Impact of Endofungal Bacteria on Infection Biology, Food Safety, and Drug Development
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Published in the journal:
. PLoS Pathog 7(6): e32767. doi:10.1371/journal.ppat.1002096
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Pearls
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https://doi.org/10.1371/journal.ppat.1002096
Summary
article has not abstract
The filamentous mould Rhizopus microsporus is a member of the zygomycetes (lower fungi). While some strains serve as food fermenting fungi, others represent infamous plant pathogens and opportunistic human pathogens. Recently, it was shown that some strains of R. microsporus are associated with symbiotic bacteria. Here, we outline why these organisms are important for human health and how they can be exploited for drug development. Furthermore, we illustrate what the investigation of bacterial–fungal symbiosis can teach us about the evolution of pathogenicity factors in general.
Rhizopus microsporus Harbors Bacterial Endosymbionts as Intracellular Toxin Factories
R. microsporus (ATCC 62417) attacks rice plants and illicits rice seedling blight, a severe crop disease affecting rice fields in Asia [1]. Symptoms of the infection include abnormal swelling of the roots and finally death of the affected tissue. Plant pathogenic R. microsporus strains live as necrotrophic pathogens, i.e., they derive energy from killed host cells. For that purpose they secrete rhizoxin (see Figure 1A), a toxin that blocks mitosis in eukarytotic cells by binding to β-tubulin [2]. Rhizopus itself is resistant to the toxin due to an amino acid exchange in the tubulin protein [3]. The production of toxins by plant pathogenic fungi is a widespread virulence mechanism [4]. In the case of R. microsporus, however, the search for biosynthetic genes coding for rhizoxin biosynthesis led to an unexpected discovery: it is not the fungus itself that produces the pathogenicity factor rhizoxin. Instead, the fungus harbors bacterial symbionts, which are the actual producers of the virulence factor [1]. Toxin formation by bacteria has been demonstrated in analogy with Koch's postulates in classical microbiology: it was discovered that rhizoxin-producing strains of R. microsporus contained symbionts, while nontoxinogenic strains did not. Removal of symbionts by antibiotics indeed abolished rhizoxin production. The bacteria could be isolated and grown in axenic (pure) culture. Finally, reintroduction into cured hosts clearly reestablished rhizoxin production [1]. Intriguingly, microscopic investigations revealed that the bacteria are true endosymbionts, i.e., they inhabit the intracellular space of fungal mycelium (see Figure 1B). The first endosymbiont of R. microsporus that could be isolated was named Burkholderia rhizoxinica for its capability to produce rhizoxin [5].
A New Role for Endobacteria in Mycotoxin Research and Food Safety
The discovery of endobacteria as producers of “mycotoxins” was relevant in the areas of natural product research and microbiology, since similar symbionts in other antibiotic producing fungi might have been overlooked in the past [6]. Indeed, another dangerous “mycotoxin”, rhizonin (see Figure 1C), originally isolated from R. microsporus (CBS 112285) is produced by endobacteria as well [7]. These endobacteria are related to B. rhizoxinica, but represent another species, Burkholderia endofungorum [5]. Rhizonin is a cyclopeptide, which is highly toxic for mammals. Tested animals exhibited serious hepatic lesions and died from chronic failure of the liver [8]. The fact that the producing strain was found on ground nuts in Mozambique underlines the relevance of the Burkholderia–Rhizopus symbiosis for food safety and human health. Concerning food safety, it is even more distressing that another Burkholderia–Rhizopus association was isolated from a tempe/sufu starter culture in Vietnam [1]. Tempe and sufu are traditional soy preparations in Asia that are fermented with R. microsporus. It could be demonstrated that rhizoxin is indeed produced during sufu fermentation, thus revealing a potential threat to human health [9].
