Marcos Monteiro

2018-19 McNair Faculty Research Mentor: Dr. Andrea Castillo – Biology

Research Title: Manuka Honey Chelates Iron to Impact Iron Regulation in Pseudomonas Aeruginosa

Abstract: The gram-negative bacterium, Pseudomonas aeruginosa, is a leading cause of infections in burn patients and demonstrates a high level of antibiotic resistance. For this reason, Manuka honey (MH) has been investigated as an alternative antimicrobial and proven effective against 152 different strains of P. aeruginosa. Additionally, exposure to sub-minimum inhibitory concentrations (MICs) of MH has not bred MH resistant bacteria. This is likely due the multiple antimicrobial mechanisms of MH, a major one being methylglyoxyl (MGO) production. Recent studies suggest that iron limitation may be an additional MH antimicrobial mechanism. Herein, we used a colorimetric ferrozine-based assay to demonstrate that MH chelates ferrous iron from solution. To determine if bacteria cultured with MH experienced iron limitation, iron regulated siderophore production was quantitated in P. aeruginosa cultured with sub-MICs of MH; siderophores are small molecules that bind ferric iron with high affinity and are produced by bacteria when iron is limited. MH treated P. aeruginosa produced significantly more siderophores than the untreated controls. Finally, we examined whether supplemental iron could offset the antimicrobial effects—growth inhibition—of MH. Growth of P. aeruginosa supplemented with either ferrous (Fe2+) sulfate or ferric (Fe3+) citrate increased significantly over the non-supplemented controls. Our results support that iron limitation through iron chelation contributes to MH’s antimicrobial activity. Our future experiments will investigate which components of MH act to chelate iron and how iron uptake mutants respond to MH with iron supplementation.

2019 Summer Research Mentor: Dr. Neelendu Dey, MD at Fred Hutch

Research Title: Bioengineering Escherichia coli as a Tool for Understanding Carcinogenic Products of Bile Acid Metabolism by Gut Bacteria

Abstract: Conjugated bile acids (CBA) are synthesized by the liver from cholesterol and released into the small intestine to emusify fats and insoluble vitamins. Bacteroides vulgatus is a gut microbe that efficiently metabolizes CBA into unconjugated bile acids (UBA); its genome encodes three distinct bile salt hydrolase (bsh) genes. UBA can be further modified by gut bacteria to produce deoxycholic acid (DCA), high levels of which are linked to colorectal cancer and colorectal adenomas. It is unclear how multiple encoded bsh genes relate to robust deconjugation by B. vulgatus. We hypothesize that one or more bsh genes encode enzymes that are efficient in deconjugation and that co-expression of multiple bsh genes enables efficient deconjugation by B. vulgatus. Our goal is to functionally characterize each B. vulgatus bsh genes via recombinant expression in Escherichia coli using the pBAD plasmid system. To validate our use of the pBAD plasmid system, we cloned the red fluorescent protein (RFP) gene into pBAD. Our results show that bsh 2, bsh 3, and RFP were cloned into pBAD in the desired region and successfully transformed into E. coli. We verified our results through plasmid extraction followed by PCR targeting the gene of interest, and via capillary sequencing. Future work will involve assessing the deconjugation ability of each bsh gene by culturing these E. coli constructs with conjugated bile acids. This work will further our understanding of B. vulgatus and other bsh-expression microbes contribute to the production of DCA and the development of colorectal cancer.