Active Invasion of Bacteria Into Living Fungal Cells

1 Subject category: Microbe-microbe and microbe-host interactions 2 3 Active Invasion of Bacteria into Living Fungal Cells 4 5 Nadine Moebius1, Zerrin Üzüm1, Jan Dijksterhuis2, Gerald Lackner1 and Christian Hertweck1,3* 6 7 Affiliations: 8 1Leibniz Institute for Natural Product Research and Infection Biology (HKI), Department of 9 Biomolecular Chemistry, Beutenbergstr.11a, 07745 Jena, Germany. 10 2Applied and Industrial Mycology, CBS/Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT 11 Utrecht, the Netherlands. 12 3Friedrich Schiller University, Jena, Germany. 13 *Correspondence to: [email protected] 14 15 Running Title: Bacterial Invasion into Fungal Cells 16 17 Abstract: The rice seedling blight fungus Rhizopus microsporus and its endosymbiont 18 Burkholderia rhizoxinica form an unusual, highly specific alliance to produce the highly potent 19 antimitotic phytotoxin rhizoxin. Yet, it has remained a riddle how bacteria invade into the fungal 20 cells. Genome mining for potential symbiosis factors and functional analyses revealed that a type 21 2 secretion system (T2SS) of the bacterial endosymbiont is required for the formation of the 22 endosymbiosis. Comparative proteome analyses show that the T2SS releases chitinolytic 23 enzymes (chitinase, chitosanase) and chitin-binding proteins. The genes responsible for 24 chitinolytic proteins and T2SS components are highly expressed during infection. Through 25 targeted gene knock-outs, sporulation assays and microscopic investigations we found that 26 chitinase is essential for bacteria to enter hyphae. Unprecedented snapshots of the traceless 27 bacterial intrusion were obtained using cryo-electron microscopy. Beyond unveiling the pivotal 28 role of chitinolytic enzymes in the active invasion of a fungus by bacteria, these findings grant 29 unprecedented insight into the fungal cell wall penetration and symbiosis formation. 30 31 Keywords: Symbiosis, Chitinase, Rhizopus, Burkholderia, Type 2 secretion system 32 33 One Sentence Summary: Bacterial invade into living fungal cells using secreted chitinolytic 34 enzymes that allow for a traceless entry, as shown by microscopic snapshots. 35 36 2 37 Introduction 38 Interactions between bacteria and fungi are widespread in nature and play pivotal roles in 39 ecological and medicinal processes (Frey-Klett et al 2011). Moreover, fungal-bacterial 40 associations are widely used for the preservation of the environment (e.g. mycorrhizae in 41 reforestation), agriculture (e.g. food processing), and biotechnology (e.g. pharmaceutical 42 research) (Scherlach et al 2013). Beyond the most commonly observed microbial cell-cell 43 interactions, there is a growing number of known endosymbioses where bacteria dwell within 44 fungal hyphae (Bonfante and Anca 2009, Frey-Klett et al 2011, Kobayashi and Crouch 2009, 45 Lackner et al 2009b). Symbioses with endofungal bacteria are often overlooked, yet they may 46 have a profound effect on the host's lifestyle. Bacterial endosymbionts of AM-fungi, for 47 example, might be implicated in the vitamin B12 provision for the fungus (Ghignone et al 2012). 48 Endobacteria, isolated from the mycorrhiza fungus Rhizobium radiobacter, exhibit the same 49 growth promoting effects and induce systemic resistance to plant pathogenic fungi in the same 50 way that the fungus harboring the endobacteria does. Thus, it was proposed that the beneficial 51 effects for the plant result directly from the presence of bacteria (Sharma et al 2008). The rice 52 seedling blight fungus, Rhizopus microsporus, and its endosymbiont bacterium, Burkholderia 53 rhizoxinica represent a particularly noteworthy example of a bacterial-fungal endosymbiosis 54 (Lackner and Hertweck 2011, Partida-Martinez and Hertweck 2005). The fungus harbors 55 endosymbionts of the genus Burkholderia, which reside within the fungal cytosol, as shown by 56 confocal laser scanning microscopy, transmission electron microscopy (EM) and freeze–fracture 57 EM (Partida-Martinez et al 2007a, 2007b, 2007c). The bacteria are harnessed by the fungus as 58 producers of highly potent antimitotic macrolides (Scherlach et al 2006), which are then further 59 processed by the host into the phytotoxin rhizoxin (Scherlach et al 2012). The toxin represents 3 60 the causative agent of rice seedling blight, which weakens or kills the rice plants (Lackner et al 61 2009b). Both the saprotrophic fungus and the endofungal bacteria benefit from the nutrients 62 released, and R. microsporus provides a protective shelter for the bacterial partner. The 63 Rhizopus-Burkholderia association also stands out as it employs an elegant mechanism that 64 allows the persistence and spreading of the symbiosis through spores containing the 65 endosymbionts (Partida-Martinez et al 2007b) (Fig. 1). Yet it is unknown how the vegetative 66 reproduction of the fungus has become totally dependent upon the presence of the endobacteria 67 (Partida-Martinez et al 2007b). Insights into the genome of B. rhizoxinica and mutational studies 68 have unveiled several symbiosis factors (Lackner et al 2011a, Lackner G et al 2011b, Leone et al 69 2010). 