Endosymbionts of Rhizopus Were Detected in Human Pathogens but Are Not Essential for Zygomycoses
Rhizopus species including R. microsporus are regularly involved in zygomycoses (mucormycoses), disastrous fungal infections that affect immunocompromised patients. These diseases have high mortality rates [10] and are hard to treat by antifungal agents. Consequently, surgical debridement of infected tissue is often necessary [11]. After the discovery of B. rhizoxinica it was speculated whether toxin production by endosymbionts might enhance the virulence of fungal strains involved in human disease. The bacteria would then provide a promising target for the treatment of Rhizopus infections. Indeed, it has been shown that rhizoxin-producing strains are frequently involved in zygomycosis [11].
However, several lines of evidence suggest that rhizoxin production seems not to be essential for Rhizopus pathogenicity [11], [12]. First of all, clinical cases of zygomycosis are frequently caused by non-toxinogenic Rhizopus species [12]. Furthermore, when R. microsporus was cured from symbionts with antibiotics, it still retained its ability to infect mice under laboratory conditions [11]. Although these results show that endosymbionts are not the key players in zygomycosis, they might still raise the potential health threat caused by R. microsporus: it is well conceivable that endosymbionts that are released from fungal mycelium might cause sepsis or further complications. In particular, B. rhizoxinica and B. endofungorum could cause potential long-term damage by secretion of toxins into the human body. In fact, related Burkholderia strains have been isolated from clinical specimen [13]. Consequently, the existence of toxinogenic endosymbionts should be kept in mind when it comes to treatment of zygomycosis.
Investigation of Rhizoxin Biosynthesis Can Promote Antitumor Therapy
Despite all theses dangers emanating from toxin-producing Rhizopus/Burkholderia strains, rhizoxin could also assist in antitumor therapy: due to its ability to block mitosis via binding to β-tubulin, rhizoxin exhibits strong activity against tumor cell lines in vitro [14]. The substance has already tested in phase II clinical trials [15]. However, its in vivo activity was unsatisfactory, probably due to low activity and rapid elimination from plasma [15], and thus it would be desirable to produce more potent derivatives. Rhizoxin belongs to the family of macrolide antibiotics (as e.g., erythromycin) and is produced by a modular polyketide synthase. The enzymatic assembly line catalyzes repetitive condensation of activated acetate (acetyl-CoA and malonyl-CoA) units, in a similar way as in fatty acid biosynthesis [16]. In contrast to the latter, polyketide biosynthesis allows for variable degrees of chain processing, and the polyketide backbone is further modified by tailoring enzymes. Through genetic manipulation of the biosynthesis genes it is possible to engineer polyketide biosynthesis, and thus create new derivatives. Indeed, it was possible to identify the genes coding for rhizoxin biosynthesis [17] and to modify these genes in isolated symbionts. Thus, the function of some enzymes could be investigated in detail [18], [19]. Furthermore, it is promising that cultivation of B. rhizoxinica in pure culture could significantly increase the yield of rhizoxin production and lead to the isolation of new, significantly more active rhizoxin derivatives [20].
Endosymbionts of Rhizopus: Former Pathogens of Pathogens?
