Even More Antibiotics In The Nursery!
by Christoph
We had learned earlier that Philanthus triangulum beewolves – solitary digger wasps that prey on bees, hence their name – employ a "cocktail" of nine different antibiotics to protect their offspring from detrimental fungal infections. The antibiotics are produced – I repeat from the earlier post – by a bacterial symbiont, 'Candidatus Streptomyces philanthi' (Figure 1), which the females cultivate in antennal glands and secrete into the brood cells prior to oviposition (=egg-laying). They are taken up by the larvae and incorporated into their cocoons, thereby protecting them against fungal infections. Weeks or, after hibernation, months later, eclosing adult females acquire the bacteria from the cocoon's surfaces, thus completing the vertical transmission of the symbiont.
Figure 1. Streptomyces bacteria from the antennae of a female beewolf (Philanthus triangulum ) (false colors). Frontpage: male beewolf (Philanthus pulcherrimus ) on a perch in its territory. Credit: M. Kaltenpoth, MPI for Chemical Ecology, Jena, Germany. Source
A recent study by the Kaltenpoth lab at the Institute of Molecular Biology in Mainz, Germany, revealed that the Streptomyces philanthi symbionts of 25 studied beewolf species produce a stunningly rich assortment of altogether 49 compounds belonging to three classes of antibiotics. How is this diversity of antimicrobial compounds generated at the molecular level? Can synergistic or antagonistic interactions of compounds present in the mixture affect their activity against potential pathogens? Answers to these two questions would allow the researchers to address an even more interesting topic: how did the antibiotic mixture change over evolutionary timescales and to what extent do the patterns reflect phylogenetic constraints and ecological adaptations? But first things first...
Stocking-up a pharmacy (economically)
There are 130+ described Philanthus species that hunt bees – some prefer bumblebees – on all continents. Yet, an earlier study had shown that each of 28 analyzed Philanthus species harbor one strain, or lineage, of the Streptomyces philantri symbiont. To assess the full spectrum of antibiotics synthesized by the various S. philantri lineages when symbiosing with "their" beewolf species, Engl et al. collected cocoons and females from various locations in the Americas, Africa and Europe, determined 25 species, and analyzed extracts by mass spectrometry. State-of-the-art mass spectrometers can detect minute amounts of virtually any chemical substance in complex mixtures, qualitatively and quantitatively. But assigning peaks in mass spectra to particular compounds still requires detective skills and, of course, access to extensive reference spectra libraries. In several instances, the researchers had to actually count the ions to determine concentrations because reference substances for calibration weren't commercially available.
Figure 2. Known derivatives of Piericidin A. Source
They found, in total, 49 symbiont-produced (putatively bioactive) compounds, 9 of which had been found previously in other streptomycetes, 9 already identified in Philanthus triangulum, and 31 newly identified. Of these compounds, 45 were piericidin derivatives, 3 streptochlorin derivatives, and one was nigericin. The astonishingly varied "bouquet" of piericidin derivatives prompted the researchers to compare, at the sequence level, the piericidin biosynthetic gene cluster (PKS) of Philanthus triangulum with corresponding gene clusters of other Streptomycetes that had been studied for piericidin synthesis. They found a perfect match of gene order, and protein similarities in the 65% range. This suggested to them that the S. philantri piericidin synthesis machinery works in a more relaxed fashion, with the derivatives brought about by variations of the "normal" pathway. Such 'variations' could include incorporation of different precursors (for biochemistry experts: methylmalonyl-CoA and/or malonyl-CoA) in the initiation step as well as in the elongation steps of the linear polyketide chain, or skipping some steps entirely, and, lastly, variations in the post-PKS 'tailoring steps' of amidation, cyclization, hydration, and methylation of the polyketide chain (Figure 2). That's clearly a more economic approach to stock‑up a pharmacy's assortment than running a separate production pipeline for each drug! Interestingly, the beewolves' pharmacies are not equally well stocked: while the researchers detected 46 compounds in the Philanthus triangulum subsp. triangulum sample, there were only 6 in the Philanthus albopilosus sample. However, most samples contained between 10 and 20 compounds at varying concentrations. In conclusion: the compound mixtures were similar but no exactly alike, each specific for a single beewolf–S. philanthri symbiosis rather.
The art of mixing antibiotics
Figure 3. Bioactivity of different combinations of piericidin A1 (PA) and B1 (PB) and streptochlorin (SC) in agar diffusion assays against A M. guilliermondii, B Y. lipolytica, and C A. oryzae. Antagonistic effects (i.e., significantly lower inhibition zones of mixtures than one of the single substances) are highlighted by magenta boxes (according to ANOVA and Tukey HSD post hoc tests; *P < 0.05, **P < 0.01, ***P < 0.001). Source
Not only the number of compounds and their amounts affect a mixture's biological activity but also their synergistic and/or antagonistic interactions. To learn more about such interactions, Engl et al. performed agar diffusion assays with combinations of the three most abundant ingredients, piericidin A1, B1, and streptochlorin, against three different fungi, Aspergillus oryzae, and the yeasts Yarrowia lipolytica and Meyerozyma guilliermondii. Piericidin B1 did not influence the activity of piericidin A1, but it enhanced inhibition of streptochlorin against Y. lipolytica, and decreased the combined activity of piericidin A1 and streptochlorin against M. guilliermondii. Streptochlorin, on the other hand, generally increased the activity of piericidin B1 but reduced the inhibition of yeasts in mixtures containing piericidin A1 (Figure 3). Although they used just three fungi as proxy for the unknown nature of the fungal microbiota at the sample sites and tested them with only 3 of the 40+ compounds in combinations, the authors found both synergistic and antagonistic interactions. They can thus safely 'extrapolate' that even subtle changes in the composition of the antimicrobial mixture are likely to differentially affect opportunistic pathogens, and may allow for fine-tuning the antimicrobial defense to local pathogen communities. Also – and this may partly explain why the beewolf–Streptomyces symbiosis has not not run into an 'Antibiotic Resistance Crisis' during the past ~70 million years: a combination of antibiotics can slow down the evolution of resistance in potential pathogens for two main (known) reasons. First, if at least two compounds in a mixture act antagonistically, a microorganism that evolves resistance to one of the compounds experiences a higher effective inhibition by the remaining compound and thus has a decreased fitness compared with nonresistant cells. Synergistically acting compounds, on the other hand, favor a faster evolution of resistance because any arising resistance mutation against one compound quenches the synergistic inhibition. Second, intrinsically nontoxic compounds in a mixture can boost the activity of inhibitory compounds by neutralizing resistance mechanisms (a well known example: the non-antibiotic β‑lactam analog clavulanic acid inhibits certain β‑lactamases).
