Bioactivities by a crude extract from the Greenlandic Pseudomonas sp. In5 involves the nonribosomal peptides, nunamycin and nunapeptin

Bacterial and fungal strains

The Pseudomonas sp. strains, In5 (wild type) and Gr1 (nunamycin knock-out mutant described in Michelsen et al. (2015)), were routinely grown in liquid or on solid (1.5% agar) Luria-Bertani medium at 20 °C. For strain Gr1, the medium was supplemented with kanamycin (50 µg/ml). The basidiomycete R. solani Ag3 (provided by LKF Vandel, Denmark) and the oomycete P. aphanidermatum (provided by the Section for Microbial Ecology and Biotechnology, Department of Plant and Environmental Sciences, Faculty of Sciences, University of Copenhagen) were used as target strains in the in vitro antimicrobial assay. R. solani and P. aphanidermatum were maintained on standard potato dextrose agar (PDA) medium at 20 °C.

Cancer cell lines

The cancer cell lines, Jurkat T-cells (T-cell leukemia), Granta cells (mantle cell lymphoma), and the melanoma cell line FM-86 were kindly provided by Dr. Søren Skov, Section of Experimental Animal Models, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. The cells were grown in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), 2 mmol/L glutamine (Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 IU)/streptomycin (100 µg/ml) (Sigma-Aldrich, St. Louis, MO, USA). All cells were incubated at 37 °C and 5% CO₂ and passaged every second and third day.

Preparation of crude extracts

Plates with 1/5 PDA medium were inoculated with Pseudomonas sp. In5 or the nunamycin mutant, Gr1. After incubation for 48 h at room temperature, the agar was sliced into small pieces and extracted with acidified deionized water (pH 2) for 1 h at 20 °C and 225 rpm in a 2.8-liter Erlenmeyer flask followed by addition of n-butanol and incubation for 18 h at 20 °C as previously described (Michelsen et al., 2015). The n-butanol extract was separated from agar by cheesecloth filtration and subsequently from cell debris by centrifugation (6,440 × g, 10 min). n-butanol was removed with a rotary evaporator (Buechi R-200).

The presence of the two NRPs, nunamycin (m/z 1,138) and nunapeptin (i.e., the nunapeptin family of m/z 2,023–2,075), in the extracts was evaluated by matrix-assisted laser desorption/ionization—time-of-flight mass spectrometry (MALDI-TOF MS) analysis as described by Liu et al. (2010). n-butanol was removed with a rotary evaporator (Maxi Dry Lyo; Holm & Halby, Brøndby, Denmark) and the dry extracts were stored at −20 °C until further use.

Purification of nunamycin and nunapeptin

Nunamycin and nunapeptin were purified from the Pseudomonas sp. In5 derived extract by size-exclusion chromatography using a Sephadex™ LH-20 column followed by high performance liquid chromatography as previously described (Michelsen et al., 2015). In brief, the Pseudomonas sp. In5 derived extract was dissolved in 1 mL methanol and eluted through a 0.7 × 30 cm column containing Sephadex LH20 resin equilibrated in methanol. The first 7.5 mL of methanol was discarded as waste and the next 10 mL were collected and concentrated. The resulting brown residue was taken up in 200 µL of methanol and purified by reverse phase HPLC on a Vydac C18 column with 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B). The gradient started with a 5 min hold in 10% B followed by a 45 min linear ramp to 90% B. Nunamycin eluted at 27.5 min and nunapeptin eluted as a group of peaks between 34 and 37 min. The presence of each compound was confirmed by MALDI-TOF MS. Respective fractions from HPLC were combined and lyophilized to yield 1 mg of each compound. The purified compounds were stored at −20 °C until further use.

In vitro antimicrobial assay

For analyzing the antimicrobial activity of extracts or purified NRPs, 10 µg solid/ml of the Pseudomonas sp. In5 or Gr1 derived extracts or 10 µg of the purified nunamycin and nunapeptin compounds was added to a sterile filter disc in the center of a 1/5 strength PDA plate. Four 6 mm plugs of target fungal or oomycete mycelia were placed 2 cm from the center of the plate. 23% EtOH in Milli-Q H₂O was used as negative control. Plates were incubated at 20 °C and antimicrobial activity was determined as percent inhibition of radial growth (PIRG) as described by Whipps (1987) after 3 days of incubation. Tukey’s HSD test was used in conjunction with ANOVA in order to evaluate which means were significantly different from one another. P-values are considered significant at p < 0.05. The tests were carried out with R version 3.0.3 (R Development Core Team, 2009). When applicable, the data are presented as mean ± SEM.

