The mitochondrial toxin produced by Streptomyces griseus strains isolated from an indoor environment is valinomycin

Abstract

Actinomycete isolates from indoor air and dust in water-damaged schools and children's day care centers were tested for toxicity by using boar spermatozoa as an indicator. Toxicity was detected in extracts of four strains which caused a loss of sperm motility, and the 50% effective concentrations (EC50) were 10 to 63 ng (dry weight) ml of extended boar semen-1. The four strains were identified as Streptomyces griseus strains by 16S ribosomal DNA and chemotaxonomic methods. The four S. griseus strains had similar effects on sperm cells, including loss of motility and swelling of mitochondria, but we observed no loss of plasma membrane integrity or depletion of cellular ATP. None of the effects was observed with sperm cells exposed to extracts of other indoor actinomycete isolates at concentrations of >/=5,000 to 72,000 ng ml-1. The toxin was purified from all four strains and was identified as a dodecadepsipeptide, and the fragmentation pattern obtained by tandem mass spectrometry was identical to that of valinomycin. Commercial valinomycin had effects in sperm cells that were identical to the effects of the four indoor isolates of S. griseus. The EC50 of purified toxin from the S. griseus strains were 1 to 3 ng ml of extended boar semen-1, and the EC50 of commercial valinomycin was 2 ng ml of extended boar semen-1. To our knowledge, this is the first report of the presence of ionophoric toxin producers in an indoor environment and the first report of valinomycin-producing strains identified as S. griseus.

FIG. 1

Thin sections of midpieces of boar spermatozoa exposed to extracts of S. griseus 8/ppi (A through C), extracts of S. griseus DSM 40236^T (D), and commercial valinomycin (E) for 7 days. (A) Midpiece of a spermatozoon with mitochondrial damage. The frequency of swollen mitochondria in the spermatozoan midpiece was >60% after exposure to 20 μg (dry weight) of strain 8/ppi crude extract per ml. After exposure to 2 μg ml⁻¹ the frequency of swollen mitochondria was <20% (data not shown). (B) Thin section of the midpiece of a boar spermatozoon exposed to a toxic strain 8/ppi HPLC fraction, showing swollen mitochondria with disrupted outer membranes. (C) Midpiece of a spermatozoon exposed to a nontoxic strain 8/ppi HPLC fraction. (D) Section of a midpiece exposed to a similarly prepared S. griseus DSM 40236^T extract (20 μg [dry weight] per ml of extended boar semen). (E) Midpiece after exposure to 200 ng of commercial valinomycin ml⁻¹. Bars = 200 nm.

FIG. 2

HPLC fractionation and ESI MS-MS fragmentation of the toxin from S. griseus 8/ppi and commercial valinomycin. (A) HPLC elution profiles of the extract from S. griseus 8/ppi and commercial valinomycin ESI-MS-MS fragmentation patterns of an ammonium adduct of the toxin purified from S. griseus 8/ppi (ion m/z 1,128.84) (B) and of an ammonium adduct of commercial valinomycin (ion m/z 1,128.64). (C) Samples were dissolved in 50% methanol containing ammonium acetate for the MS analysis. The peak numbers correspond to the fragment ions assigned in Table 2. The peaks marked with asterisks represent loss of water.