CRP protein levels in E. coli expressing crs81 and E. coliΔcrp::kan Crp4 resistance confirmed the significance of decreased crp expression in antimicrobial resistance. Since CRP regulates a multitude of genes involved in bacteria metabolism, I screened single carbon sources to identify the major metabolic changes in Crp4-resistant E. coli. Major single carbon sources with altered utilization in E. coli expressing crs81 were alpha-ketoglutaric acid and glutamine. Notably, provision of potassium chloride, alpha-ketoglutarate, and glutamine upon Crp4 challenge lead to increased E. coli survival. Both alpha-ketoglutarate and glutamine are closely related in metabolism to glutamate, a key amino acid accumulated as an initial response to osmotic upshock. Crp4-treated E. coli had elevated glutamate levels, similar to E. coli exposed to high sodium chloride concentrations, suggesting that Crp4 antimicrobial activity may involve osmotic stress. This study identifies a role for metabolism in antimicrobial peptide resistance and defines a select set of elements sufficient for bacterial defense against alpha-defensins.
Language
English (en)
Chair and Committee
Thaddeus Stappenbeck
Committee Members
Scott Hultgren, Michael Caparon, Gautam Dantas, Christina Stallings,
Recommended Citation
Luo, Christine Tzy-Yuh, "Metabolic Modulation of Bacterial Resistance to Alpha-Defensins" (2017). Arts & Sciences Electronic Theses and Dissertations. 1128.
https://openscholarship.wustl.edu/art_sci_etds/1128
Comments
Permanent URL: https://doi.org/10.7936/K7PR7TD0