LC–MS/MS based observation of Clostridium difficile inhibition by Lactobacillus rhamnosus GG

Abstract

Clostridium difficile is a spore-forming obligate anaerobe that most commonly causes nosocomial disease, such as diarrhea and colitis. Although antibiotic treatment has been used for Clostridium difficile infection (CDI), its use can also lethally cause antibiotic-associated CDI with the risk of antibiotic resistance. Recently, to avoid these problems, probiotics therapies for CDI have been introduced and studied. However, the molecular mechanisms of C. difficile induced by probiotics, such as Lactobacillus rhamnosus GG (LGG), and associated pathways, have rarely been studied. Here, we co-cultured C. difficile and LGG, which is a promising candidate for treating CDI, in a transwell platform to profile metabolic and proteomic changes of C. difficile with mass spectrometric methods. In the co-cultured condition with LGG, energy generation pathways related to C. difficile growth, such as Stickland reactions and butyrate metabolism, were significantly inhibited, resulting in the decrease of growth rate. In particular, the inhibition of Stickland reactions had a negative effect on the toxin production of C. difficile. In addition, metabolic changes in purine biosynthesis that is known to play an important role in the life of bacteria were observed. Finally, we briefly discussed proteins involved in major iron uptake and electron transfer, as well as the chaperone proteins in bacteria that showed changes by LGG. These results demonstrate that metabolic alterations in C. difficile by LGG caused growth inhibition. Our in vitro co-culture model and multi-omics approach will not only provide better understanding of pathogen-probiotics interaction, but will also contribute to functional study, such as probiotics screening or probiotics engineering, for the therapy of disease caused by pathogenic bacteria.