s2fieldsofstudy: Biology Medicine

text:
Non-thermal plasma induces immunogenic cell death in vivo in murine CT26 colorectal tumors

ABSTRACT Immunogenic cell death is characterized by the emission of danger signals that facilitate activation of an adaptive immune response against dead-cell antigens. In the case of cancer therapy, tumor cells undergoing immunogenic death promote cancer-specific immunity. Identification, characterization, and optimization of stimuli that induce immunogenic cancer cell death has tremendous potential to improve the outcomes of cancer therapy. In this study, we show that non-thermal, atmospheric pressure plasma can be operated to induce immunogenic cell death in an animal model of colorectal cancer. In vitro, plasma treatment of CT26 colorectal cancer cells induced the release of classic danger signals. Treated cells were used to create a whole-cell vaccine which elicited protective immunity in the CT26 tumor mouse model. Moreover, plasma treatment of subcutaneous tumors elicited emission of danger signals and recruitment of antigen presenting cells into tumors. An increase in T cell responses targeting the colorectal cancer-specific antigen guanylyl cyclase C (GUCY2C) were also observed. This study provides the first evidence that non-thermal plasma is a bone fide inducer of immunogenic cell death and highlights its potential for clinical translation for cancer immunotherapy.

Introduction
Cancer treatment strategies in the past have focused on reducing tumor burden through delivery of cytotoxic agents. These methods often do not rely on the patient's adaptive immune responses for the resolution of cancer. Thus, once cells escape treatment, they continue to grow, resulting in tumor recurrence and resistance to therapy. [1][2][3] Immunogenic cell death (ICD), initially described by Zitvogel, Kroemer, and co-workers, is a modality of death where dying cells stimulate immune responses against dead-cell antigens. [4][5][6] In cancer therapy, this is advantageous as tumor cells undergoing ICD activate an anti-tumor immune response that is specific for that cancer. Therefore, developing treatments that elicit immunogenic cell death and facilitate the active participation of the patient's adaptive immune system, offer the potential to improve clinical outcomes of cancer therapy.
Plasma, the fourth state of matter, is ionized gas composed of charged particles, active neutral gas species, electric fields, and low amounts of ultraviolet (UV) light. [7][8][9][10][11] Development of plasma systems that can be sustained in atmospheric pressure and at room temperature has opened doors for biomedical applications, including, but not limited to, cancer therapy. [12][13][14] Mounting evidence demonstrates that these 'non-thermal plasmas' (NTP) can be optimized to destroy tumors with minimal damage to neighboring healthy tissue. [14][15][16][17] Decreased tumor burden and prolonged animal survival following direct plasma treatment have been reported, suggesting that plasma should be further explored as a viable candidate for cancer treatment. 18 The potential of NTP to induce immunogenic cancer cell death is only recently being explored. Bekeschus, et al. has demonstrated that NTP treatment of two murine cell lines in vitro, the B16F10 melanoma cells and the CT26 colorectal cancer cells, increased immunogenic cell surface molecules such as major histocompatibility complex I (MHC-I) and surfaceexposed calreticulin (ecto-CRT). 19,20 We have reported successful in vitro ICD induction in two human cell lines, a radiationresistant primary nasopharyngeal carcinoma cell line (CNE-1) and the A549 lung carcinoma cell line in response to NTP exposure. 21,22 The mechanism is postulated to be reactive oxygen and nitrogen species (RONS) dependent. NTP-generated RONS rapidly change the oxidative status of cells and induce endoplasmic reticulum (ER) stress pathways in these cells. [19][20][21][22] Upregulation of two proteins associated with ER stress and upstream of CRT emission, activating transcription factor 4 (ATF4) and stanniocalcin (STC2), was also demonstrated. 21 Moreover, abrogation of NTP-generated and cell-stimulated RONS tempered the effect of NTP on CRT emission. These reports indicate that NTP-induced ICD is not specific to a single cancer cell type, and merits further investigation into its clinical relevance as an anti-cancer modality. Plasma treatment in animal models of cancers is needed to assess if plasma-induced ICD could benefit patient outcome.
In this study, we used the CT26 murine colorectal tumor model to explore the potential of NTP to induce ICD in vivo. NTP generated by a nanosecond-pulsed dielectric barrier discharge (nspDBD) plasma system induced the expression of two key surrogate markers of ICD in these cancer cells: ecto-CRT and secreted adenosine triphosphate (ATP). A vaccination assay, used to determine if a stimulus is a bone fide ICDinducer, showed partial protective immunity against tumor challenge in syngeneic Balb/c mice immunized with NTPtreated CT26 cells. Furthermore, treatment of subcutaneous colorectal tumors expressing the cancer antigen guanylyl cyclase C (GUCY2C) resulted in higher expression of ICD markers in tumors, recruitment of antigen presenting cells (APCs), and generation of more GUCY2C-specific T cells. Together, our findings are the first report that establish the potential of plasma for cancer immunotherapy via ICD.

Plasma induces emission of surrogate markers of ICD
To measure cell death in response to nspDBD plasma, the CT26 colorectal carcinoma cell line was exposed to several plasma energies. Cell viability, quantified with a Muse Cell Analyzer 24 hours after plasma treatment, decreased in an energy dependent manner (Figure 1(a)). As previously described, not all modalities of cell death are immunogenic and capable of initiating anti-tumor effects. The identification of ICD in vitro mainly relies on detection of associated damage associated molecular patterns (DAMPs). Therefore, we examined the effect of plasma on cell viability and two DAMP signals in CT26 cells: externalization of CRT and secretion of ATP. 23 Immunogenicity of dying cancer cells is strongly dictated by surface exposure of CRT. 24 Normally located on the ER membrane, exposed CRT on the cell surface acts as an 'eat me' DAMP signal that facilitates recognition, engulfment, and processing of tumor cells by APCs, [25][26][27] a critical step for the initiation of an adaptive anti-cancer response. [28][29][30] Surface-exposure of CRT in response to 10 second plasma exposure was measured 24 hours after treatment. Intact cells were labeled with anti-CRT antibodies, stained with fluorescent secondary antibodies and analyzed using flow cytometry. Our results show that the emission of ecto-CRT on CT26 cells increased in an energy dependent manner, suggesting plasma may increase the immunogenicity of tumor cells (Figure 1(b,c)).
ATP, the most abundant intracellular molecule required for metabolism, is secreted from cells undergoing ICD. 31 It has even been suggested that secretion of ATP follows overlapping pathways with externalization of CRT. 32 Once ATP reaches the extracellular space, it becomes another hallmark of ICD and functions as a 'find me' DAMP signal for recruitment and activation of APCs. [32][33][34] To detect this secreted DAMP by cells exposed to plasma, the cell culture media was collected 10 minutes after treatment and extracellular ATP was quantified. ATP levels were low at baseline (8.2 nM) and increased 70-fold (582.1 nM) following 300 mJ plasma treatment (Figure 1(d)).

Vaccination with plasma-induced ICD cells provides protection against tumor challenge in mice
To ascertain whether the DAMP signals elicited by plasma could enhance immune responses against cancer, we performed a vaccination assay. Balb/c mice were immunized with CT26 Figure 1. Plasma-induced cell death, surface emission of CRT, and secretion of ATP in CT26 cells. A) Cell viability is indicated by the percentage of live CT26 cells normalized to untreated (0 mJ), 24 hours after plasma treatment. B, C) CRT was detected on the surface of intact CT26 cells 24 hours after plasma exposure. B) Representative histograms and C) mean fluorescence intensity showed increased surface CRT following plasma treatment. D) ATP content was detected in the media 10 minutes after plasma treatment using a chemiluminescent kit. CRT, ATP, and viability data are presented as means ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, Dunnett's multiple comparison test). cells treated in vitro with plasma at the ICD-inducing regime (29 kV, 30 Hz, 1 mm gap distance, 10 seconds). Cells were prepared for inoculation and injected into the left flank as a whole-cell vaccine to allow an immune response to develop. One week after immunization, mice were challenged with live CT26 cancer cells on the opposite flank and tumor growth was monitored twice a week until day 26 when the study was terminated as a subset of the animals reached IACUC-approved endpoints (Figure 2(a)). CT26 cells treated with media only or Cisplatin (50 μM for 24 hours), a non-ICD inducer, 23 were used as controls.
Challenge tumors in the media and Cisplatin groups grew rapidly while tumors in the plasma group developed relatively slowly ( Figure 2(b-d)). The mean tumor volume for the plasma immunized group was significantly smaller compared to that of the media group (414.7 ± 104.3 mm 3 vs 847.4 ± 141.5 mm 3 ; p < 0.001) or the Cisplatin group (1041.8 ± 208.3 mm 3 ) at day 26 ( Figure 2(e)). Indeed, 90% of the mice in the plasma immunized group had tumor volumes smaller than the mean tumor volume of the media group (850 mm 3 ), suggesting that these mice were partially protected by vaccination. Moreover, 3 out of the 10 mice in the plasma group did not develop subcutaneous tumors on the challenge site ( Figure 2(f)).

