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°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°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.Mycobacterium marinum is a globally distributed pathogen of marine and freshwater fish which also causes skin infections in humans (7, 9). M. marinum is phenotypically distinguished from other mycobacteria by its low optimal growth temperature, light-induced carotenoid production, and relatively rapid growth rate compared to other slow-growing Mycobacterium species. There is considerable heterogeneity among M. marinum isolates, and several subgroups have been described (28,(33)(34)(35).Mycobacteriosis was first diagnosed in fish from the Red Sea in 1990 (5). The infection was initially found in cultured sea bass (Dicentrarchus labrax) in Eilat and has since been found in over 20 different fish species and a hawksbill sea turtle. The Red Sea isolates differed phenotypically from other M. marinum strains by being scotochromogenic (having constitutive pigment production). Whereas most M. marinum strains form colonies on mycobacterial media within 8 days, initial growth was not obtained from these isolates for at least 2 weeks. Similar isolates have also been found in the Mediterranean Sea in Greece and Italy. Molecular characterization of the Israeli isolates from fish confirmed their identity as M. marinum, but analysis of the 16S rRNA gene showed that the isolates formed clades within the species (33, 34). Molecular comparison of the fish isolates with human isolates of...

Evolution of Mycobacterium ulcerans and Other Mycolactone-Producing Mycobacteria from a Common Mycobacterium marinum Progenitor

“…Based on some phenotypic traits that distinguished it from M. ulcerans, such as photochromogenicity, absence of growth at 37°C, and scant growth at 30°C on Middlebrook 7H10 agar, this mycobacterium was given a new species designation, Mycobacterium pseudoshottsii. Subsequent analysis of the strain showed that it too contained a pMUM-like plasmid with the mycolactone mls genes and that it made yet another mycolactone, called mycolactone F (26). In the same study, a cluster of M. marinum strains that had been isolated from diseased fish in the Red and Mediterranean Seas (38) were also positive for mls gene sequences, and they too produced mycolactone F (26).…”

“…Subsequent analysis of the strain showed that it too contained a pMUM-like plasmid with the mycolactone mls genes and that it made yet another mycolactone, called mycolactone F (26). In the same study, a cluster of M. marinum strains that had been isolated from diseased fish in the Red and Mediterranean Seas (38) were also positive for mls gene sequences, and they too produced mycolactone F (26). One of these strains (DL240490) shared identical hsp65 gene sequences with M. pseudoshottsii and another fish pathogen, Mycobacterium seriolae (28).…”

Total synthesis of mycolactones A and B

“…For this reason, the deprotection was quenched at approximately 60% completion, and the recovered triol was recycled. After one recycle, synthetic mycolactones A and B were isolated in 67% yield as an approximately 3:2 mixture of 4′-Z (mycolactone A) and 4′-On comparison of 1 H NMR (Figure 2), 13 C NMR (Table 1), and TLC [silica gel, CHCl 3 -MeOH-H 2 O (90:10:1)], the synthetic mycolactones A and B were found to be superimposable on the natural mycolactones A and B, respectively. Rigorously speaking, however, these comparisons could not eliminate the possibility that the synthetic mycolactones A and B might be the remote diastereomers 25 of the natural products.…”

“…Flash chromatography (100:1 hexanes/EtOAc) gave the vinyl iodide 25 (1.413 g, 65% yield). 1 , 9H), 0.056 (s, 3H), 0.054 (s, 3H), 0.04 (two overlapped singlets, 6H); 13 Synthesis outlined in Scheme 10-To a solution of t-BuOK (6.56 g, 58.5 mmol) in THF (400 mL) at -78 °C was added Z-2-butene (8.2 g, 146 mmol) followed by n-BuLi in hexanes (2.16 M, 27.1 mL, 58.5 mmol). The bright yellow suspension was stirred at -78 °C for 5 min, -45 °C for 10 min, and then -78 °C for 15 min.…”

“…Flash chromatography (3:1 hexanes/ EtOAc) gave the alcohol 29 (0.662 g, 83% yield for 2 steps). 67 (m, 2H), 1.47-1.55 (m, 1H), 0.87 (d, J = 7.0, 3H); 13 …”