Mucin-derived O-glycans supplemented to diet mitigate diverse microbiota perturbations.

Microbiota-accessible carbohydrates (MACs) are powerful modulators of microbiota composition and function. These substrates are often derived from diet, such as complex polysaccharides from plants or human milk oligosaccharides (HMOs) during breastfeeding. Host-derived mucus glycans on gut-secreted mucin proteins serve as a continuous endogenous source of MACs for resident microbes; here we investigate the potential role of purified, orally administered mucus glycans in maintaining a healthy microbial community. In this study, we liberated and purified O-linked glycans from porcine gastric mucin and assessed their efficacy in shaping the recovery of a perturbed microbiota in a mouse model. We found that porcine mucin glycans (PMGs) and HMOs enrich for taxonomically similar resident microbes. We demonstrate that PMGs aid recovery of the microbiota after antibiotic treatment, suppress Clostridium difficile abundance, delay the onset of diet-induced obesity, and increase the relative abundance of resident Akkermansia muciniphila. In silico analysis revealed that genes associated with mucus utilization are abundant and diverse in prevalent gut commensals and rare in enteric pathogens, consistent with these glycan-degrading capabilities being selected for during host development and throughout the evolution of the host-microbe relationship. Importantly, we identify mucus glycans as a novel class of prebiotic compounds that can be used to mitigate perturbations to the microbiota and provide benefits to host physiology.

The phosphoinositide-dependent kinase, PDK-1, phosphorylates conventional protein kinase C isozymes by a mechanism that is independent of phosphoinositide 3-kinase.

Phosphorylation by the phosphoinositide-dependent kinase, PDK-1, is required for the activation of diverse members of the AGC family of protein kinases, including the protein kinase C (PKC) isozymes. Here we explore the subcellular location of the PDK-1-mediated phosphorylation of conventional PKCs, and we address whether this phosphorylation is regulated by phosphoinositide 3-kinase. Pulse-chase experiments reveal that newly synthesized endogenous PKC alpha is primarily phosphorylated in the membrane fraction of COS-7 cells, where it is processed to a species that is phosphorylated at the activation loop and at two carboxyl-terminal positions. This "mature" species is then released into the cytosol. Deletion of the plekstrin homology domain of PDK-1 results in a 4-fold increase in the rate of processing of PKC indicating an autoinhibitory role for this domain. Autoinhibition by the plekstrin homology domain is not relieved by binding 3'-phosphoinositides; PKC is phosphorylated at a similar rate in serum-treated cells and serum-starved cells treated with the phosphoinositide 3-kinase inhibitors, LY294002 and wortmannin. Under the same conditions, the PDK-1-catalyzed phosphorylation of another substrate, Akt/protein kinase B, is abolished by these inhibitors. Our data are consistent with a model in which PDK-1 phosphorylates newly synthesized PKC by a mechanism that is independent of 3'-phosphoinositides.