Evolution of a pharmacy
Figure 4. Phylogenetic and geographic influence on richness (number of compounds) and evenness (Shannon's E, roughly: proportional distribution of the components in a mixture) of the beewolf symbiont-produced antibiotic mixture. A Symbiont phylogeny (CaSp: 'Candidatus Streptomyces philanthri'), B host phylogeny, and C dendrogram based on a logarithmic distance matrix of sampling locations, all in comparison with heat maps displaying compound richness and evenness. Species are color-coded by sampling location (purple, North America; blue, South America; orange, Europe/ Africa). Richness and evenness heat maps are presented in color if a significant phylogenetic (Blomberg's K) or geographic (PGLS) influence was detected; otherwise, they are presented in gray. Branch numbers in the phylogenies are Bayesian posterior probability values. Source
By molecular phylogenetics, Kaltenpohl et al. had traced the origin of the beewolf–S. philanthri symbiosis and its coevolution back to an ancestor of the Philanthini tribe some 70 million years ago (and had, by the way, obtained indications for rare horizontal symbiont exchange among different beewolf species). This allowed Engl et al. to assess the intriguing questions: how did the symbiont-produced antibiotic mixture change over evolutionary timescales and to what extent do the patterns reflect phylogeny constraints and ecological adaptations? Such questions can be tackled by applying a battery of statistical methods that pull out significant correlations in multivariate data tables, that is, distance matrices. One of the distance matrices represented the Philanthini phylogeny, the second the phylogeny of the symbionts, and the third the sample sites (expressed as logarithmic distances of the sampling locations). The antibiotic mixtures were converted into distance matrices to account for the different numbers of compounds (richness) and their proportional abundance (evenness). They found that while the number of compounds (richness) was influenced by the phylogenetic background of the symbiont (Figure 4 A), the evenness of the mixture was significantly influenced by geographic location, suggesting an adaptation to the local environment (Figure 4 C). The authors summarize: "The first finding is consistent with evolutionary changes in the genetic basis of secondary metabolite biosynthesis that limit or expand the number of produced compounds. The second finding suggests an environmental influence on the quantitative expression of compound biosynthesis. Such expression changes presumably allow for rapid adaptations to the local environment (temperature, humidity, soil microbial community) by quantitatively adjusting the composition of the antibiotic mixture."
Beewolves et al.: 'defensive symbioses'
Figure 5. Cladogram of selected animal groups highlighting those with described defensive microbial symbionts and the corresponding symbiont taxa. Colored circles represent the major biosynthetic pathways reported for symbiont-produced compounds. Branch lengths are not to scale and branch order is adapted from previous phylogenetic analyses for the deep branches, hexapods and all other bilaterians. Common names in brackets denote selected groups within the respective taxon that harbor known defensive symbionts. PKS, polyketide synthase gene(s); NRPS, non-ribosomal peptide synthetase gene(s). Source
The term 'defensive symbiosis' was not coined in contrast to 'nutritional symbiosis' (for example, the aphid–Buchnera symbiosis), but rather to emphasize a somewhat different "goal." The beewolf–Streptomyces symbiosis is an example for such a 'defensive symbiosis' among Eukaryotes and Bacteria, in which a cocktail of compounds produced by a single symbiotic bacterial strain is capable of providing efficient protection for the eukaryotic host against an array of opportunistic bacterial and fungal pathogens. But this is not a unique evolutionary somersault. Flórez et al. reviewed in 2015 the then known 'defensive symbioses' – 19 and counting – and point to an interesting feature found repeatedly among animal–microbe protective associations: the simultaneous employment of multiple defensive chemicals, produced by either a single or several symbiotic partners (Figure 5). Call this 'combination prophylaxis', an approach that starkly contrasts with the conventional prophylactic approach to prevent infections in surgery patients by using a single broad sprectrum antibiotic.
And there is another, more application-oriented aspect that Flórez et al. mention: "Animal–microbe defensive symbioses ... are a promising research target for the field of natural products discovery, due to their immense chemical potential and the advantages of studying the microbial producers directly embedded in an ecological context (i.e. fulfilling a role for their eukaryotic host), as opposed to free-living microorganisms. Furthermore, discovered natural products are more likely to be applicable in medical contexts, since they have been naturally tested for side effects on, at least some, eukaryotes." Which is, in my opinion, a pretty good argument when it comes to developing promising strategies to tackle the antibiotic resistance crisis (see here and here for a fairly recent two-part 'situation report').