From a biologist's point of view, the Burkholderia–Rhizopus symbiosis has some intriguing aspects that go beyond zygomycosis and natural product research. For instance, the question was raised as to how the association between Rhizopus and Burkholderia has evolved and how it is maintained. Microscopic investigations with GFP-labeled endobacteria revealed that endosymbionts enter fungal spores to be “inherited” during vegetative reproduction [21]. Intriguingly, the reproduction of the host has been hijacked by the symbionts: the host is unable to sporulate when endosymbionts are removed [21]. Thus, they ensure their own propagation alongside the host lineage (vertical transmission, see Figure 1C). In addition, endobacteria are able to infect compatible host organisms in laboratory cultures [22]. Spread of symbionts by release and infection of another host is called horizontal transmission (see Figure 1C). Both vertical and horizontal transmission could be shown to be relevant during evolution of the Burkholderia–Rhizopus alliance: comparison of phylogenetic trees of host strains and the corresponding symbiont strains revealed that the topologies of these trees resemble each other [23]. This means that host fungi and symbionts have undergone “cospeciation”—symbiont lineages have been associated with their fungal host lineage over a long period of time. However, some host switching events could be detected, which must be due to horizontal transmission. These results suggest that endosymbionts have actually evolved from former parasites (pathogens) of Rhizopus. It looks like both partners benefit from their close relationship: while the fungus obtains powerful chemical weapons produced by B. rhizoxinica, the latter is supplied with nutrients and a safe niche. Any mutually beneficial symbiosis is called mutualism. However, the line between mutualism and parasitism is thin and shifts might occur during evolution. This parasitism–mutualism shift hypothesis is further supported by genome sequencing of B. rhizoxinica [24], [25]: while the genome retains only a reduced number of genes compared to free-living Burkholderia species, it encodes a large repertoire of typical virulence factors. It could be demonstrated that a type III secretion system (T3SS) [22] and the lipopolysaccharide O-antigen [26] are essential to maintain the symbiosis. Both factors are known pathogenicity factors of animal and plant pathogens. T3SSs are giant protein export machineries anchored in both membranes of Gram-negative bacteria (see Figure 2A). Usually, they accomplish export of effector proteins that manipulate cellular processes of the host [27]. Symbionts lacking these systems are unable to reinfect their host fungus and cannot illicit its sporulation like the wild type [22]. Similar results were obtained for mutants lacking the O-antigen—a long polysaccharide chain anchored in the outer membrane (see Figure 2B) [26]. It is plausible that the O-antigen is needed for protection against fungal defense mechanisms or recognition of the symbionts.
These results teach us that bacterial symbionts of fungi employ similar factors as plant parasites or human pathogens. These mechanisms seem to be universal host control tools that can be adapted to highly versatile hosts and cellular targets.
Zdroje
1. Partida-MartinezLPHertweckC 2005 Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature 437 884 888
2. SatoZNodaTMatsudaIIwasakiSKobayashiH 1983 Studies on rhizoxin, a phytotoxin produced by Rhizopus chinensis causing rice seedling blight. Annu Phytopathol Soc Japan 49 128
3. SchmittIL.P.P-MWinklerRVoigtKEinaxE 2008 Evolution of host resistance in a toxin-producing bacterial-fungal alliance. ISME J 2 632 641
4. MoebiusNHertweckC 2009 Fungal phytotoxins as mediators of virulence. Current Opinion in Plant Biology 12 390 398
5. Partida-MartinezLPGrothISchmittIRichterWRothM 2007 Burkholderia rhizoxinica sp. nov. and Burkholderia endofungorum sp. nov., bacterial endosymbionts of the plant-pathogenic fungus Rhizopus microsporus. Int J Syst Evol Microbiol 57 2583 2590