Microscopy of R. solani Ag3 fungal hyphae

A thin layer of 1/10 strength PDA was spread on top of a 76 × 26 mm sterile object glass (Menzel-Gläser, Braunschweig, Germany) in sterile Petri dishes. Two µg solid/ml of the Pseudomonas sp. In5 derived extract was added 2 cm away from a fungal mycelia plug placed in the center of the slide and incubated at 28 °C for 48 h. Slides containing fungal hyphae treated with four µl of 23% EtOH in Milli-Q H₂O served as control treatments. The experiment was carried out in triplicates. Following incubation, the slides were cut out of the Petri dishes and placed in a Zeiss Axioplan 2 Imaging fluorescence microscope (Brock & Michelsen A/S, Birkerød, Denmark). Preparations were visualized by 40× magnification and recorded with the AxioCam MRc program. In order to perform vital staining of mitochondria in R. solani Ag3 fungal hyphae with the fluorescent dye, DiOC₇(3) (3, 3′-Diheptyloxacarbocyanine iodide) (Duckett & Read, 1991), 1 µg/ml of DiOC₇(3) was added to the samples and incubated for 5 min at 30 °C before visualization by fluorescence microscopy (Zeiss Axioplan 2 Imaging fluorescence microscope (Brock & Michelsen A/S, Birkerød, Denmark)).

Isolation and stimulation of CD4 T-cells

CD4 T-cells were isolated from peripheral blood mononuclear cells obtained from healthy blood donors (The State Hospital, Copenhagen, Denmark) using CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD4 T-cells were left untouched (naïve) or activated with one Dynabead CD3/CD28 T-cell expander (Invitrogen, Carlsbad, CA, USA) per cell for three days in RPMI-1640 medium, supplemented with 5% human serum (Lonza, Basel Switzerland), 2 mmol/L glutamine, 2 mmol/L penicillin/streptomycin.

Detection of Annexin V and PI staining by flow cytometry

For the bacterial extracts: the cancer cells (i.e., Jurkat T-cells, Granta cells, and the melanoma cells) as well as naïve CD4 T-cells, or activated CD4 T-cells were incubated with 23% EtOH in Milli-Q H₂O (control) at a final dilution of 1:200, the anticancer agent, FR901228 (20 ng/ml, obtained from the National Cancer Institute, Bethesda, MD), Pseudomonas sp. In5 (1.5 or 2.5 µg solid/ml) or Pseudomonas sp. Gr1 (2.5 µg solid/ml) derived crude extracts for 18 h at 37 °C and 5% CO₂. For the purified nunamycin and nunapeptin compounds: Jurkat T-cells were incubated with DMSO (control) or 10 µg/ml compounds either alone or in combination for 24 h at 37 °C and 5% CO₂. Cells were stained with FITC-labeled Annexin V (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufactures protocol. PI staining of cells was done by adding 1 µg/ml propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA) to the samples followed by incubation at room temperature for 1 min. Data acquisition was done on a BD LSR II or Accuri C6. Data was analyzed using FlowJo software. All group comparisons were performed using a Students t-test. P-values are considered significant at p < 0.05. When applicable, the data are presented as mean ± SEM.

A crude extract derived from Pseudomonas sp. In5 possesses strong antimicrobial activity by inducing a mitochondrial stress-response