Plasma induces ICD in vivo and stimulates immune cell recruitment
To directly validate whether plasma could induce ICD in vivo, CT26 colorectal tumors were established subcutaneously in Balb/c mice and exposed to plasma when they became palpable ( Figure 3). A safe operating plasma regimen was first identified by exposing subcutaneous CT26 tumors in Balb/c mice to plasma for various treatment times (10, 25, 50 sec) at set plasma parameters (29 kV and 750 Hz) once daily for five consecutive days. One day and 3 days after the final treatment, tumors were resected with overlaying skin, fixed, and stained with hematoxylin and eosin (H&E) to assess the structural changes in the dermal and epidermal layers of the skin. Ten seconds of plasma treatment resulted in minimal epidermal damage but no changes to the tumor were observed (Figure 4(b,f)). When plasma treatment duration was increased to 25 seconds, thermal and necrotic damage in the epidermis was observed one day after plasma (Figure 4(c)) but not in the underlying tumor. By the third day after treatment, the epidermis appears improved but not fully healed, suggesting that the damage was reversible (Figure 4(g)). The 50-second treatment resulted in considerable damage through all the layers of the skin, and, in fact, tumors just below the skin showed thermal and necrotic damage ( Figure 4 (d)). By the third day thermal damage was still apparent and changes in collagen had begun to appear. There were also more neutrophils in the skin signifying an inflammatory response (Figure 4(h)).
Based on the results of our safety studies, we chose to investigate if the 10 second plasma treatment over five days induced ICD in the subcutaneous tumors. Tumors were exposed to plasma using three different treatment procedures: i) the same area each day (Plasma 1 Spot), ii) two areas of the tumor each day (Plasma 2 Spots), or iii) different areas of the tumor each day (Plasma Multiple Spots). Three days after the last plasma treatment, tumors were resected, fixed, and sectioned. Immunofluorescence microscopy was performed on tumor sections to identify ICD markers. CRT expression increased in all plasma treatment groups ( Figure 5(a)) and was maximum following the "Plasma Multiple Spot" regimen (1.6 ± 0.2 fold, p < 0.05). Tumor sections were also stained for High mobility group box 1 (HMGB1), another DAMP signal much like ATP, that recruits inflammatory immune cells and mediates signals between APCs. [35][36][37][38] HMGB1 has been observed to translocate from the nucleus to the cytoplasm and extracellular space. [37][38][39] Here, we observed an increase in immunofluorescence intensity of HMBG1 in all plasma treatment groups ( Figure 5(b)), suggesting HMGB1 protein concentration may be elevated following plasma exposure.
To determine if emitted DAMPs stimulated the recruitment of immune cells into the tumor environment, we also stained for CD45+ (leukocytes) and CD11c+ (APCs) immune cells. Indeed, increased CRT and HMGB1 were associated with more CD45+ (2.0 ± 0.4 fold, p < 0.05) and CD11c+ cells (1.8 ± 0.2 fold, p < 0.05) following the Plasma Multiple Spots regimen ( Figure 5(c,d)). Because CD45 is expressed by all leukocytes, including T cells, B cells, neutrophils, NK cells and others, 40,41 it is possible that other immune cell subsets are also being recruited as a downstream consequence of ICD induction. Representative immunofluorescence images of the multi-spot treatment compared to the untreated are shown in ( Figure 5(e)).
Altogether, Plasma Multiple Spots treatments enhanced both emission of DAMPs and recruitment of immune cells in the tumor compared to the other application methods. Therefore, treatment of different spots may be more beneficial compared to repeat treatment of the same area. This treatment condition was used to investigate plasma-induced ICD effects on downstream T-cell response.

Plasma amplifies specific T-cell responses against CT26-GUCY2C tumors
To analyze whether plasma-induced ICD could stimulate an adaptive anti-tumor response, we treated subcutaneous CT26 tumors expressing the colorectal cancer antigen GUCY2C 42,43 (CT26-GUCY2C) in Balb/c mice. Mice were treated with either plasma alone or with plasma in combination with the Ad5-GUCY2C-S1 vaccine. The Ad5-GUCY2C-S1 vaccine was previously shown to safely induce GUCY2C-specific immune responses and antitumor immunity in mice [44][45][46][47][48][49][50][51] and has been translated to human clinical trials. 52 Mice were treated with plasma for five consecutive days and one group was vaccinated with the Ad5-GUCY2C-S1 vaccine, one week after the last plasma treatment (Figure 6(a)). An untreated group and a vaccine only group (Ad5-GUCY2C-S1 vaccination) served as our negative and vaccine controls, respectively.
Splenic GUCY2C-specific T-cell responses were analyzed by IFNγ ELISpot assay 28 days after the initial tumor inoculation. Plasma treatment alone had a marginal effect on GUCY2C-specific responses (22.1 ± 11.9 spots vs 3.0 ± 1.8 spots in Untreated; p = ns). However, plasma treatment prior to vaccination amplified GUCY2C-specific T-cell responses (167.8 ± 41.5 spots vs 109.7 ± 22.3 spots with vaccine alone, p < 0.05) (Figure 6(b)). This observation supports the potential of plasma to increase the immunogenicity of cancer cells and stimulate canonical pathways required for tumor control.
Typically, immune responses are generated against one or two dominant epitopes of an antigen. When the specificity of immune responses spreads to include subdominant epitopes, new populations of T cells may be generated against the antigen. 53, 54 We tested whether plasma and vaccination treatment exposes neoantigens unrelated to the original vaccination target, GUCY2C. 55 We measured T-cell responses against AH1, an endogenous immunodominant MHC class I-associated epitope from gp70 expressed by CT26 tumor  Figure 6(c)). [55][56][57] Mice receiving both plasma and peptide vaccine exhibited a modest increase in the AH1-specific T-cell response (24.2 ± 8.3 vs 11.0 ± 4.3 spots; p = ns). While the study was not powered to detect a statistically-significant change, epitope spreading was observed. Further optimization of plasma treatment to alter tumor microenvironment may  produce conditions favorable for epitope spreading. Altogether, we provide compelling evidence that plasma acts as an adjuvant for cancer therapy as shown by the amplification of GUCY2C-specific and AH1-specific T-cell responses in mice. Increased efficacy with the addition of vaccine suggests that plasma may prime the host's immune system and may allow for its use in other combination therapies.

