How Bacteroides species coordinate glycogen metabolism and global gene expression to colonize the mammalian intestine

Pennsylvania State Univ Hershey Med Ctr, Hershey PA

Investigators

Abstract

PROJECT SUMMARY The human gut is colonized by trillions of microbial symbionts that defend against pathogenic invasion, regulate host immunity, and convert nutrients into host-absorbable metabolites. Gut microbes have evolved adaptations to conserve energy during colonization and survive in this highly competitive ecological niche. For example, members of the Bacteroidetes family employ pyrophosphate, a byproduct of DNA, protein, and glycogen synthesis, to drive central metabolic reactions and conserve nucleotide triphosphate (NTP) pools. Adaptations such as these contribute to the prevalence of Bacteroidetes, constituting up to half of all bacteria in the human intestine. Our lab has extensively studied a conserved Bacteroides transcription factor, called Cur, that encodes products necessary for colonization and regulating T-cell populations in the intestine. For example, the cur- dependent gene, fusA2, is a unique translation elongation factor that facilitates GTP-independent protein synthesis, a process that conserves NTPs and is necessary for intestinal fitness. Furthermore, Cur is directly responsible for the transcription of BT4295, an outer membrane protein that induces T-cell differentiation. We have previously demonstrated that the abundant dietary sugars, glucose and fructose, dominantly inhibit Cur activity. I have recently established that these simple sugars inhibit Cur via hierarchically governed ATP- and pyrophosphate (PPi)-dependent fructose bisphosphate (FBP) biosynthetic pathways. Remarkably, the canonical ATP-dependent glycolytic pathway is dispensable for in vitro growth and only necessary to control Cur during in vivo colonization because PPi-dependent enzymes are primarily responsible for the glycolytic load. These findings demonstrate that the role of ATP-dependent FBP synthesis is to regulate Cur during intestinal colonization, rather than producing energy equivalents via glycolysis; however, the mechanism by which FBP governs Cur activity has not been established. Other members of the Bacteroides family require FBP to synthesize intracellular glycogen, a process that is necessary for (i) the utilization of cur-dependent, but not - independent, sugars, (ii) Cur activation during carbon limitation, and (iii) fitness in a murine intestine. Collectively, the preliminary findings suggest that FBP and glycogen biosynthetic pathways form a cyclical regulatory pathway that controls Cur based on nutrient availability in the intestine. Therefore, I hypothesize that FBP governs Cur activity by regulating glycogen metabolism in response to intestinal nutrient availability. In Aim 1.1, I will investigate the role of FBP in promoting glycogenesis in Bt using an FBP-conjugated affinity resin coupled with enzymatic assays. In Aim 1.2, I will identify glycogenic genes that govern Cur activity during nutrient limitation by engineering Bt mutants and measuring effects on cur- dependent genes. In Aim 2, I will elucidate the signal that is produced by glycogen metabolism during nutrient limitation using a recombinant, affinity-tagged Cur protein to capture endogenous metabolites.