We have previously shown that the Greenlandic biocontrol bacterium Pseudomonas sp. In5 possesses strong antimicrobial activity against various filamentous fungi and oomycetes by using in vitro co-culture experiments with either bacterium, extract or the purified NRPs nunamycin and nunapeptin (Michelsen & Stougaard, 2011; Michelsen & Stougaard, 2012; Michelsen et al., 2015). To expand our knowledge about the pharmaceutical potential of the bioactive metabolites derived from Pseudomonas sp. In5 we prepared a crude extract by organic solvent extraction and tested the extract for antimicrobial activity using an in vitro agar diffusion assay. The crude extract exhibited strong antimicrobial activities with 66% and 34% mean inhibition of mycelial radial growth of R. solani Ag3 and P. aphanidermatum, respectively (Fig. 2A), which was consistent to the activities previously observed with Pseudomonas sp. In5 (Michelsen & Stougaard, 2011; Michelsen & Stougaard, 2012; Michelsen et al., 2015). A more detailed microscopic analysis of the in vitro effect of the crude extract against R. solani hyphae was employed by vital staining of mitochondria with the fluorescent dye DiOC₇(3) (Duckett & Read, 1991). Notably, treatment with the Pseudomonas sp. In5 derived crude extract caused changes in growth and cell morphology of the R. solani hyphae, including intense vacuolization in the hyphae tips and increased branching and swelling of the hyphae (Fig. 2B). In contrast, the control treatment did not show any abnormal morphology of the R. solani hyphae (Fig. 2C). Furthermore, intracellular activities of mitochondria in the hyphae treated with the crude extract were reduced compared to the control treatment and the mitochondria became randomly organized with an altered morphology from longitudinal shaped (i.e., control treatment) to small and circular (i.e., In5 extract treatment) (Figs. 2C and 2B, respectively). The crude extract was analyzed by MALDI-TOF MS and confirmed the presence of the two antimicrobial NRPs, nunamycin (m/z 1,138) and nunapeptin (i.e., the nunapeptin family of m/z 2,023–2,075) (Fig. 3A).

We additionally prepared a crude extract from the mutant strain Pseudomonas sp. Gr1, which is deficient in producing the NRP, nunamycin (Michelsen et al., 2015). As confirmed by MALDI-TOF MS analysis, nunapeptin, but not nunamycin, was detected in the Pseudomonas sp. Gr1 derived crude extract (Fig. 3B). When tested for antimicrobial activity this crude extract showed decreased antimicrobial activity against R. solani with a mean value of 27% inhibition of mycelial radial growth (Fig. 2A),  whereas the activity against P. aphanidermatum was comparable to that observed with the Pseudomonas sp. In5 derived crude extract (i.e., mean value of 38% and 34% inhibition, respectively) (Fig. 2A). These results are consistent with our previous results obtained from co-culture experiments with the Pseudomonas sp. Gr1 strain (Michelsen et al., 2015) and suggest that the strong antimicrobial effect against R. solani by the Pseudomonas sp. In5 derived crude extract involves the activity of nunamycin as well as nunapeptin and/or other metabolites present in the extract, whereas the antimicrobial effect against P. aphanidermatum is not dependent on nunamycin.

The antimicrobial crude extract derived from Pseudomonas sp. In5 increases Annexin V staining of cancer cells, but not of naïve or activated T-cells

The antimicrobial crude extract derived from Pseudomonas sp. In5 was further analyzed for anticancer activity by testing the ability to induce Annexin V staining in three different cancer cell lines, i.e., the Jurkat T-cell line (T-cell leukemia), the Granta cell line (mantle cell lymphoma), and a melanoma cell line. In cells undergoing apoptosis, loss of cell membrane asymmetry is one of the earliest morphological features, which causes the membrane phospholipid phosphatidylserine (PS) to translocate from the inner to the outer leaflet of the cell membrane and thereby exposes it to the extracellular environment. Annexin V is a phospholipid-binding protein that binds PS with high affinity and can therefore be used to detect cells undergoing apoptosis (Vermes et al., 1995). Interestingly, a significant increase of Annexin V staining of all three cancer cell lines tested was observed with the In5 derived crude extract compared to the control treatment (Figs. 4A–4C). A mean value of 40.1% (melanoma cells), 80.7% (Granta cells), or 75.6% (Jurkat T-cells) Annexin V positive cells was observed at a concentration of 1.5 µg solid/ml (Figs. 4A–4C, respectively), which was further increased at a concentration of 2.5 µg solid/ml (Figs. 4A–4C). Numerous chemotherapeutic drugs target proliferating cells and hence can affect the viability of cancer cells as well as normal healthy cells (Lake & Robinson, 2005). We therefore extended our analysis of the anticancer potential and examined the effect of the Pseudomonas sp. In5 derived crude extract on non-proliferating naïve and proliferating CD3/CD28 stimulated CD4 T-cells. Strikingly, the crude extract did not induce Annexin V staining of neither naïve nor activated CD4 T-cells (Fig. 5). In contrast, treatment with the known anticancer agent FR901228 (VanderMolen et al., 2011) induced Annexin V staining of all the cancer cells tested (Figs. 4A–4C) as well as activated CD4 T-cells, but not in naïve CD4 T-cells (Fig. 5). Together these results suggest that the Pseudomonas sp. In5 derived crude extract contain one or more metabolites with anticancer potential.

