Mutanofactins are a family of natural products produced by Streptococcus mutans from the human oral microbiome. We report a unified approach to all mutanofactins by developing a total synthesis amenable to diversification. The key to success for the most complex members, mutanofactins 607 and 697, was an acyl ketene based strategy. Access to the family enabled comprehensive biological profiling, where we demonstrate that all mutanofactins are biofilm promoting in Streptococcus mutans. Experiments were extended to other inhabitants of the oral microbiome for the first time: Streptococcus gordonii and Streptococcus oralis, two early colonizers, were similarly affected with mutanofactins being biofilm promoting. Conversely, Veillonella dispar and Fusobacterium nucleatum showed little to no reaction to mutanofactins. Biophysical investigations based on quartz crystal microbalance with dissipation monitoring and atomic force microscopy reveal a previously unknown mucin-mutanofactin 697 interaction. Incubation of a mucin layer with mutanofactin 697 induces a morphology change within the mucin layer, which promotes bacterial adhesion and biofilm formation. This unique property of mutanofactin 697 might be key to early stages of biofilm formation in the human oral microbiome. Combined, an interdisciplinary approach consisting of total synthesis, microbiology and biophysical characterization provides insight into the roles of mutanofactins in the oral microbiome.

Bacterium-triggered carious lesions implicate dental hard tissue destruction and the simultaneous initiation of regenerative events comprising dental stem cell activation. Streptococcus mutans (S. mutans) is a prominent pathogen of the oral cavity and the principal cause of caries. S. mutans generates complex products involved in interbacterial interactions, including Mutanobactin-D (Mub-D), which belongs to a group of non-ribosomal cyclic lipopeptides. In the present study, we aimed to analyse the potential role of the synthetic Mub-D peptide in cell populations involved in tissue regenerative processes. To this end, we assessed the in vitro effects of Mub-D in human dental pulp stem cells (hDPSCs) and human bone marrow stem cells (hBMSCs). Our data demonstrated a concentration-dependent effect of Mub-D on their viability and a significant increase in their proliferation and osteogenic/odontogenic differentiation. These events were associated with specific changes in gene expression, where CCDN-1, RUNX-2, OSX, OCN, DMP-1, DSPP, and BMP-2 genes were upregulated. The ability of Mub-D to modulate the osteogenic/odontogenic differentiation of both hDPSCs and hBMSCs and considerably enhance mineralisation in a controlled and concentration-dependent manner opens new perspectives for stem cell-based regenerative approaches in the clinics.

Mutanobactin D is an interkingdom communicator derived from the human oral microbiome. The lipopeptide prevents yeast-to-hyphae morphogenesis in Candida albicans, notably without fungicidal or fungistatic activity. The mode of action and structure-activity relationship of mutanobactin D are unknown and prompt an interdisciplinary program of study. Stereoselective synthesis of designed mutanobactin D analogs reveals that the C26 configuration is crucial for bioactivity associated with inhibition of pathogenesis, or yeast-to-hyphae transition, in C. albicans. To shed light on this finding, we employ molecular dy-namics simulations of mutanobactin D and selected analogs in increasingly complex environments: Monophasic (water or CHCl3), interfacial (water/CHCl3), and explicit lipid membrane (phosphatidylcholine) models. Monophasic MD simulations do not distinguish between bioactive and inactive compounds. In contrast, at a polar/apolar interphase, a dominant, stable conformation emerges for mutanobactin D and bioactive analogs. Explicit lipid membrane simulations reinforce these results and further reveal the formation of a continuous, structured water cushion, which is not found for inactive analogs. Our studies collectively reveal how the stereodefined attachment of the lipid in the C26-C28 motif governs activity against C. albicans and provide a framework for understanding the membrane behavior of mutanobactin D, which may be coupled to its role in the human oral microbiome.