Globally distributed mycobacterial fish pathogens produce a novel plasmid-encoded toxic macrolide, mycolactone F

Abstract

Mycobacterium ulcerans and Mycobacterium marinum are closely related pathogens which share an aquatic environment. The pathogenesis of these organisms in humans is limited by their inability to grow above 35 degrees C. M. marinum causes systemic disease in fish but produces localized skin infections in humans. M. ulcerans causes Buruli ulcer, a severe human skin lesion. At the molecular level, M. ulcerans is distinguished from M. marinum by the presence of a virulence plasmid which encodes a macrolide toxin, mycolactone, as well as by hundreds of insertion sequences, particularly IS2404. There has been a global increase in reports of fish mycobacteriosis. An unusual clade of M. marinum has been reported from fish in the Red and Mediterranean Seas and a new mycobacterial species, Mycobacterium pseudoshottsii, has been cultured from fish in the Chesapeake Bay, United States. We have discovered that both groups of fish pathogens produce a unique mycolactone toxin, mycolactone F. Mycolactone F is the smallest mycolactone (molecular weight, 700) yet identified. The core lactone structure of mycolactone F is identical to that of M. ulcerans mycolactones, but a unique side chain structure is present. Mycolactone F produces apoptosis and necrosis on cultured cells but is less potent than M. ulcerans mycolactones. Both groups of fish pathogens contain IS2404. In contrast to M. ulcerans and conventional M. marinum, mycolactone F-producing mycobacteria are incapable of growth at above 30 degrees C. This fact is likely to limit their virulence for humans. However, such isolates may provide a reservoir for horizontal transfer of the mycolactone plasmid in aquatic environments.

FIG. 1.

Pathology showing infection with M. marinum DL240490 in sea bass (Dicentrarchus labrax) from an outbreak of mycobacterial disease in the Red Sea. (A) Severe splenomegaly and granulomatous spleen and kidney. (B) Massive load of acid-fast bacilli (Ziehl-Neelsen staining) within a splenic granuloma (total magnification, ×100).

FIG. 2.

Mass spectroscopy of ASLs from mycolactone F-producing M. marinum strains. (A) MS of ASLs from M. marinum DL240490, showing the mycolactone F sodium adduct at m/z 723. (B to D) MS/MS of the m/z 723 peak showing the core (*) and side chain (**) in M. marinum DL180892 from the Red Sea (B), M. marinum 045 Thalassa from the Mediterranean Sea (C), and BB170200 from a freshwater pond, Israel (D). The MS/MS peak for m/z 723 from DL240490 was identical to that shown for DL180892.

FIG. 3.

Reverse-phase HPLC of ASLs derived from mycolactone F-producing strains. (A) M. marinum DL240490; (B) M. pseudoshottsii L15; (C) M. marinum SA200695; (D) M. marinum BB170200. The major isomer of mycolactone F at a retention time of 20.5 ± 0.05 min is indicated with an asterisk.

FIG. 4.

1D H¹ NMR spectrum of mycolactone F.

FIG. 5.

Structural comparison of mycolactone A/B and mycolactone F.

FIG. 6.

Analysis of mycolactone-mediated cytopathicity. (A) Cytopathicity on L929 murine fibroblasts, showing untreated cells (left; total magnification, ×200) versus cells treated with 100 ng mycolactone A/B (middle; total magnification, ×200) versus cells treated with 100 ng mycolactone F (right; total magnification, ×200). (B) Cytotoxicity measured by LDH release. Culture supernatants were collected from wells containing L929 cells 4 h after mycolactone treatment, and the amount of LDH was measured using a CytoTox 96 assay kit (Promega). Data are means and standard deviations of the values obtained from triplicate samples; P > 0.05 for all concentrations (Student's t test). (C) Apoptosis was assessed at 24 h with the cell death detection enzyme-linked immunosorbent assay kit (Roche) and expressed as fold enrichment of nucleosomes. Data are means and standard deviations of the values obtained from triplicate samples; P < 0.05 for 15 ng, 150 ng, and 1.5 μg (Student's t test).