Extracellular Streptomyces lividans vesicles: composition, biogenesis and antimicrobial activity

Abstract

We selected Streptomyces lividans to elucidate firstly the biogenesis and antimicrobial activities of extracellular vesicles that a filamentous and highly differentiated Gram-positive bacterium produces. Vesicle types range in diameter from 110 to 230 nm and 20 to 60 nm, respectively; they assemble to clusters, and contain lipids and phospholipids allowing their in situ imaging by specific fluorescent dyes. The presence of the identified secondary metabolite undecylprodigiosin provokes red fluorescence of a portion of the heterogeneous vesicle populations facilitating in vivo monitoring. Protuberances containing vesicles generate at tips, and alongside of substrate hyphae, and enumerate during late vegetative growth to droplet-like exudates. Owing to in situ imaging in the presence and absence of a green fluorescent vancomycin derivative, we conclude that protuberances comprising vesicles arise at sites with enhanced levels of peptidoglycan subunits [pentapeptide of lipid II (C55)-linked disaccharides], and reduced levels of polymerized and cross-linked peptidoglycan within hyphae. These sites correlate with enhanced levels of anionic phospholipids and lipids. Vesicles provoke pronounced damages of Aspergillus proliferans, Verticillium dahliae and induced clumping and distortion of Escherichia coli. These harmful effects are likely attributable to the action of the identified vesicular compounds including different enzyme types, components of signal transduction cascades and undecylprodigiosin. Based on our pioneering findings, we highlight novel clues with environmental implications and application potential.

Fig 1

Macroscopic and microscopic inspection of S. lividans exudates.A. Streptomyces lividans was grown on agar-containing medium for 7 days. Using a macro lens, a photo was taken from the top of the S. lividans lawn containing red droplets. Bar: 5 mm.B–D. Aliquots from the droplets (see, A) were inspected microscopically by phase contrast under visual light, and for the presence of endogenous red fluorescence. Then, pictures were merged (B–D). Regions from undiluted (B, C) and diluted (D) samples are presented. Bars: 5 μm.E, F. Aliquots of the sample (corresponding to Fig. 1C) were prepared on grids (see, Experimental procedures), and inspected by TEM at two different magnifications (E and F). Bars: 200 nm.

Fig 2

Analyses of droplet samples following staining by NAO and Nile red, or by transmission electron microscopy (TEM).A-CoA″. A sample of a droplet (see Fig. 1A) was treated with the dye NAO (10-N-Nonyl Acridine Orange), and inspected microscopically under visual light (A), for the presence of green fluorescence (A′), or analysed after merging (A″). The control was done without staining, and is presented as merged picture (CoA″). Bars 2.5 μm.B-CoB″. A sample (corresponding to Fig. 1D) was treated with Nile red, and it was analysed microscopically under visual light (B), for the presence of red fluorescence (B′), and after merging (B″). The endogenous undecylprodigiosin derived fluorescence was considerably fainter than that one obtained after Nile red staining, and hence, undetectable in the unstained reference (control CoB″) under the set photographical conditions that were used to detect specifically Nil red derived fluorescence. Bars 2.5 μm.

Fig 3

Formation of vesicle-like structures during development of S. lividans.A–F″. Spores were seeded onto agar plates, and incubated as described under Experimental procedures. Samples were inspected by phase contrast after 2.7 (A), after 4–5 (B–E) and after 6 (F) days within areas containing a few (A, B), moderate (C, D) or high numbers (E, F) of substrate hyphae. In addition, the samples were scored for endogenously derived red fluorescence (A′–F′), and merged with A–F to result in A″–F″. Different types of arrows mark particles that have a red fluorescence (), or none (). Bars: 2.5 μm.G. Aerial hyphae (←) lack intensive red fluorescence (G′ and G″). Bars: 2.5 μm.

Fig 4

Detection of nascent vesicles using lipid-specific dyes. Streptomyces lividans was grown (see Fig. 3) for 4 days.A, B′. Two samples of hyphae were treated with NAO, and inspected microscopically under visual light (A, and B) as well as for the presence of green fluorescence (A′ and B′). Pronounced bulged structures are marked ().C,C‴. Hyphae were treated with NAO and Nile red, and inspected microscopically under visual light (C), for the presence of NAO derived green fluorescence (C′), for Nile red derived strong red fluorescence (C″), and following merging (C‴) of both types of fluorescence. Pronounced protuberance-structures are marked ().Co–Co*. Untreated control hyphae (Co) lacked green fluorescence (Co′). If analysed under the same gain value that corresponded to C″, the hyphae were not red fluorescent (Co″) showing that the detection of Nile-red dependent staining (C″) was specific. However, if four times higher gain value were applied, some endogenous red fluorescence (Co*) was detectable. Bars: 5 μm.

