Bacillus amyloliquefaciens strains isolated from moisture-damaged buildings produced surfactin and a substance toxic to mammalian cells

Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day. Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 g dry wt/ml). Two bioactive substances were purified from biomass of the fungicidal isolates. One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm. In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (_m) and the plasma membrane (_p) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K⁺:Na⁺:Ca²⁺ of 26:15:3.5. The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin. Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca. 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w). The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.Abbreviations RP-HPLC Reversed-phase high-performance liquid chromatography - BLM Black lipid membrane - DAD Diode-array detector - _m Mitochondrial membrane potential - _p Plasma membrane potential - JC-1 5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenz-imidazolo carbocyanine iodide - AM Calcein acetoxymethyl ester - PI Propidium iodide - MALDI-TOF-MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry - ESI-IT-MS Electrospray ionization ion trap mass spectrometry - EC₅₀ Endpoint concentration which caused 50% change in the viability parameters - FCCP Carbonyl cyanide 4-trifluoromethoxyphenylhydrazone