Discussion
Although significant advancement in conventional tumor-targeted cancer therapies (e.g. surgery, chemotherapy, radiation therapy, etc.) has reduced cancer related morbidity and mortality, this comes at the cost of significant toxicity. Another major challenge is relapse from cells that escape treatment and eventually develop resistance to therapy. [58][59][60] In contrast, immunotherapy aims to activate the patient's natural defenses to selectively target tumors for resolution of cancer 60-63 with reduced non-specific damage to normal tissue. While current strategies (e.g. adoptive T-cell transfer, checkpoint inhibitors, etc.) are clinically efficacious, several instances of serious adverse effects, including pneumonitis and enterocolitis, have been reported. 64,65 To address these, an ICD-mediated immunotherapy approach is being explored. These treatments stimulate the release of DAMP signals in cancer cells which engage APCs to expose neoantigens and facilitate the initiation of adaptive immune responses. 66,67 The methods to induce ICD include certain chemotherapeutic agents, irradiation, photodynamic therapy with hypericin (PDT-hypericin), and high hydrostatic pressure. 68 Here, we have demonstrated that non-thermal plasma may be operated for ICD induction and utilized in immunotherapeutic strategies. In vitro, nspDBD plasma elicited CRT emission and secretion of ATP from CT26 colon carcinoma cells. Our vaccination study showed that mice immunized with cancer cells treated with ICD-inducing plasma were partially protected against tumor challenge. In agreement with in vitro studies, in vivo plasma treatment of subcutaneous tumors in mice induced immunogenic cancer cell death. The treatment of multiple spots within the tumor elicited the greatest emission of DAMPs and recruitment of APCs into the tumor area. This treatment condition also led to a tumor-antigen-specific T-cell response. While this is the first report of plasma inducing ICD in an animal model, these results should be confirmed in other cancer models to validate, broaden, and enhance the relevance of plasma as an ICD inducer.
Overall, our results highlight the potential of plasma development for cancer immunotherapy. Further optimization of plasma parameters (applied voltage, pulse frequency, application time, etc.) and treatment schedules must be performed to improve its efficacy. This could be accomplished, in part, by determining the physical and chemical mechanism by which plasma elicits ICD. In vivo, it has been reported that antitumor effects of plasma are associated with plasma-generated RONS, but the link to immunogenic cell death has not been made. 16 In vitro, RONS produced by plasma were demonstrated to be the major effectors for eliciting ecto-CRT and ATP secretion in cancer cells, although potential synergy with the associated pulsed electric fields or UV radiation should not be discounted. 22 Therefore, an in-depth delineation of the specific RONS essential for ICD could help the development of improved plasma systems.
The study of the extracellular microenvironment generated by plasma should be paralleled with investigation of the intracellular mechanisms of induced ICD and the mode of cell death. In 2018, a comprehensive review on the molecular mechanisms of cell death was published by the Nomenclature Committee on Cell Death (NCCD). 69 Twelve major cell death subroutines (intrinsic apoptosis, extrinsic apotosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, and immunogenic cell death) were defined from morphological, biochemical, molecular, and functional perspectives. To date, six DAMPs have been linked with cell death that is immunogenic [CRT, ATP, HMGB1, type I interferon (IFN), cancer cell-derived nucleic acids, and annexin A1], but not all the underlying mechanisms are clear, and some are dependent on the specific ICD-inducer. [70][71][72][73][74][75][76] As these mechanisms become elucidated, understanding the molecular processes following plasma-induced ICD will be possible and will help the development of this potential therapy.
The scheduling of plasma treatment will also influence clinical outcome, as plasma may also induce bystander effects on other resident or recruited cells in the tumor environment (e.g. macrophages, dendritic cells, effector T cells, etc.). Indeed, in a separate study where mini pigs were exposed to plasma, recruited myeloid cells were detected in the treated areas of the skin one week later, 77 suggesting that local plasma exposure may influence immune cells even in the absence of cancer. Furthermore, several studies have demonstrated that within a defined range of treatment parameters, plasma may stimulate immune cell function (e.g. migration, secretion of cytokines, etc.) in vitro. 21,[78][79][80][81] In depth studies in animal models are required to determine direct plasma effect on cells of the immune system, as the host microenvironment may affect biological outcome. However, this could potentially provide an advantage over radiation or PDT, known ICD inducers, as they are reported to be detrimental to immune cells. [82][83][84] An indicator that plasma may be immunomodulatory at physiologically safe doses was reported in a study where Drosophila exposure to plasma caused differentiation of hematocytes without affecting development or fecundity of the organism. 85 While the present studies provide preclinical proof-of-concept for plasma immunotherapy in cancer, clinical administration of plasma will be a major challenge for its use in cancer treatment. For superficial cancers such as melanomas, administration of plasma is relatively straight-forward, as cancerous tumors/lesions are easily accessible for direct deposition of plasma-generated RONS. However, treatment of non-superficial cancers is a challenge for the plasma medicine field. One approach to deliver plasma species to deep tumors is through the use of plasma treated liquid (PTL), 86,87 in which media is treated with NTP to enrich dissolved RONS and injected locally in the tumor or perfused through body cavities. 88, 89 Utsumi and co-workers demonstrated that injection of PTL locally into subcutaneous tumors can inhibit growth of malignant tumors in mice though the anti-tumor effects are not as prominent as direct plasma treatment. 89 This is likely due to the instability of plasma-dissolved species in the media and the animals' antioxidant capacity. 90 Optimization of PTL generation and storage is required before it finds a role in clinical cancer treatment. A more direct, but invasive approach may involve intraoperative plasma treatment following surgical tumor excision to eliminate cancer cells remaining in the surgical margins. Physicists and engineers are designing different plasma sources and geometries for a less invasive and more focused approach to deliver plasma inside the body. [91][92][93] Plasma has been shown to propagate along tubes up to several meters in length and with diameters as small as 15 μm. 91,92 The effectiveness of some of these endoscopic plasma devices is being tested in an in vivo pancreatic cancer model. 93 For successful clinical application with this approach, a detailed understanding of the RONS delivered to the target from the plasma aperture is critical. Finally, as we show here, immunization with a plasma-created whole-cell vaccine provided protective anti-tumor effects. With optimization of vaccine development and delivery this could be a feasible strategy for plasma-mediated cancer control where plasma acts as an adjuvant.
Ultimately, it is unlikely that a single treatment will be the solution to any type of cancer; a combination of different therapies may be required. Our data suggests that combining plasma with other immunotherapeutic agents may provide additional clinical value ( Figure 6). Development of these strategies should be considered based on their effect on the different steps of adaptive immune response progression. 94 For example, plasma-induced ICD could prime the host immune response against tumor antigens, which could be boosted by a targeted vaccine, while checkpoint inhibitor blockade may enhance the therapeutic effect of plasma immunotherapy.

Conclusion
We recently proposed a new paradigm of plasma treatment for cancer: 'plasma onco-immunotherapy'. 95 This approach not only debulks tumors, but also engages the innate immune system via ICD to initiate adaptive immune responses. 95,96 In this study, we demonstrated that plasma is a bona fide ICD inducer and can be used alone or in combination with other immunotherapies to generate tumor-specific T-cell responses. With further development of plasma delivery systems and administration protocols, it has potential for clinical translation as a standalone treatment modality or an adjuvant for cancer immunotherapy.

Materials and methods
nspDBD plasma system and treatment parameters NTP was generated in vitro by applying high voltage pulses to a dielectric barrier discharge (DBD) electrode. DBD electrodes used in this study were fabricated in our lab and have a quartz dielectric covering a copper electrode. This prevents current build-up and creates an electrically safe plasma without heating surrounding gas and tissue. A nanosecond pulser (FPG-20-05NM, FID GmbH, Germany) was used to generate high voltage pulses, characterized in our previous work. 8 Briefly, our system produced: 29 kV pulses, 2 ns rise times, 20 ns total pulse duration and 0.9 mJ/pulse.

