D-alanine (D-Ala) and several other D-amino acids (D-AAs), unusual amino acids present in mammals, act as hormones and neuromodulators in nervous and endocrine systems. Unlike the endogenously synthesized D-serine in animals, D-Ala may be from exogenous sources, e.g., diet and intestinal microorganisms. However, it is unclear if the capability to produce D-Ala and other D-AAs varies among different microbial strains in the gut. We isolated individual microorganisms of rat gut microbiota and profiled their D-AA secretion in vitro, focusing on D-Ala. Serial dilutions of intestinal content from adult male rats were plated on agar to obtain clonal cultures. Using MALDI-TOF MS for rapid strain typing, we identified 38 unique isolates, grouped into 11 species based on 16S rRNA gene sequences. We then used two-tier screening to profile bacterial D-AA secretion, combining a D-amino acid oxidase-based enzymatic assay for rapid assessment of overall D-AA amount, followed by chiral LC-MS/MS to quantify individual D-AAs, revealing 19 out of the 38 isolated strains as D-AA producers. LC-MS/MS analysis of the eight top D-AA producers showed high levels of D-Ala in all strains tested, with substantial inter- and intra-species variations. Though results from enzymatic assay and LC-MS/MS analysis aligned well, LC-MS/MS further revealed the existence of D-glutamate and D-aspartate, which are poor substrates for enzymatic assay. We observed large inter- and intra-species variation of D-AA secretion profiles from rat gut microbiome species, demonstrating the importance of chemical profiling of gut microbiota in addition to sequencing, furthering the idea that microbial metabolites modulate host physiology.
Quantification of amino acids and peptides in an ionic liquid based aqueous two-phase system by LC–MS analysis