Since a robust induction of Annexin V staining was observed for all three cancer cell lines tested, the Jurkat T-cell line was selected for further investigations of anticancer activity by the Pseudomonas sp. In5 crude extract. We included PI staining in our flow cytometry analysis of Jurkat T-cells treated with 2.5 µg solid/ml of the Pseudomonas sp. In5 crude extract and found that almost all cells were double positive for Annexin V and PI staining (i.e., Annexin V⁺PI⁺) (Fig. 4D). Necrotic cells or late apoptotic cells undergoing secondary necrosis (Silva, 2010) are quickly stained by short incubations with propidium iodide (PI) due to extensive loss of cell membrane integrity, whereas cells undergoing apoptosis show a much lower uptake of PI than necrotic cells. By using PI staining together with Annexin V staining it is therefore possible to distinguish living (i.e., Annexin V⁻ PI⁻), early apoptotic (i.e., Annexin V⁺ PI⁻), late apoptotic (i.e., Annexin V⁺ PI^dim) and late apoptotic/necrotic (i.e., Annexin V⁺ PI^bright) cell populations from each other (Overbeeke et al., 1998; Vitale et al., 1993). When taking a closer look at the Annexin V⁺ PI⁺ Jurkat T-cells in the upper right quarter, an Annexin V⁺ PI^dim (grey arrow) and an Annexin V⁺ PI^bright (black arrow) cell population could be detected, indicating the occurrence of a late apoptotic and a late apoptotic/necrotic cell population, respectively (Fig. 4D).

In contrast to the crude extract derived from Pseudomonas sp. In5, treatment of the Jurkat T-cells with 2.5 µg solid/ml of the Pseudomonas sp. Gr1 derived crude extract showed a much weaker induction of Annexin V staining with a mean value of 15.2% Annexin V positive cells  compared to the 95.0% induction by the Pseudomonas sp. In5 derived crude extract (Figs. 4C and 4D). These results suggest that the anticancer effect of the Pseudomonas sp. In5 derived crude extract involves the activity of nunamycin.

Nunamycin and nunapeptin purified from the Pseudomonas sp. In5 derived crude extract display distinct antimicrobial activities and a synergistic anticancer activity

To test the antimicrobial and anticancer activities of the two NRPs, nunamycin (m/z 1,138) and nunapeptin (m/z 2,023–2,075), we purified the NRPs from the Pseudomonas sp. In5 derived crude extract by HPLC. We observed distinct antimicrobial activities against R. solani and P. aphanidermatum between nunamycin and nunapeptin, which were comparable to our previous observations (Fig. 2A and Michelsen et al., 2015). Nunamycin potently inhibited mycelial radial growth of R. solani with a mean value of 52% inhibition, whereas no growth inhibition of P. aphanidermatum was observed (Fig. 2A). In contrast, nunapeptin strongly inhibited the growth of P. aphanidermatum with a mean value of 48% inhibition, but did not affect the growth of R. solani (Fig. 2A).

Surprisingly, we found that treatment with nunamycin alone was not able to induce apoptosis in the Jurkat T-cell line (Fig. 6A), despite the strong indications from our investigations with the crude extracts. In addition, no anticancer activity was observed by treatment with nunapeptin alone (Fig. 6A). However, when the two NRPs were combined, a significant induction of Annexin V staining in the cancer cells was observed with a mean value of 25.5% Annexin V positive cells relative to the control treatment (Fig. 6A), which included an increase in both early (i.e., 22.8% Annexin V⁺, PI⁻) and late (i.e., 3.9% Annexin V⁺, PI^dim/bright) apoptotic cells (Fig. 6B). Thus, despite the individual potent antimicrobial activities observed with nunamycin and nunapeptin, treatment with the two compounds simultaneously was essential to obtain robust anticancer activity. Accordingly, a synergistic anticancer effect between nunamycin and nunapeptin would explain the much lower induction of Annexin V staining of the Jurkat T-cells observed by the crude extract derived from the nunamycin deficient strain Pseudomonas sp. Gr1 (Figs. 4C and 4D).