70 A plausible scenario for the evolution of the symbiosis is a shift from antibiosis or 71 antagonism to mutualism. The rhizoxin complex secreted by the bacteria arrests mitosis in 72 almost all eukaryotic cells. Yet, Rhizopus, amongst other zygomycetes, has gained resistance to 73 this toxin due to a mutation at the β-tubulin binding site (Schmitt et al 2008). Furthermore, 74 phylogenetic analyses point to host switching events during evolution (Lackner et al 2009a), 75 which is also supported by the engagement of an hrp locus of B. rhizoxinica (Lackner et al 76 2011a). In addition to this, the LPS layer of the B. rhizoxinica is known to be unique to its niche, 77 due to high resemblance to fungal sugar content (Leone et al 2010). Although there is ample 78 knowledge on the persistence of the symbiosis, it has remained fully enigmatic how the bacteria 79 enter the fungal cells. Interestingly, there is no sign of endo-/phagocytosis, which rules out a 80 major avenue of bacterial colonization (Partida-Martinez and Hertweck 2005, Partida-Martinez 81 et al 2007b, Partida-Martinez et al 2007c). 4 82 Bacterial invasion of eukaryotic cells is a major area of research in infection biology, and 83 a large body of knowledge has been gathered on the pathogen's strategies to invade host cells 84 (Cossart and Sansonetti 2004). In addition to induced phagocytosis, a number of enzymes have 85 been described that act locally to damage host cells and to facilitate the entry of the pathogen 86 into the tissue (Harrison 1999, King et al 2003). Yet, this knowledge is limited to the invasion of 87 human, animal and plant cells. It has been reported that some bacteria employ extracellular 88 enzymes for mycophagy (Leveau and Preston 2008). However, despite a growing number of 89 described fungal endobacteria (Frey-Klett et al 2011, Lackner et al 2009b), there is a striking 90 lack of knowledge about the avenues and active mechanisms that permit fusion with or entry into 91 fungal hyphae, where the fungus is left intact to serve as a host for the endobacteria. Here we 92 report the genomics- and proteomics-driven discovery of a new bacterial invasion process that 93 involves the secretion of chitinolytic enzymes. Furthermore, we present the first electron 94 microscopic snapshots of the actual infection process. 95 96 97 5 98 Results 99 A type 2 secretion system (T2SS) of the Burkholderia endosymbiont is essential for the 100 formation of the endosymbiosis 101 Both pathogens (or antagonists) and mutualists often employ the same mechanisms 102 during the infection process (Dale and Moran 2006). Thus, we mined the gene repertoire of B. 103 rhizoxinica (Lackner G 2011) for potential molecular infection mechanisms known from 104 pathogenic bacteria. A type 2 secretion system (T2SS), also called general secretion pathway 105 (gsp), encoded by a 12 kb gene cluster on the B. rhizoxinica chromosome seemed to be a 106 promising candidate to enable the bacterium to enter the host. T2SS are typically involved in the 107 secretion of various toxins and lytic enzymes (Cianciotto 2005, Korotkov et al 2012) and the 108 overall organization of T2SS gene clusters is well conserved between related species (Fig. 2). 109 We generated targeted deletion mutants to investigate the role of the T2SS in the infection 110 process. Specifically, we selected gspC and gspD, since their gene products are essential proteins 111 of the type 2 secretion machinery in related bacteria (DeShazer et al 1999, Korotkov et al 2011). 112 The outer membrane pore is formed presumably by the multimeric secretin GspD, and GspC 113 appears to link the inner and outer membranes by providing the contact to GspD via a homology 114 region (Korotkov et al 2011). Although it is notoriously difficult to genetically modify the 115 symbiotic bacteria, we succeeded in generating ΔgspC and ΔgspD mutants using a double- 116 crossover strategy. 117 To begin, we addressed the proteolytic potential of B. rhizoxinica wt and the T2SS 118 defective mutants to evaluate the effect of the knock-outs (Fig. 3, Supp. Fig 1). Using a skim 119 milk plate assay we detected strong proteolytic activity in the wt supernatant, while the T2SS 120 mutants showed no activity (Fig. 5, Supp. Fig 1). The ability of the isolated endobacteria to re- 6 121 infect the fungus and to control fungal sporulation was examined using a sporulation bioassay. 122 The appearance of mature sporangia that form sporangiospores is seen as an indication of a 123 successful establishment of the symbiosis. In co-cultures of wild-type (wt) B. rhizoxinica and the 124 cured fungal host, sporulation is visible after 2-3 days. In contrast, there was absolutely no 125 visible spore formation upon co-cultivation with B. rhizoxinica ΔgspD::Kanr or ΔgspC::Kanr 126 (Fig. 3). Furthermore, fluorescence microscopy proved to be most helpful to distinguish between 127 mutants defective in colonization or induction of fungal sporulation.

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