https://orcid.org/0000-0002-7213-5014
Abstract
The environmental distribution of the neurotoxic amino acid, 3-N-methyl-2,3-diaminopropanoic acid (BMAA), first isolated in 1967, was initially believed to be limited to tropical and subtropical plants of the genus Cycas. The seeds of one such species, which had been used historically on the Pacific island of Guam as a foodstuff, had a reputation for neurotoxicity. Some 40 years later the amino acid was detected in terrestrial and aquatic cyanobacteria and in other aquatic organisms. Overlooked was the discovery of BMAA in peptides of bizarre structure that had been isolated in 1975 from Paenibacillus pulvifaciens during a search for antibiotics. More recently (2014), peptides of similar structure were isolated from Paenibacillus larvae; this organism is causative of American Foulbrood, a lethal disease of honeybee colonies. These are interesting chemical and environmental observations, but knowledge of the bacterial distribution of BMAA is limited to just these two species of Paenibacillus, while more than 200 Paenibacillus spp. are known. Paenibacillus spp. are ever present naturally in the environment and are used agriculturally; recent research reports that some species infect human foods – including cow's milk – and have been isolated from human body fluids. We wish to stimulate interest in the environmental distribution of the neurotoxic BMAA in Paenibacillus spp. by drawing together previously isolated streams of research and by proposing experimental approaches by which this matter might be resolved.
The neurotoxic amino acid, 3-N-methyl-2,3-diaminopropanoic acid (BMAA), known in plants of the genus Cycas and in aquatic cyanobacteria and other organisms, is a component of antibiotic peptides in two species of Paenibacillus.
Introduction
Paenibacillus spp., of which more than 200 are known, are endospore-forming bacteria, which are widely distributed in soils, where they promote plant growth by fixing nitrogen, solubilising soil phosphate, influencing the production of phytohormones and, in some species, by biosynthesising siderophores.1Paenibacillus spp. infect human foods, including raw and heat-treated milk, and commercial ready-made meals;2,3 some Paenibacillus spp. have been isolated from human body fluids.4 There is world-wide concern regarding American Foulbrood, a lethal condition to colonies of the honeybee (Apis mellifera), which is spread by Paenibacillus larvae.5
Paenibacillus spp. are used in agriculture as a means of biological control against phytopathogens and insect herbivores,1 an activity that probably results from the wide range of compounds of diverse structure that Paenibacillus spp. produce.6 Some of these compounds are peptides, which are assembled by non-ribosomal peptide synthetase (NRPS) mechanisms and contain non-encoded amino acid residues not found in peptides that are assembled ribosomally.6
Discussion
Many antimicrobial peptides have been isolated from strains of Paenibacillus spp.; Bacillus polymyxin, (reclassified as Paenibacillus polymyxin), has been known as a major source since 1947. Two antibiotic peptides of bizarre structure, named galantins (Fig. 1a), which were isolated in 1975 from Paenibacillus pulvifaciens (then classified as Bacillus pulvifaciens7), contain, among other amino acid residues, one residue each of l-2,3-diaminopropanoic acid (l-DAP) and its methylated derivative l-3-N-methyl-2,3-diaminopropanoic acid (l-BMAA) per molecule of peptide. Both are non-encoded amino acids; the former is a common secondary metabolite of bacteria and higher plants,8 but the latter was known only in seeds of the Cycadacae,9–11 which are ancient gymnosperms. Consumption of these seeds by the native Chamorro people of Guam, an island in the western Pacific, is considered causative of the profound neurological disease: motor neurone disease (ALS)/Parkinson's disease/dementia (ALS/PD) once common on the island; l-BMAA has been implicated in this disorder12
Structures of galantins and paenilamicins. All structures are shown as the protonated forms that exist at physiological pH values. (a) Galantin I. The amino acid sequence of the core peptide (functional groups in black) is: d-ALA/l-2,3-diaminopropanoic acid/d-LYS or d-ornithine (shown: 5-amino group in mid-blue)/galantinic acid (a polyhydroxyaminoacid)/l-3-N-methyl-2,3-diaminopropanoic acid (BMAA: methyl group in red)/GLY. The N-terminal group of the core peptide is derivatised by the carboxyl group of galantinamic acid (a polyhydroxyaminoacid derived from LYS: functional groups in purple); the C-terminal group is derivatised by the C-3 amino group of spermine (functional groups in dark-blue). Redrawn from ref. 7. (b) Paenilamicins A1/A2. The structures of these peptides which, like galantins may contain either d-LYS or d-ornithine (shown: 5-amino group in mid-blue), are identical to galantins, except for the methylation of the l-2,3-diaminopropanoic acid (l-DAP) residue, yielding a second residue of l-3-N-methyl-2,3-diaminopropanoic acid (BMAA: methyl group in red). Redrawn from ref. 13. (c) Paenilamicins B1/B2. The structures are identical to those of paenilamicins A1/A2, except that the N-terminal group is modified by a derivative of ARG, not LYS (functional groups in violet). For chemical simplicity we suggest that this compound be named paenilamic acid, by analogy with the LYS derivative galantinamic acid in galantins and paenilamicins A1/A2. A residue of d-LYS is shown in the central core (6-amino group in light-blue). Compare with the d-ornithine derivative (5-amino group in mid-blue) in galantin I and paenilamicins A1/A2. Redrawn from ref. 13.