6. LacknerGPartida-MartinezLPHertweckC 2009 Endofungal bacteria as producers of mycotoxins. Trends Microbiol 17 570 576
7. Partida-MartinezLPde LoossCFIshidaKIshidaMRothM 2007 Rhizonin, the first mycotoxin isolated from the zygomycota, is not a fungal metabolite but is produced by bacterial endosymbionts. Appl Environ Microbiol 73 793 797
8. WilsonTRabieCJFinchamJESteynPSSchipperMA 1984 Toxicity of rhizonin A, isolated from Rhizopus microsporus, in laboratory animals. Food Chem Toxicol 22 275 281
9. RohmBScherlachKMobiusNPartida-MartinezLPHertweckC 2010 Toxin production by bacterial endosymbionts of a Rhizopus microsporus strain used for tempe/sufu processing. Int J Food Microbiol 136 368 371
10. AntoniadouA 2009 Outbreaks of zygomycosis in hospitals. Clin Microbiol Infect 15 Suppl 5 55 59
11. IbrahimASGebremariamTLiuMFChamilosGKontoyiannisDP 2008 Bacterial endosymbiosis is widely present among zygomycetes but does not contribute to the pathogenesis of mucormycosis. J Infect Dis 198 1083 1090
12. Partida-MartinezLPBandemerSRuchelRDannaouiEHertweckC 2008 Lack of evidence of endosymbiotic toxin-producing bacteria in clinical Rhizopus isolates. Mycoses 51 266 269
13. GeeJEGlassMBLacknerGHelselLODaneshvarM 2011 Characterization of Burkholderia rhizoxinica and B. endofungorum isolated from clinical specimens. PLoS ONE 6 e15731 doi:10.1371/journal.pone.0015731
14. TsuruoTOh-haraTLidaHTsukagoshiSSatoZ 1986 Rhizoxin, a macrocyclic lactone antibiotic, as a new antitumor agent against human and murine tumor cells and their vincristine-resistant sublines. Cancer Res 46 381 385
15. McLeodHLMurrayLSWandersJSetanoiansAGrahamMA 1996 Multicentre phase II pharmacological evaluation of rhizoxin. Eortc early clinical studies (ECSG)/pharmacology and molecular mechanisms (PAMM) groups. Br J Cancer 74 1944 1948
16. HertweckC 2009 The biosynthetic logic of polyketide diversity. Angew Chem Int Ed Engl 48 4688 4716
17. Partida-MartinezLPHertweckC 2007 A gene cluster encoding rhizoxin biosynthesis in “Burkholderia rhizoxina”, the bacterial endosymbiont of the fungus Rhizopus microsporus. Chembiochem 8 41 45
18. KusebauchBBuschBScherlachKRothMHertweckC 2009 Functionally distinct modules operate two consecutive alpha,beta→beta,gamma double-bond shifts in the rhizoxin polyketide assembly line. Angew Chem Int Ed Engl 49 1460 1464
19. KusebauchBBuschBScherlachKRothMHertweckC 2009 Polyketide-chain branching by an enzymatic Michael addition. Angew Chem Int Ed Engl 48 5001 5004
20. ScherlachKPartida-MartinezLPDahseH-MHertweckC 2006 Antimitotic rhizoxin derivatives from a cultured bacterial endosymbiont of the rice pathogenic fungus Rhizopus microsporus. J Am Chem Soc 128 11529 11536
21. Partida-MartinezLPMonajembashiSGreulichKOHertweckC 2007 Endosymbiont-dependent host reproduction maintains bacterial-fungal mutualism. Curr Biol 17 773 777
22. LacknerGMoebiusNHertweckC 2011 Endofungal bacterium controls its host by an hrp type III secretion system. ISME J 5 252 261
23. LacknerGMoebiusNScherlachKPartida-MartinezLPWinklerR 2009 Global distribution and evolution of a toxinogenic Burkholderia-Rhizopus symbiosis. Appl Environ Microbiol 75 2982 2986
24. LacknerGMoebiusNPartida-MartinezLPHertweckC 2011 Complete genome sequence of Burkholderia rhizoxinica, the endosymbiont of Rhizopus microsporus. J Bacteriol 193 783 784
25. LacknerGMoebiusNPartida-MartinezLPBolandSHertweckC 2011 Evolution of an endofungal lifestyle: deductions from the Burkholderia rhizoxinica genome. BMC Genomics In press
26. LeoneMRLacknerGSilipoALanzettaRMolinaroA 2010 An unusual galactofuranose lipopolysaccharide warrants intracellular survival of toxin-producing bacteria in their fungal host. Angew Chem Int Ed Engl 49 7476 7480
27. CornelisGR 2006 The type III secretion injectisome. Nat Rev Microbiol 4 811 825
Štítky
Hygiena a epidemiologie Infekční lékařství LaboratořČlánek vyšel v časopise
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