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 ...