Cell culture and in vitro plasma treatment
Colorectal cancer cell line CT26.WT obtained from ATCC (CRL-2638). Generation of CT26-GUCY2C cells was described previously. 97 Cells were cultured in complete media: DMEM with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Corning Life Sciences, USA). All cells were grown in a humidified environment at 37°C with 5% CO 2 (Panasonic, MCO-19AICUVH-PA, USA). Cells were plated one day prior to plasma treatment in 24-well plates at 3.0x10 5 cells/mL (0.5 mL/ well). Before treatment with plasma, media was removed from each well and cells were washed twice with phosphate buffered saline (PBS). PBS from the second wash was removed from the well right before cells were exposed to plasma in the absence of any liquid. Fresh, complete media (0.5 mL) was immediately added back into the well following exposure to plasma.
For treatment with plasma, a DBD electrode (1.3 cm diameter) was placed 1 mm above cells in the 24-well plate on top of a grounded metal plate with a z-positioner. Since all liquid was removed, plasma was generated in the gap between the electrode and the plate, directly on the cells by applying high voltage pulses from the nanosecond pulser. Treatment time was fixed to 10 seconds, and pulse frequency was controlled by an external function generator (TTi, TG5011LXT, USA). A range of pulse frequencies were used (50,15,30 and 75 Hz), and the combination of plasma treatment parameters produced in the following plasma treatment energies: 50, 100, 300 and 700 mJ, respectively. 8

Mice and in vivo plasma treatment
Balb/c mice were obtained from Jackson Laboratory (USA), and animal protocols were approved by The Thomas Jefferson University Institutional Animal Care and Use Committee. Subcutaneous tumors were established by injecting 1 × 10 5 CT26-GUCY2C cells in the flanks of mice, and monitored for growth. Prior to plasma treatment, hair over the tumor area was removed using a chemical depilatory agent to avoid obstruction with plasma generation and treatment. Tumors were treated once daily with plasma beginning on day 7 (for effector T cell development studies) or day 18 (for ICD and recruitment studies) and continued for 5 consecutive days. A smaller DBD electrode (3 mm diameter) was fabricated and used for treatment of mouse tumors. The nanosecond pulser and function generator used for in vitro treatment were also used here. Pulse frequency was adjusted to 750 Hz and treatment time was 10, 25 or 50 seconds. Mice were anesthetized with 5% isoflurane and treated on the grounding plate with the electrode positioned approximately 1 mm above the tumor with the z-positioner. Tumor volumes were monitored by measuring 3 orthogonal diameters and calculated using 4 3 π Â r 1 Â r 2 Â r 3 .

Cell viability assay
Cell viability was determined 24 hours after plasma treatment using a Muse Cell Analyzer (Millipore, USA). Cell suspensions were collected, diluted 1:20 with Muse Count & Viability Reagent (Millipore, USA) and analyzed according to the manufacturer's instructions. The percent viability was determined for each sample.

ATP release assay
Cell supernatant was collected 10 minutes after plasma treatment and extracellular ATP was measured using a luciferin and luciferase-based chemiluminescent kit (Sigma-Aldrich, USA). All procedures were performed and reagents were prepared following manufacturer instructions. Luminescence value was measured by a Photon-Master luminometer (LuminUltra, USA) which was calibrated with the provided UltraClear calibration solution. The measured relative light units were converted into ATP concentration (pgATP/mL). Data were represented at ATP concentration (nM).
Fluorescence detection of surface-exposed calreticulin CT26 cells were collected 24 hours after plasma treatment and washed twice with blocking buffer (PBS+ 1% heat-inactivated FBS). Cells were then incubated with rabbit anti-mouse calreticulin antibody (ThermoFisher Scientific, USA) in blocking buffer (1:200) for 30 minutes in the dark at room temperature. Following incubation, cells were washed twice with blocking buffer and stained with Alexa Fluor 488 conjugated goat antirabbit IgG secondary antibody (ThermoFisher Scientific, USA) at 1:500 in blocking buffer. Cells were incubated at room temperature, in the dark, for 40 minutes. Following staining, cells were washed and fixed with 4% PFA and analyzed by FACS.

Anti-tumor vaccination assay
To prepare the whole-cell vaccine, CT26 cells were treated with either: 1) 300 mJ of plasma, 2) Cisplatin (50 μM) or 3) complete media. Plasma-created vaccine was prepared from cells treated in 24-plates with plasma and cultured in regular media for 24 hours. Cisplatin-vaccine was prepared from cells incubated for 24 hours in Cisplatin media. Media-Vaccine was prepared from cells cultured in regular, complete media for 24 hours. Cisplatin-and media-vaccines were used as controls.
After a 24-hour incubation 3 × 10 6 cells in 100 μL of PBS were inoculated subcutaneously into the left flank of Balb/c mice. Seven days later, mice were challenged with 3 × 10 5 live CT26 cells subcutaneously injected into the right flank. Tumors were measured twice weekly with calipers and all mice were euthanized on day 28.

H&E staining of tissue sections and damage assessment
Tumors, with overlaying skin, were resected 1 or 3 days after the final plasma treatment and fixed in 10% formalin for at least 48 hours. Tissue was then paraffin-embedded, sectioned with a microtome, deparaffinized and stained with hematoxylin and eosin. Images of stained sections were captured using the EVOS FL Auto Cell imaging system. Sections were evaluated for damaged by a blinded pathologist.

Immunofluorescence staining of tumor tissue
Tumor sections from day 3 after final plasma treatment were used for fluorescence detection of DAMP signals and immune cell recruitment. For antigen retrieval, slides were transferred to a Dako Target Retrieval buffer pH9 (1:10 dilution in H 2 O) and boiled for 15 min in a pressure cooker. Slides were then cooled and blocked with blocking solution (10% milk in PBS + 0.3% v/ v TritonX + 15 μL Fab donkey anti-mouse IgG (H + L) fragments) for 1 hour at room temperature in a humidified chamber. Following blocking, tumor sections were stained the following antibodies (1:100 in blocking solution) overnight at 4°C in a humidified environment: anti-mouse CRT (PA3-900, ThermoFisher Scientific, USA) and anti-mouse HMGB1 (MA5-16,264, ThermoFisher Scientific, USA) or anti-mouse CD45 (103,101, Biolegend, USA) and anti-mouse CD11c (33,483, Abcam, USA). Tissue samples were then washed four times with PBS + 0.1% v/v Tween (PBST). Secondary antibodies (1:1000 in blocking bluffer) were added: donkey anti-rabbit IgG Alexa Fluor 594 for CRT (A21207, Life Technologies, USA), goat anti-mouse IgG Alexa Fluor 488 for HMGB1 (115-545-205, Jackson Immuno, USA), donkey anti-rat IgG (H + L) Alexa Fluor 594 for CD45 (A21209, Life Technologies, USA) and goat anti-armenian hamster IgG (H + L) Alexa Fluor 488 for CD11c (127-545-160, Jackson Immuno, USA). Tissue sections were stained for 1.5 hours at room temperature in a humidified environment, protected from light. Following secondary staining, tissue sections were washed four times with PBST and mounted with DAPI (P36935, Molecular Probes, USA). A glass cover slip was placed on top of each tissue section and cured overnight. Sections were viewed under an EVOS FL Auto Imaging System (Life Technologies, USA). Using ImageJ software, mean fluorescence intensity of DAMPs and CD45 +/CD11c+ signals were determined by measuring the intensity of three representative areas on the tissue. Data are presented as normalized mean fluorescence intensity of individual resected tumors.


Purification and Characterization of Active Aggregates of an Organic Solvent Tolerant Lipase from Marinobacter sp. EMB5

Background: Marinobacter is a halophilic bacterial genus and a bio-geochemically important group in sea water, is generally associated with the bioremediation of hydrocarbon-rich effluents released into the salt-rich environment by various anthropogenic activities. It is a well-known lipase producer-the NCBI Protein database shows nearly 520 Marinobacter lipases. In this context, its lipase and esterase are considered important. However, their enzymatic properties in purified form have been scantily studied. Since hydrocarbons are acted upon on solvent water interface, the stability and catalytic behavior of Marinobacter lipase in non-aqueous media needs to be more specifically investigated. Aim: Marinobacter sp. EMB5 was isolated from Kozhikode, Kerala, India. It was found to be potent lipase producer. The aim of the present work was to obtain a purified preparation and undertake its detailed characterization with respect to above aspects. Work: The crude Marinobacter sp. lipase was purified by Sephacryl S200 gel filtration chromatography. The lipase eluted as a single activity peak in void volume and had a very high molecular mass. Aggregation has not been reported in case of Marinobacter lipase, so far, although it is known in other lipases. The aggregated lipase was mixed with 2-propanol (70%, v/v) and disaggregated lipase was obtained as a single activity peak in Sephacryl S200 gel filtration chromatography corresponding to 82 kDa molecular mass. This monomeric species was less active than aggregated purified preparation. Hence, further characterization and studies were carried out on purified active aggregate lipase. It was stable in a wide range of organic solvents at high concentration up to 24 h. The organic solvent stability was further investigated by Far UV CD spectroscopy and fluorescence spectroscopy. Conclusion: Due to the fact that Marinobacter sp. EMB5 lipase remained stable in organic solvents for prolonged incubation periods, it has potential to be used in industrial organic synthesis.

