Bacillamide D produced by Bacillus cereus from the mouse intestinal bacterial collection (miBC) is a potent cytotoxin in vitro

The gut microbiota influences human health and the development of chronic diseases. However, our understanding of potentially protective or harmful microbe-host interactions at the molecular level is still in its infancy. To gain further insights into the hidden gut metabolome and its impact, we identified a cryptic non-ribosomal peptide BGC in the genome of Bacillus cereus DSM 28590 from the mouse intestine (www.dsmz.de/miBC), which was predicted to encode a thiazol(in)e substructure. Cloning and heterologous expression of this BGC revealed that it produces bacillamide D. In-depth functional evaluation showed potent cytotoxicity and inhibition of cell migration using the human cell lines HCT116 and HEK293, which was validated using primary mouse organoids. This work establishes the bacillamides as selective cytotoxins from a bacterial gut isolate that affect mammalian cells. Our targeted structure-function-predictive approach is demonstrated to be a streamlined method to discover deleterious gut microbial metabolites with potential effects on human health.


Figure S4 .
Figure S4.Western blot of subcellular fraction samples after 24h treatment for the presence of marker proteins: Lprotein ladder; Wwhole cell fraction; Mmembrane fractions with 1-integrin bands; Ccytosolic fraction with tubulin band; Nnuclei with lamin a/c bands.

Figure S5 .
Figure S5.Uptake of bacillamide D into cytosolic fractions of HCT116 cells at different time points, compared to negative EtOH control.

Figure S6 .
Figure S6.Detection of bacillamide D in pellet sample of Bacillus cereus.The strain was grown over 24 h in BHI and the pellet extracted with methanol as described previously.HR-MS (ESI+): m/z = 315.1278[M+H] + , calc.: 315.1274; m/z = 337.1093[M+Na] + , calc.: 337.1094.The retention time of the identified signals matched a bacillamide D reference sample.

Figure S7 .
Figure S7.Taxonomic spread of the bac BGC in Bacillus cereus group strains.All Bacillus cereus group genomes in the proGenomes v2 database were downloaded.5The quality

Figure S8 .
Figure S8.Schematic representation of the plasmid sequencing results of pET28b-ptetO::bac::gfp.The pairwise identity within the insert region is 100%.

Table S1 .
Bacillamide D did not show antibacterial activity against the tested panel of bacterial pathogens.