Fig 5

In situ effect of vancomycin.A–C2. Spores were seeded on agar plates as described under Experimental procedures, and incubated for different time periods: 20 h (A1-a2″), 42 h (B1-b2″), and 5 days (C1-c2″). Samples were treated with Van-FL, analysed under visual light by phase contrast, and scored for red fluorescence (due to endogenous undecylprodigiosin), merged (A1, A2, B1, B2, C1 and C2) and presented as magnified subareas (a1, a2, b1, b2, c1 and c2). In addition, the Van-FL-derived green fluorescence of the same samples was recorded, and merged with their red florescence (endogenous undecylprodigiosin, see above); this procedure resulted in the pictures A1′, A2′, B1′, B2′, C1′ and C2′ with corresponding magnified regions (a1′, a2′, b1′, b2, c1′, and c2′). Additionally, the magnified areas showing only Van-FL derived fluorescence are shown (a1″, a1″, b1″, b2″, c1″ and c2″). Positions containing droplet-like structures with a fluorescence () or none () are marked. Fluorescence sites (*) that were additionally enlarged are marked. Bars: 2.5 μm.CoA-CoC′. Control (Co) samples corresponding to A, B, and C, which were not treated with Van-FL were analysed under visual light by phase contrast (CoA, CoB and CoC), and inspected for red and green fluorescence light. The resulting pictures were merged. (CoA′, CoB′ and CoC′). Bars: 2.5 μm.

Fig 6

Applications of vesicle-containing droplets to E. coli and B. subtilis.A–D. An aliquot (500 μl) of logarithmically grown E. coli was supplemented with a sample (20 μl) of vesicle-containing droplets (see Fig. 1A, and C), incubated at 30°C, and analysed microscopically after 2 min [A and its framed enlarged (a), area], and respectively after 40 min (B, and enlarged area, b). Controls were done (C, c, and D, d) without the addition of droplet. Bars 5 μm.E, F. A portion of logarithmically grown B. subtilis was incubated at 30°C for 2 h with a sample of vesicle-containing droplets (E, and e), or without them as a control (F, and f), and presented in two different magnifications. Bars 5 μm:

Fig 7

Effect of vesicle-containing droplets on A. proliferans.A–C. Conidia of A. proliferans were incubated 4 h in liquid medium. Then, an aliquot (20 μl) of vesicles containing droplets (see Fig. A and C) was added to a culture aliquot (500 μl), and incubation continued for additional 3 h incubated for 2 h at 30°C. (A) or for 8 h (B, C). All samples were inspected microscopically by phase contrast as well as for undecylprodigiosin-derived red florescence, and are shown in a merged fashion. Vesicle-like particles that are red fluorescent () are marked. Regions from the hyphae (B) with high red intracellular fluorescence (*1, or that had developed blown up (*2), or strongly deformed (*3) areas are presented in an enlarged fashion (b¹, b² and b³). Bars 10 μm.D–F. Controls were without the addition of droplets, and inspected as outlined above. Bars 10 μm.

Fig 8

Effect of vesicle-containing droplets on V. dahliae.A,B. An aliquot (20 μl) of vesicle-containing droplets (see Fig. 1A and C) was added to an aliquot (500 μl) of a V. dahliae culture, and incubated for 2 h at 30°C. Samples were inspected microscopically by phase contrast, and for the presence or absence of undecylprodigiosin-derived red florescence. Subsequently, pictures were merged. Selected areas (*) from A, and B were enlarged, and are presented in a and b. Vesicles adhering to fungal hyphae had either a red () or no () fluorescence. Bars 5 μm.C–E. Pre-grown fungal hyphae were incubated for 20 h with vesicles containing droplets, and inspected as described above. An area (*) of C is presented additionally in an enlarged fashion in c.F–G. Controls correspond to cultures C and E without the addition of droplets, and these were inspected as outlined above. Bars 5 μm.