Introduction
Halophilic lipases are interesting for biotechnological applications as they retain their functionality even under high salt and lowwater conditions. Low-water conditions predominate within the halophilic cell, due to excessive accumulation of salts within the cytoplasm as an adaptation mechanism. In the industry, organic synthesis reactions carried out under solvent-rich conditions mimic such low-water conditions. Since the activity

Insights in Enzyme Research
of halophilic lipases is not compromised even in presence of very high concentration of organic solvents, they are very promising candidates for carrying out lipase-catalyzed organic biotransformation [1][2][3].
Although a large number of halophilic lipases have been detected and listed on the NCBI's protein database (http://www.ncbi.nlm. nih.gov/protein), very few have been purified and characterized in detail. These include Oceanobacillus rekensis PT-11 lipase [4], Haloarcula sp. G41 lipase [5], Idiomarina sp. W33 lipase [2] and LipBL from Marinobacter lipolyticus SM19 [6]. Moreover, not much is known about the correlation of their solvent stability with structural features. Structural studies further require purified form of lipase.
Purification of halophilic lipase, in general, poses problem for two reasons, (i) Requirement of incorporating the salt (necessary to maintain native structure of halophilic proteins) throughout the purification process. Otherwise, protein is denatured leading to loss of activity [7]. But the addition of salt disrupts the chromatographic procedure. (ii) Propensity of aggregation in lipase causes formation of aggregates during purification, making purification quite unpredictable.
Most halophilic lipases are also organic solvent tolerant owing to a similarity in the micro-environment created by the presence of organic solvents and high salt concentration. LipBL from Marinobacter lipolyticus displayed remarkable stability in presence of both hydrophobic as well as hydrophilic solvents, with maximum stability in presence of methanol [6]. The Idiomarina sp. W33 and Haloarcula sp. G41 lipases were stable in presence of hydrophobic organic solvents [2,5]. Oceanobacillus rekensis PT-11 lipase was stable in presence of long-chain alcohols [4]. Lipase from Idiomarina sp. W33 and Haloarcula sp. G41 have been successfully used for biodiesel production. Protein structural studies to support organic solvent tolerant stability in halophilic lipases, are however not available. The present study addresses this gap.
Halophilic Marinobacter sp. EMB5 showing extracellular lipolytic activity was reported from our lab previously which showed significant levels of lipase secretion [8]. The present work describes the purification and characterization of the naturally aggregated lipase of this strain that showed remarkable organic solvent stability. The organic solvent stability of the lipase was further investigated by secondary and tertiary structure studies in presence of a stabilizing as well as destabilizing organic solvent.

Materials
Sephacryl S200 and p-nitrophenyl esters were a product of the Sigma Chemical Company (St. Louis, MO, USA). Polyacrylamide and bis-acrylamide were procured from GE Healthcare Life Sciences (Bangalore, India). Sodium dodecyl sulphate (SDS) and Bovine serum albumin (BSA) was purchased from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). Other media components were purchased from Hi-Media Laboratories (Mumbai, India). All other chemicals used were of analytical grade. All the solvents used in the present study were treated with molecular sieve (4 Å) prior to use.

Strain isolation, identification and lipase production:
Marinobacter sp. EMB5 was isolated fromthe sea coast of Kozhikode (Kerala, 11°25′N 75°77′E) by salt enrichment. It was found to be related to Marinobacter sp. by the 16S rDNA sequence analysis which was submitted to GenBank, NCBI, USA with accession number FJ525429 [8]. Lipase was produced in a medium containing (g/l): peptone 5.0; yeast extract 5.0; NaCl 50.0; gum arabic 1.0 and olive oil 5.0 ml/l. The pH of the medium was maintained at 9.0. The production medium was seeded with 4% (v/v) inoculum raised overnight in nutrient broth with 5% (w/v) NaCl (A 660~1 .0) and incubated at 30°C and 160 rpm for 48 h. Cell-free supernatant (CFS), containing the extracellular lipase was obtained by pelleting down the cells by centrifugation of the culture broth at 8,000 rpm for 15 min.
Lipase assay: Lipase was assayed following the method of Kilcawley et al.
[9] using p-nitrophenyl octanoate as the substrate. Briefly, 1.8 ml of Tris-HCl (50 mM, pH 8.5) containing 0.5% (v/v) Triton X-100 and 200 µL of diluted enzyme was pre-incubated for 10 min following which 20 µL of 50 mM p-nitrophenyl octanoate (PNPO) dissolved in acetonitrile was added to the reaction mixture. PNPO hydrolysis was carried out for 20 minutes at 50°C. The p-nitrophenol released by enzyme action was measured at 400 nm. One unit (U) of lipase activity was defined as the amount of enzyme releasing 1 nmol of p-nitrophenol equivalent per minute from the ester under standard assay conditions.

Lipase purification and characterization:
The crude lipase was subjected to ultrafiltration using 10 MWCO and retentate, so obtained was subjected to size-exclusion chromatography by loading onto a Sephacryl S200 column (1.5 cm × 50 cm) equilibrated with Tris-HCl buffer (50 mM, pH 8.5, containing 2% (w/v) NaCl) and eluted at a flow rate of 24.0 ml/h. Fractions of 2 mL each were collected using the fraction collector (BioRad Model 2110, California, USA). Each fraction was assayed for lipase activity. The active fractions were pooled and characterized.

Disaggregation of EMB5 lipase:
The Marinobacter sp. EMB5 lipase aggregates were treated with 70% 2-propanol, which was included in the SEC elution buffer as per the method of Dunhaupt et al.

Protein determination, electrophoresis and zymogram analyses:
Protein content was determined using the Bio-Rad Bradford Assay Kit (BioRad, California, USA) with bovine serum albumin as the standard. In order to determine the molecular mass of the lipase, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli [11]. A 12.5% SDS-PAGE gel was used on a BioRad Model Mini Protean gel electrophoresis unit (BioRad, California, United States). Silver staining was used to visualize protein bands on gel. Relative molecular mass (M r ) was estimated by comparison with molecular mass standard (14-97.4 kDa) (Bangalore Genie, Bangalore, India). Zymography was done by detection on lipolytic bands on the SDS-PAGE gel using tributyrin as substrate [12].

Effect of temperature and pH on lipase activity and stability
The optimum temperature for enzyme activity was determined by assaying at various temperatures ranging between 30°C and 60°C. Thermal stability was determined by pre-incubating the enzyme at various temperatures (50°C-70°C) for different time periods and then measuring the residual lipase activity. Effect of pH on lipase activity was determined by assaying in buffers (50 mM) of varying pH viz. sodium acetate (pH 4.0-5.0), sodium phosphate (pH 6.0-7.0), Tris-HCl (pH 8.0-10.0) and Borax-NaOH buffer (pH 10.0-12.0). The effect of pH on stability was investigated by preincubating the lipase at various pH (ranging from 4.0 to 12.0) for 12 h at 25°C. The residual activity was determined under standard assay conditions.

Determination of kinetic constants
K m and V max values of the purified lipase were determined by assaying at different p-nitrophenyl octanoate (PNPO) concentrations. Kinetic constants were calculated using the Line weaver-Burk plot.

Surfactant stability
The effect of detergents, Sodium dodecyl sulfate (SDS), Cetyl trimethylammonium bromide (CTAB), Tween 80, Triton X-100 and Brij 35 at 1% (v/v) concentration, was examined by incubating the enzyme with these detergents at 30°C for 5 h. The residual activity was then measured by standard assay procedure.

Organic solvent stability
One milliliter of lipase was mixed with equal volume of organic solvents (ethanol, isopropanol, butanol, dichloromethane, benzene, toluene, cyclohexane, hexane and decane) in screw capped tubes. The mixture was incubated at 30°C with constant shaking at 200 rpm for 24 h. Samples were withdrawn from aqueous phase and the residual enzyme activity estimated. Enzyme incubated without solvent was treated as control and its activity was taken as 100%.

