Jikang Wu (Presenter)
Ohio State University
Bio: I obtained my bachelor degree in biology from Wuhan University, China in 2013 and in the same year started as a graduate student in the chemistry PhD program in the Ohio State University in the United States. I joined Dr.Vicki Wysocki's group since January 2014.
Authorship: Jikang Wu, Anindita Sengupta, Anice Sabag-Daigle, Brian Ahmer, Venkat Gopalan, Edward Behrman and Vicki Wysocki
The Ohio State University
| Short Abstract Fructose-asparagine (F-Asn), an amadori product from food, has been found to play an essential role for Salmonella"s growth in the inflamed intestine. Determining the role of enzymes involved in F-Asn metabolism in Salmonella will facilitate the discovery of therapeutic targets. And quantification of F-Asn from mouse feces is necessary for the study of effects on Salmonella"s F-Asn metabolism in mice model. Here a Salmonella deglycatase, FraB is verified to catalyze the deglycation of F-6-phosphate-Asp producing glucose 6-phosphate and aspartate. Also the method to extract F-Asn from mouse feces is developed and isotope-labelled F-Asn is used for its quantification. |
Long Abstract
Salmonella is a worldwide foodborne disease, causing tens of millions of human cases globally [1], and 1.2 million illnesses with 450 deaths in the United States [2] every year. Recently, fructose-asparagine (F-Asn), an amadori product from food, has been found to play an essential role for Salmonella"s growth in the inflamed intestine. [3] The facts that Salmonella heavily relies on this single nutrient and no other organism is found to synthesize or utilize F-Asn provide a novel target for therapies. [3] Among many genes located on fra locus which is found to be responsible for the utilization of F-Asn for Salmonella, fraB encodes a deglycase FraB, whose function is unidentified.[3] Understanding the metabolism of F-Asn in Salmonella will facilitate the knowledge of Salmonella"s growth in intestine. Besides determining the role of enzymes involved in F-Asn"s metabolism could benefit the discovery of therapeutic targets. Furthermore, development of the method to extract F-Asn from mouse feces would enable the further study of multiple factors" effect on Salmonella"s consumption on F-Asn. 13C heavy labelled F-Asn with multiple reaction monitoring (MRM) mode on a triple quadrupole is used as a tool for quantification. The goal of the study is to identify the role of a Salmonella enzyme, FraB in F-Asn"s metabolism and develop a method for the extraction and quantification of F-Asn.
The transitions of a series of possible F-Asn metabolites and some of the analogs are constructed on Thermo Finnigan TSQ Quantum Mass Spectrometer, including F-Asn, 13C F-Asn with six heavy carbon labeled carbohydrate ring, F-Asp, F-Lys, F-6-phosphate-Asn, F-6-phosphate-Asp, aspartate, glucose 6-phosphate, and fructose 6-phosphate. FraB is proposed to catalyze the deglycation of F-6-phosphate-Asp to glucose 6-phosphate and aspartate. After being constructed and purified, FraB is mixed with F-6-phosphate-Asp and incubate for 5, 20 or 60 minutes following by deactivation. The same reaction without FraB is conducted as controls. 13C F-Asn is spiked as the external standard. In MRM mode, the transitions for 13C F-Asn, F-6-phosphate-Asp, aspartate, glucose 6-phosphate are acquired. The abundances of F-6-phosphate-Asp and possible products at three-time points are quantified after normalization to 13C F-Asn. In addition, methods are optimized to extract and quantify F-Asn, a highly polar compound, from mouse chow which contains a low concentration of F-Asn and mouse feces, collected from mouse chow fed germ-free or conventional mouse. Either mouse chow or mouse feces are frozen in -80 oC, dried in vacuum concentrators, weighted, spiked with a known amount of 13C F-Asn, and ground. They are then resuspended in H2O and sonicated, following by centrifugation. After the supernatant flowing through a 0.45 micron filter cartridge, the concentration of F-Asn is quantified using MRM mode. Besides, an ultra-performance liquid chromatography system coupled with a triple quadrupole mass spectrometer is being developed for the separation of F-Asn. Waters nanoACQUITY UPLC System is used with M-Class Column ACQUITY UPLC M-Class BEH Amide Column 1.7um, 75um x 100 mm, coupling with Xevo TQ-S Waters.
Under a collision induced dissociation (CID) voltage of 15 volts in negative mode, the precursors m/z=259 of standard glucose 6-phosphate and fructose 6-phosphate show slightly different fragmentation pattern. In FraB reaction system, m/z= 259 precursor gives similar fragmentation pattern as standard glucose 6-phosphate does, which suggests glucose 6-phosphate is the main product of FraB catalyzed deglycation of F-6-phosphate-Asp. The concentration of F-6-phosphate-Asp decreases by 11% at 20 minutes and 28% at 60 minutes. The concentration of glucose 6-phophate increases by 8% at 20 minutes and 17% at 60 minutes. And the concentration of aspartate increases by 13% at 20 minutes and 100% at 60 minutes. Thus, the deglycation of F-6-phosphate-Asp to glucose 6-phosphate and aspartate by FraB is verified. For standard F-Asn, the protonated form with m/z= 295 loses two H2O molecules successively, forming m/z= 277 and 259, and then loses H2O and CH2O forming m/z= 211 at a collision induced dissociation (CID) voltage of 10 volts. For 13C F-Asn, under the same condition, the protonated form with m/z= 301 loses two H2O molecules successively, forming m/z= 283 and 265, and then loses H2O and 13C H2O forming m/z= 216, which shows identical intensity ratios between fragmentation products as F-Asn. Using 13C F-Asn, the amount of F-Asn in 100 mg mouse chow is quantified to be 12 ug and the amounts of F-Asn in 100 mg conventional and germ-free mouse feces are 1.7 ug and 5.1 ug respectively. These result shows the ability of the developed method to extract F-Asn and measure its concentration at low level and it also verifies the expectation that there are some microorganisms in conventional mouse intestine consuming F-Asn coming from mouse chow.
References & Acknowledgements:
References
[1] http://www.who.int/mediacentre/factsheets/fs139/en/
[2] http://www.cdc.gov/nationalsurveillance/PDFs/NationalSalmSurveillOverview_508.pdf
[3] Ali MM, Newsom DL, Gonzalez JF, Sabag-Daigle A, Stahl C, Steidley B, Dubena J, Dyszel JL, Smith JN, Dieye Y, Arsenescu R, Boyaka PN, Krakowka S, Romeo T, Behrman EJ, White P, Ahmer BM. 2014. Fructose-asparagine is a primary nutrient during growth of Salmonella in the inflamed intestine. PLoS Pathog 10:e1004209. http://dx.doi.org/10 .1371/journal.ppat.1004209
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