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Trends in Ecology & Evolution

Forum
Resisting Antimicrobial One is a tripartite mutualistic relationship,
within which the ants host antimicrobial-
by 14 gene clusters (ranging from 15 to
17 genes). The clusters share a region
Resistance: Lessons producing bacteria on their bodies to which synthesises the dilactone core
from Fungus Farming protect their fungal cultivar. Many of these
bacteria have coevolved with their hosts,
common to all antimycin analogues [5,6].
Therefore, such gene clusters (originating
Ants producing antimicrobials to inhibit the para- from one ancestral cluster) would support
sitic fungi, while in return, the ants provide the synthesis of different antimicrobial variants
Ayush Pathak,1 Steve Kett,2 and them with nutrition and a microclimate [5,6]. A similar organisation has been
Massimiliano Marvasi3,* suitable for growth [3]. The parasitic fungi identified in the Pseudonocardia spp., which
compete with the ant-associated bacteria, produce multiple, structurally similar antimi-
as both depend upon the same fundamen- crobials such as dentigerumycin, gerumycin
Attine ants use antimicrobials pro- tal source from which they directly, or A, gerumycin B, and gerumycin C, as well
duced by commensal bacteria indirectly, derive nutrition (Box 1). Thus, as a polyene antifungal, nystatin P1 [4,7].
to inhibit parasites on their fungal a question arises: why are these antimicro- The structural similarity of dentigerumycin,
gardens. However, in this agricul- bials still effective in controlling the parasitic gerumycin A, gerumycin B, and gerumycin
tural system, antimicrobial use fungi even after millions of years, while C, along with region-specific similarity of two
does not lead to overwhelming pharmaceutical antimicrobials are rendered of the three clusters responsible for their
ineffective within a few decades? synthesis, suggests they derive from a single
resistance, as is typical in clinical
ancestral cluster that diversified in response
settings. Mixtures of continually
Antimicrobial Heterogeneity to selection from antimicrobial-resistant
evolving antimicrobial variants could
through Evolution of Gene Clusters organisms (Figure 1). Several regions of the
support these dynamics.
To control the parasitic Escovopsis, the gene clusters producing these antimicrobials
ants house antimicrobial-producing strains are flanked by mobile genetic elements
Symbiotic Antiparasite Defence of Pseudonocardia and Streptomyces (transposases, integrases, and endonucle-
Strategies bacteria on their cuticle. Streptomyces ases), indicating horizontal gene transfers
In an antimicrobial-mediated evolutionary strains inhabiting attine clades are acquired play a role in their recombination [7]. Such
arms race, the synthesis and release of via environmental sampling and are, thus, mobile genetic elements could also generate
antimicrobials evolves in one group of or- not associated with specific taxa, but the variation within the gene cluster of one
ganisms and their competitor organisms Pseudonocardia are nonrandomly associ- clonal line via transference in and out of the
counter this by evolving antimicrobial re- ated with attine species and display cluster. Similarly, in the genomes of the
sistance. Within such a milieu of intense cospeciation at higher taxonomic levels Pseudonocardia phylotypes Ps1 and Ps2,
selection and counterselection, not only [4]. Ant-associated Streptomyces produce novel nystatin P1-like compounds are
will participating organisms evolve, but so the antimicrobials candicidin and antimycin encoded by at least 14 biosynthetic gene
too will the antimicrobials themselves. ([5] and references within). Variants of clusters sharing multiple common genes
antimycin produced by Streptomyces [8]. Thus, it seems likely that variability in ef-
Fungus-growing ants (tribe: Attini) have culti- differ in UV absorbance profile, liquid chro- fectiveness of variants of antimycin pro-
vated specific fungi as food for 60 million matography retention time, and mass-to- duced by Streptomyces and variants of
years [1]. Their coevolutionary interdepen- charge (m/z) ratio [5], and are produced nystatin and dentigerumycin produced by
dence is so refined that, for many attine spe-
cies, their fungal cultivars are not found
outside this symbiotic association [1,2]. Box 1. Attine Ants, Allies, Enemies and Coevolved Crypts
The fungi need specific microclimates and More derived clades of attine ants such as the leaf-cutting ants (Atta spp. and Acromyrmex spp.) specialise in the
cultivation of Leucoagaricus spp. fungi. In this mutualistic association Leucoagaricus gongylophorus is an
nutrition provided by the ants and, in turn, obligate cultivar, and forms the ant colony’s dominant food source [1]. The fungus is vertically transmitted from
constitute the ants’ sole food source. Other colony to colony by the gynes (female reproductive ants) when they first establish their own colonies. The
fungi, of the genus Escovopsis, can invade Leucoagaricus cultivar may be parasitised by fungi of the genus Escovopsis [2]. In response, the ants house
the cultivated fungus and, because the multiple antimicrobial-producing bacteria on their cuticle in coevolved cuticular crypts and specialised exocrine
glands. The two Actinobacteria predominantly associated with the ants are Pseudonocardia and Streptomyces
ants rely entirely on cultivated fungus for [4]. Bacteria are also carried by the gynes on their mating flights and transmitted to offspring colonies [3].
food, this parasitism is detrimental to the A phylogenetic rooted-tree reconstruction of all known fungus-growing ants showed that the crypts are
ants [2]. To counteract these parasitic specifically evolved to house the Actinobacteria. They are morphologically different and differently located on
fungi, ants have evolved multiple strategies. the body of ancient paleo-attines, more basal attine genera and in the later evolved leaf-cutting attines [3].

Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx 1

Trends in Ecology & Evolution

Figure 1. Summary of the Interactions among Symbionts of Acromyrmex spp. Leaf-Cutting Ants and Microorganisms Living in Their Nest. Ants grow the
mutualist (green arrows) fungi Leucoagaricus spp. on cut leaf fragments to provide their sole food source. The parasitic fungus Escovopsis also feeds off Leucoagaricus
(antagonism, red lines). Mutualistic bacteria, Pseudonocardia (Phylum Actinobacteria) live on the ants, fed via subcuticular glands and, in return, provide antimicrobial
compounds to kill the parasite Escovopsis (antagonism). Escovopsis counteracts the defensive mutualists by either evolving resistance to the antimicrobials produced
by the Pseudonocardia or via producing antimicrobials such as melinacidin and shearinine that inhibit Pseudonocardia. The evolution of resistance in Escovopsis to
antimicrobials produced by Pseudonocardia induces selection pressures on the bacterial gene clusters (dotted pale blue arrow), causing the evolution of antimicrobial
gene clusters harboured in Pseudonocardia, via rearrangement of the gene clusters or via positively selected mutations in the gene clusters, thus inducing synthesis of
novel antimicrobial compounds.

Pseudonocardia is a result of constant varia- antagonism between parasite and mutualist on the bacteria to evolve new variants.
tion in the gene clusters producing them. triumvirate [2]. Both metabolites inhibit They have coevolved with ants that
Pseudonocardia growth and one, shearinine provide their nutrition and microclimate,
Escovopsis has evolved countermeasures; D, degrades ants’ Escovopsis weeding so the ants’ continuing existence is
in vitro tests suggesting it can develop efficiency and, at high concentrations, is necessary for their survival. In this coevo-
resistance to antimicrobials produced by lethal to them. It may well be that a similar lutionary arms race, novel bacterial anti-
Pseudonocardia, even if the majority of pattern of gene cluster evolution might be microbial compounds can be formed via
wild populations remain susceptible to present in the Escovopsis as described in novel gene cluster rearrangement or
them [9]. Escovopsis is an obligate parasite Pseudonocardia and Streptomyces. mutations. Novel compounds so gener-
of the fungus cultivar. This habitat specificity ated achieve greater or lesser evolution-
exerts further selection pressure on the Comparing Arthropod and Human ary success based upon the Escovopsis
parasite to develop resistance to antimicro- Strategies of Antimicrobial Usage strain antimicrobial susceptibility. This
bials produced by the ant-associated If modification of antimicrobial-synthesising mechanism is best explicated by Red
bacteria. Furthermore, recent discovery of gene clusters is amplified in the presence Queen Dynamics [10], by which in
two specialised secondary metabolites of the parasite, the diversity of bacterially the long term, continual evolution of
produced by Escovopsis has offered new synthesised antimicrobials suggests that novel antimicrobial compounds would be
insight regarding antimicrobial-mediated there is continuous selection pressure encouraged thus preventing sympatric

2 Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx

1
Department of Life Sciences, Imperial College London,
populations of Escovopsis from acquiring When comparing the attine model of London, UK
effective antimicrobial resistance. natural selection and diversification 2
Department of Natural Sciences, Middlesex University London,
London, UK
of antimicrobials to antimicrobial usage 3
Department of Biology, University of Florence, Florence, Italy
In the last decades several models and ex- in clinical settings, the contrast is strik-
perimental studies based upon them have ing. Humans use diverse antimicrobials, *Correspondence:
massimiliano.marvasi@unifi.it (M. Marvasi).
been developed, lending some support to but they are structurally discrete com-
https://doi.org/10.1016/j.tree.2019.08.007
this hypothesis. Mathematical and clinical pounds rather than the diverse range
trials show that mixing antibiotics results in of subtle variants utilised by the © 2019 Elsevier Ltd. All rights reserved.

resistance reduction [11]. Equally, other ants and their mutualists. The humans’
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