Far UV CD spectroscopy
In order to study the effect of solvents on the secondary structure, the far UV CD spectrum of the control, cyclohexane and 2-propanol treated enzyme samples was recorded on a JASCO J-815 spectrometer (Jasco Corporation, Tokyo, Japan) using a cell with a path length of 0.1 cm and a scanning rate of 50 nm min −1 with a 2 nm bandwidth over the wavelength range 200-250 nm for far-UV with five scans averaged for each spectrum using a protein concentration of 0.5 mg/ml in the respective buffers. All CD spectra were corrected for buffer contributions.

Fluorescence spectroscopy
The effect of cyclohexane and 2-propanol on the protein tertiary structure was studied from the fluorescence spectra recorded at 25°C on a Perkin Elmer LS50 spectrofluorimeter (PerkinElmer, MA, USA). The fluorescence emission spectra were recorded from 300 to 400 nm upon excitation at 295 nm in presence of cyclohexane and 2-propanol. These organic solvents were chosen based on the organic solvent stability of the lipase, which was highest in cyclohexane and least in 2-propanol. The excitation and emission slit widths were kept at 2.5 nm and 5 nm, respectively. All fluorescence spectra were normalized and corrected for buffer contributions. All the experiments were done in triplicate and the variation was within ± 5%.

Results and Discussion
Halophiles have been perceived as a potential source of industrially useful enzymes endowed with exceptional stabilities in high salt, alkaline pH and organic solvents [13,14]. The isolation of the halophile Marinobacter sp. EMB5 strain, from Kozhikode sea water, India, has been previously reported by us [8]. The isolate was Gram-negative rod and grew well at high salt concentrations (1-20%, w/v) and pH range of 6.0-9.0. Identification of the isolate by 16S rDNA analysis  The isolate was found to be a potent lipase producer. Lipase production was growth dependent. The Cell-free Supernatant (CFS) collected at 48 h was used for all purification and characterization studies. The crude was quite stable in cyclohexane and therefore an interesting system to understand the structural basis of this phenomenon in halophiles.

Lipase purification
Since very few lipases are purified from halophiles, Marinobacter sp. EMB5 lipase was first attempted to be purified by conventional gel chromatography using Sephacryl S200 ( Table 1). The purification chromatogram is shown in Figure 1. The lipase activity was eluted close to the void volume, indicating possibility of high molecular weight lipase aggregates. Aggregation in non-halophilic lipases is well known and has been widely studied [15,16]. In the extreme halophile Haloarcula marismortui, an esterase with a tendency to aggregate in presence of high salt concentration has been previously described by Rao et al. [17]. Although aggregation is less expected in halophiles because the presence of high salt concentration in the cytoplasm may prevent aggregation of proteins. The Marinobacter sp. EMB5 lipase also shows the same phenomenon wherein despite presence of 2% (w/v) NaCl in the CFS, lipase aggregation is observed. These lipase aggregates are soluble, active and have a high molecular mass of 180 kDa (Figure 2).
Previous studies on disaggregation of other lipases have shown that certain additives such as 2-propanol, sodium cholate, lauryldimethylamine-N-Oxide (LDAO) and SDS successfully dissociate them into monomeric forms ( Table 2). The Marinobacter sp. EMB5 lipase aggregates were disaggregated by the inclusion of 70% 2-propanol in the elution buffer of Sepharcyl S200 gel chromatography. The lipase peak fractions obtained from SEC were pooled, concentrated against sucrose and subjected to SDS-PAGE. A single protein band confirmed the homogeneous purification of lipase with a molecular mass of approx. 82 kDa on the SDS PAGE gel (Figure 3a). Similar molecular weight lipases have been reported from non-halophiles, Vibrio and Burkholderia species and strains of Pseudomonas fluorescens (https://www. ncbi.nlm.nih.gov/protein/?term=LIPASE). The zymography also revealed the presence of hydrolysis zone in the tributyrin agar plate confirmed it to be a lipase (Figure 3b).

Thus, our study confirms the aggregation phenomenon in
Marinobacter lipase despite the presence of 2% NaCl. The ultrafiltration (10,000 MWCO) followed by Sephacryl S200 gel exclusion chromatography in presence of 70% (v/v) 2-propanol led to disaggregation of lipase aggregates. Various combinations of chromatographic procedures have been employed for purification of halophilic lipases in the past. Two steps led to 2.5 fold purification with 15% yield in halophilic lipase from Oceanobacillus strain PT-11 [4]. Ammonium sulphate precipitation using 75% (w/v) ammonium sulphate followed by DEAE-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography resulted in 7.4 purification fold with 24.6% yield in case of Idiomarina sp. W33, extracellular lipase [2]. Thus, our strategy to purify monomer (disaggregated form) by incorporating 2-propanol and to purify aggregated form by omitting 2-propanol worked well.

Characterization of purified lipase
The purified aggregated lipase showed 16312.5 U/mg specific activity while the disaggregated showed 208.28 U/mg. Considering that (i) lipase is more active in aggregated form, as observed in Figure 1 The first step of CE-mediated hydrolysis involves the nucleophilic attack of Ser -OH group on the -CO carbon of the ester bond thereby releasing an alcohol moiety and an acylated enzyme. The second step works on nucleophilicity which increases affinity to water and releases acid moiety and the enzyme [5]. and Ba 2+ . The activation in presence of Ca 2+ ions was even more pronounced at 5 mM concentration. Lipases in general are known to be activated by Ca 2+ [13]. The lipase was less responsive to PMSF, indicating the non-involvement of serine, unlike other lipases. However, PCMB caused a decrease in activity, indicating involvement of cysteine residue.
Organic solvent and surfactant stability: Solvent stability is a generic feature of all halophilic proteins [13,21]. This is attributed to the fact that they work at high salt surroundings which is akin to low water medium. Lipase in the present study was stable in 50% (v/v) concentration of cyclohexane (Table 3). Polar solvents are known to strip off essential water of activity from active site universally causing loss in activity, so was in the present case. Among the halophilic lipases, solvent stability has been reported from Haloarcula, Idiomarina, Alkalibacillus salilacus [2,3,5].

Surfactant stability:
The lipase was found to be extremely stable in presence of CTAB, Triton X100, Tween 80 and Brij 35 except anionic SDS which is a highly desirable property for lipase from the viewpoint of detergent applications ( Table 4). The halophilic lipases have hydrophobic amino acid residues in the lid that covers the lipase active site. The organic solvent molecules interact with these residues and are likely to maintain the enzyme in the open conformation. This may contribute mainly to their stability in presence of organic solvents and surfactant stability [22]. This is the first study which reports both these properties for Marinobacter lipase, making it quite attractive for applications.

Structural Studies Far UV CD spectroscopy
In order to understand solvent stability, the CD spectra of purified lipase were recorded in presence and absence of cyclohexane at 25°C and pH 8.5 ( Figure 5). Marinobacter lipase was extremely stable in presence of cyclohexane up to 24 h. The results show changes in the peak position of negative ellipticity between 222 and 225 nm. This region corresponds to the spectral profile of α-helix [23]. In presence of cyclohexane, there was only a minor decrease in negative ellipticity as compared to the control. Thus, the CD spectra have given us an insight into the case of other lipases too [18] and (ii) that from the application point of view, the purified active lipase was characterized.

Enzymatic properties:
The enzymatic characteristics of Marinobacter have not been investigated in details before. The enzyme exhibited broad substrate specificity with maximum activity towards C8 and C10 substrates (Figure 4). Similar substrate specificity has been reported in case of Salicola sp. IC10 [19] and LipBL from Marinobacter lipolyticus lipa [6] lipases. It indicates that Marinobacter sp. lipase in the present case has better affinity for medium chain length fatty acids.
The K m and V max of the lipase towards p-nitrophenyl octanoate (PNPO) were 1.43 µM and 1.43 µmoles/ml/min respectively. Among other enzymatic properties, the EMB5 lipase had temperature optima at 50°C and pH optima at 9.0. The lipase was stable in the pH range 8.0-10.0 up to 12 h. Thus the Marinobacter lipase was alkaline in nature. These are in agreement with other halophilic lipases. Lipase LipBL from Marinobacter lipolyticus was stable over the pH range of 7.0-10.0 [6] but that from Oceanobacillus was stable only in a narrow range between pH 8.0-9.0 [4].
As a very novel feature, the Marinobacter lipase was significantly heat stable as compared to other reported halophilic lipases. It had a half-life of 90 minutes at 60°C and 30 min at 70°C. This property makes it quite attractive for applications at elevated temperatures. Among the lipases which have their temperature optimum at 50°C, Aspergillus terreus lipase is stable at 60°C for more than 12 h [20].