A group of antibiotic peptides has been isolated from P. larvae that are related structurally to galantins (Fig. 1b and c).13 These compounds, named paenilamicins, contain similar non-encoded amino acid residues to galantins, but the 3-amino group of the l-DAP residue in galantins is methylated in the paenilamicins, which therefore contain two residues of l-BMAA (Fig. 1b and c) per molecule of peptide. Like galantins, paenilamicins may contain either ornithine or lysine residues within the core peptide. Paenilamicins A1 and A2, like galantins, contain a lysine derivative at the N-terminal end of the core peptide; paenilamicins B1 and B2 contain a derivative of arginine in this position (Fig. 1b and c). Thus, P. larvae produces four forms of the paenilamicins. Lacking negative charge, but possessing seven positively-charged groups at physiological pH values (Fig. 1a–c), galantins and paenilamicins are strongly cationic. This feature may account for their antimicrobial activity against Gram-negative bacteria.6
Several toxic principles derived from P. larvae have been suggested as being causative of American Foulbrood.5 We propose that l-BMAA, one of the components of galantins and paenilamicins, should also be considered a candidate.
l-BMAA was first isolated from seeds of Cycas micronesica Hill9 and shown to be neurotoxic to small laboratory animals.14 Subsequently, the amino acid was reported in several species of cyanobacteria15 and later in diatoms, a marine dinoflagellate, molluscs and fish from several habitats.12 The distribution of BMAA in these organisms, even into human brains, has been proposed to occur via food-chains originating from cyanobacteria. For example, cyanobacteria of Nostoc sp. produce BMAA in the coralloid roots of Cycas micronesica, from where it is translocated to the aerial parts of the plant and thence into the seeds that were used for human consumption on Guam. Cyanobacteria and microalgae, including diatoms and dinoflagellates, serve as primary producers in marine and fresh water environments.12,15
The biosynthetic route to l-BMAA in cyanobacteria is unknown, but might arise from 3-N-methylation of bound or free l-DAP.8 In P. larvae the methylation of the l-DAP residue to form l-BMAA occurs within a NRPS-multienzyme complex and the genomic information required to biosynthesise the paenilamicins, the pam gene cluster, has been identified.13 This biosynthetic route implies that free l-BMAA does not occur intracellularly in P. larvae, but metabolic turnover of the paenilamicins and their hydrolysis by peptidases within the gut or gut wall of honey bee larvae would release the free neurotoxin. The proposed mechanisms of toxicity of l-BMAA to animal cells are controversial, but include excitoxicity to central nervous system neurones, association with proteins, induction of aberrant intracellular protein metabolism and the formation of formaldehyde.12
Conclusion
The presence of l-BMAA in P. larvae is of considerable interest to many scientific disciplines. It is unlikely that paenilamicins are the sole cause of the American Foulbrood pathology caused by P. larvae,13 as a mutant form of the organism, lacking the pam gene cluster, also kills honeybee larvae, but more slowly than does the wild-type bacterium. However, that l-BMAA may occur in other species of Paenibacillus, which are of widespread ecological and agricultural importance, raises further questions of the environmental abundance of the neurotoxin in these and other bacterial species. A search for galantins, paenilamicins and their analogues in Paenibacillus spp. would be a formidable analytical task. However, searching appropriate data bases for the pam gene cluster in Paenibacillus spp. (11 new species were identified only recently4) and other bacterial species should be rewarding;13 analyses of extracts of bacterial cells before and after acid hydrolysis has been successful in identifying BMAA in cyanobacteria and offers an additional, relatively rapid, investigative route.15 Thus, apart from the possible involvement of P. larvae as a causative factor of American Foulbrood, the contribution that other Paenibacillus spp. might make to the environmental load of l-BMAA, in addition to that contributed by cyanobacteria and potentially by microalgae, is worthy of investigation.
The possibility of human food becoming contaminated by Paenibacillus spp. containing l-BMAA, the likelihood of these same BMAA-producing bacteria occupying the human microbiome and the chance of BMAA-producing endospores from Paenibacillus spp. entering the human central nervous system16 present challenges to research in toxicology.
Conflicts of interest
There are no conflicts of interest.
Acknowledgements
PBN thanks Dr P. R. Brown for helpful discussions.