Effect of additives and metal ions:
The effect of metal ions and additives (oxidizing, reducing and chelating agents) was tested at 1 and 5 mM in 0.1 M Tris-HCl buffer at pH 8.5. Lipase was activated by Ca 2+ ions and was inhibited by the presence of K + , Cu 2+ , Hg 2+

Insights in Enzyme Research
secondary structure stability in presence of an organic solvent in which the lipase displays enormous stability. Despite a very high concentration of the organic solvent and a prolonged exposure to solvent, there were no distinct changes in the secondary structure.

Fluorescence spectroscopy
The effect of cyclohexane on the tryptophan fluorescence spectrum of lipase was studied by exciting the samples at 295 nm ( Figure 6). The solvated tryptophan residues in aqueous solutions have fluorescence maxima at 350 nm. Under conditions that promote protein folding, the tryptophan residues are firmly embedded in the non-polar protein core causing a characteristic blue shift of its fluorescence spectrum. Denaturing solvents, on the other hand cause an increase in fluorescence intensity with a characteristic red shift of tryptophan fluorescence in the 328 to 348 nm wavelength range. Incubation of EMB5 lipase with cyclohexane caused a red shift from 344.5 nm to 346.5 nm. This suggested a slight increase of the accessibility of these residues to the solvent, i.e., increase of the protein molecule hydrodynamic volume, evidently fluorescence quenching in presence of cyclohexane. Similar phenomenon has been previously reported by us in case of halophilic protease in presence of n-heptane and n-decane [7]. Presence of a biphasic solvent system of water and cyclohexane causes conspicuous changes in the immediate microenvironment of the lipase. However, due to the presence of salt we expect these changes to have minimal effect on the interactions that stabilize the protein structure. In fact, decreasing fluorescence intensity is suggestive of tryptophan residues still buried in the EMB5 lipase hydrophobic core which is part of the enzyme active site. This explains the stability of the halophilic enzyme even in presence of a high cyclohexane concentration. Most probably the interactions responsible for the functional tertiary structure are more or less intact and there is very little if any loss of structure. The results are in agreement with secondary structure data to suggest that the structural changes whatsoever are minimal and do not result in compromising enzyme function.

Conclusions
Halophilic isolate Marinobacter sp. EMB5 produced a lipase with interesting surfactant and organic solvent stability. The lipase is prone to aggregation. It was purified 28.2 fold by sizeexclusion chromatography on Sephacryl S200 column. The active form of lipase occurred in aggregated form. The active form lipase was stable in a wide range of organic solvents at 50% (v/v) concentration even up to 24 h. This property was well correlated to its retaining its secondary and tertiary features. It was haloalkaline in nature, having an optimum pH at 9.0 and salt 2% (w/v), respectively. It was stable across a wide pH range of 5.0 to 10.0 and temperature up to 60°C. Thus, the Marinobacter sp. EMB5 lipase emerges as a novel halo-alkaline lipase which could be successfully purified by Sephacryl S200.


extfieldsofstudy: Biology Medicine

text:
Fungal Diversity and Community Composition of Culturable Fungi in Stanhopea trigrina Cast Gibberellin Producers

Stanhopea tigrina is a Mexican endemic orchid reported as a threatened species. The naturally occurring microorganisms present in S. tigrina are unknown. In this work, we analyzed the diversity of endophytic and epiphytic culturable fungi in S. tigrina according to morphological and molecular identification. Using this combined approach, in this study we retrieved a total of 634 fungal isolates that presented filamentous growth, which were grouped in 134 morphotypes that were associated to 63 genera, showing that S. tigrina harbors a rich diversity of both endophytic and epiphytic fungi. Among these, the majority of the isolates corresponded to Ascomycetes, with Trichoderma and Penicillium as the most frequent genera followed by Fusarium and Aspergillus. Non-ascomycetes isolated were associated only to the genus Mucor (Mucoromycota) and Schizophyllum (Basidiomycota). Identified genera showed a differential distribution considering their epiphytic or endophytic origin, the tissue from which they were isolated, and the ability of the orchid to grow on different substrates. To our knowledge, this work constitutes the first study of the mycobiome of S. tigrina. Interestingly, 21 fungal isolates showed the ability to produce gibberellins. Almost half of the isolates were related to the gibberellin-producer genus Penicillium based on morphological and molecular identification. However, the rest of the isolates were related to the following genera, which have not been reported as gibberellin producers so far: Bionectria, Macrophoma, Nectria, Neopestalotiopsis, Talaromyces, Trichoderma, and Diplodia. Taken together, we found that S. tigrina possess a significant fungal diversity that could be a rich source of fungal metabolites with the potential to develop biotechnological approaches oriented to revert the threatened state of this orchid in the near future.

INTRODUCTION
Fungi play a central role in most ecosystems and they have important functions in soil and plant habitats. It is now clear that the microbiota associated with plants contribute to maintain their biological diversity in terrestrial ecosystems through different biological processes (Coats and Rumpho, 2014;Hardoim et al., 2015). In this regard, the fungal population associated with plants contributes to the adaptation process in response to biotic and abiotic stress, it is involved in the defense against pathogens and it has been related to the production of secondary metabolites with antimicrobial activity or with the synthesis of different phyto-hormones including gibberellins (Tsavkelova et al., 2008;Hajiboland et al., 2010;Contreras-Cornejo et al., 2011;Khan et al., 2012Khan et al., , 2015b. Gibberellins play an essential role in regulation of growth and development of angiospermic plants (Bahalla et al., 2010), they are associated with several plant growth and development processes as seed germination, stem elongation, flowering, and fruit development (Bilkay et al., 2010;Lu et al., 2016).
Fungi can be found as epiphytes (on the surface of the plant) or as endophytes (inside the plant tissue). Both populations are growing apart only by millimeters nevertheless they differ greatly in their composition (Santamaría and Bayman, 2005). Epiphytes grow on the surface with contact to outer environment, whereas endophytes are in contact with the inner microenvironment. It is unclear to what extent plants control which epiphytes are able to enter the leaf, and to what extent endophytes may affect this process and the mycelial growth of fungi complicates the difference between both pupulations, while comparison of endophytic and epiphytic floras may help to determine the basis for selectivity. The coexistence of epiphytic and endophytic microorganisms contributes to maintain the conditions needed for plant health, plant protection (Andrews and Harris, 2000;Santamaría and Bayman, 2005;Rodriguez et al., 2009), and microbial biodiversity (Huang et al., 2008;Kharwar et al., 2010), allowing a global homeostatic state. For this reason, further knowledge of the fungal mycobiota residing in plants is decisive to understand fungal ecology and to generate approaches for plant conservation (Yuan et al., 2009).
Mexico possess a great diversity of orchids with around 1,260 species and 170 genera (Hágsater et al., 2005; from which 40% correspond to endemic species. Unfortunately, in spite of being one of the largest families of flowering plants of the planet, it is also of the most threatened. Different factors including pollution, climate change, and human activities (Rands et al., 2010) have had a negative impact on orchid diversity. In Mexico, 188 orchid species are categorized as endangered by the government (SEMARNAT, 2010). Stanhopea is a neotropical orchid genus consisting of approximately 40 species out of which 13 occur in Mexico. Although the species belonging to this genus are widespread through Mexico, Central, and South America, some of the species are endemic in Mexico. Stanhopea tigrina is an endemic orchid in Mexico that is considered threatened at the state-level. This orchid shows beautiful massive flowers (18 cm in diameter) with a sweet and penetrating aroma that makes them very attractive for marketing (Soto-Arenas and Solano-Gómez, 2007) and due to its Mexican endemic nature, it was chosen as the symbol of the Mexican Orchid Association. Concerns about the decline of S. tigrina populations in Puebla State and in Mexico require the development of efficient and ecologically viable strategies to preserve the species. Given the importance of the microbiome in the extension of the plants' ability to adapt to many kinds of environmental conditions and changes (Vandenkoornhuyse et al., 2015), it is important to identify the associated microbiome in order to develop strategies that allow its conservation. On the other hand, fungi isolated from orchids may serve as beneficial species that could be used in orchid propagation and reintroduction, improving acclimatization and vigor (Brundrett, 2007;Yuan et al., 2009). However, to our knowledge there are no reports dealing with the diversity of fungi living in the S. tigrina, one of the most spectacular orchids in Mexico.
In this work, we analyzed the fungal diversity of both epiphytic and endophytic fungi in the threatened Mexican endemic orchid S. tigrina (collected from Cuetzalan del Progreso, Puebla, México) according to morphological and molecular identification. Additionally, we examined the ability of the isolates to produce gibberellins and we uncovered several fungal genera as gibberellin producers. Our results indicate that S. tigrina harbors a rich diversity for both epiphytic and endophytic fungi that could be a source of metabolites with a positive effect on the development of this endangered species. With this work, we want to contribute to the knowledge of the filamentous fungal diversity associated to S. tigrina but also to provide some tools that could allow the development of alternative strategies that could help to revert the threatened state of this orchid.

Study Site
Samples were taken from the Botanical Garden Xoxoctic (20 • 2 ′ 22.63 ′′ N, 97 • 30 ′ 32.16 ′′ W) located in the municipality of Cuetzalan del Progreso, Puebla, Mexico, which corresponds to a natural area devoted to preserve and investigate the biological richness of the Sierra Norte de Puebla. This location corresponds to a tropical mountain cloud forest ecosystem, to an altitude of 1000 masl, with semi-warm to warm humid climate and rainfall throughout the year (PIGEU, 2009). Considering the endangered status for this species, only six plants and one flower of S. tigrina were collected, from which one was lithophyte, two grew as epiphytes, and three were cultivated on a substrate that contained coconut fiber and soil.

Fungal Isolation and Cultivation
For fungal isolation, we took 2 g of fresh and healthy tissue for each part of the plant (leaf, pseudobulb, root, or flower). The procedure was performed for the six plants independently. To isolate fungal epiphytes, weighed and washed in 100 ml of isotonic saline solution with constant stirring for 3 min. Then, tissues were removed and with the washing suspension we performed serial dilutions (10 −1 , 10 −2 , and 10 −3 ) and 100 µl of each dilution were plated in triplicates in different media: Potato Dextrose Agar (PDA, MCD LAB), Malt Extract Agar medium (MEA, Difco), and V8 juice agar. The composition of these media is the following; PDA: potato infusion 20%, dextrose 2%, agar 1.5%; MEA: malt extract 1.2%, dextrin 0.2%, glycerol 02%, peptone 0.07%, agar 1.5%; V8 juice agar: V8 juice 200 ml/l, 3 g/l CaCO 3, and 20 g/l agar. To recover fungal endophytes, tissues were rinsed with water and surface-sterilized in a sequence of 70% ethanol for 2 min followed by 1% NaClO 2 during 2 min, and finally rinsed twice in sterile distilled water. Disinfected plant tissues were cut and macerated. The extract obtained was recovered and serial dilutions of 10 −1 , 10 −2 , and 10 −3 were performed. 100 µl of each dilution was plated on PDA, MEA, and V8 juice agar; the plating was performed in triplicate. Rhizospheric fungi were also isolated by taking 1 g of sample which was placed in 100 ml of isotonic saline and the resulting suspension was used to perform serial dilutions of 10 −1 , 10 −2 , and 10 −3 ; from each dilution, 100 µl were taken and plated on PDA, MEA, and V8 juice agar. All plates were incubated at 28 • C for 1 month. Plates were checked daily and each emerging fungal colony was transferred onto a new PDA plate until axenic cultures were obtained.

Phenotypic Identification of the Fungal Isolates
Individual strains with apparent distinct morphology were subcultured until obtaining a pure isolate. In order to identify distinct and representative morphologies, isolates were plated on PDA and incubated for 1 week at 28 • C. To induce sporulation or formation of distinctive structures that could be used to identify fungal genus, isolates were grown under microculture conditions. Briefly, a sterile PDA agar cube (1 cm) was cut and placed on a slide where the fungal isolate was inoculated and then a coverslip was placed on top. This system was deposited in a petri dish with humid chamber environment and incubated for 1 week at 28 • C. After the incubation, the coverslip was removed and the fungal growth was stained with lactophenol cotton blue. Morphological characteristics of the isolates were challenged by the identification keys. These isolates were then classified into morphotypes according to the growth rate, color, texture, and morphology of their colonies. Non-sporulating fungal morphotypes were grown in different media such as malt extract agar (MEA), Czapek agar (CZ), oat agar (OA) among others, combined with different light periods to stimulate sporulation.

Molecular Identification of the Fungal Morphotypes
To further analyze the identity of the fungal isolates recovered, different samples of each morphotype were considered according to the number of members in each group. When the morphotype included 1-3 elements, we used only one isolate to perform the molecular identification. In the same fashion, we used 3 isolates for groups with 7-10 elements and 5 isolates for groups with more than 10 elements. For these fungi, DNA was extracted as previously reported (Liu et al., 2000). Fresh mycelium was scraped from the surface of the agar plate and transferred into a clean tube with 500 µl of lysis buffer (400 mM Tris-HCl [pH 8.0], 60 mM EDTA [pH 8.0], 150 mM NaCl, 1% sodium dodecyl sulfate) and the mixture was vortexed in the presence of glass beads. After 30 min, 150 µl of precipitation solution (3 M potassium acetate, 5 M acetate, pH 4.8) were added to the tube, vortexed briefly and centrifuged at 10,000 rpm for 5 min. The supernatant was recovered and centrifuged again. Precipitation was performed with an equal volume of isopropyl alcohol at −80 • C for 20 min with centrifugation at 10,000 rpm for 5 min. DNA pellet was washed with 300 µl of 70% ethanol. After centrifugation at 10,000 rpm for 5 min, the supernatant was discarded and the DNA pellet was dissolved in 40 µl of water.
Fungal ITS-region and partial LSU were amplified using primers ITS1F (Gardes and Bruns, 1993) and LR5 (Hopple and Vilgalys, 1994;Moncalvo et al., 2000), which are specific for fungi. The resulting PCR products ranged from 1.3 to 1.7 kb in size. The LSU region is useful to identify fungi in public databases. PCR reactions were performed with 2 µl of genomic DNA in a total volume of 25 µl, using Taq DNA polymerase with ThermoPol buffer, according to the manufacturer's instructions (New England BioLabs) and further purified with the High Pure PCR purification kit (Roche). The following thermocycling program was used: 95 • C for 5 min (1 cycle); 94 • C for 35 s, 55 • C 35 s, 72 • C for 2 min (35 cycles); and 72 • C for 10 min (1 cycle). Amplification products were sequenced using primers ITS1F and LR5 using the services provided by Macrogen (Korea). Two sequencing rounds were performed in order to confirm the genetic features. Sequences obtained in this study were annotated in the GenBank (Supplementary Material).

Sequence Analysis
Forward and reverse sequence reads were analyzed using the CLC genomics workbench (QIAGEN). BLAST searches (www. ncbi.nlm.nih.gov/BLAST) were conducted to determine the close known relative for each case. Isolates were assigned as closest to a certain genus when identity was ≥95% (Sánchez et al., 2008;Chen et al., 2012;Tan et al., 2012). The evolutionary history was inferred by using the Maximum Likelihood Phylogeny 1.2 method with the Jukes Cantor nucleotide substitution model. The analysis involved 45 nucleotide sequences with a total of 100 replicates in the bootstrap analysis. Evolutionary analyses were conducted in CLC Main Workbench 7.9.1.

Qualitative Determination of Gibberellins
Pure fungal isolates were incubated in 5 ml of ICI medium, a medium with a low concentration of nitrogen that allows the production of gibberellins, for 10 days at 30 • C under dark conditions and agitation (200 rpm). After incubation, 0.2 ml of culture media were recovered and combined with 0.2 ml of ethanol (96%, v/v) and 2 ml of a cold mixture of sulfuric acid/ethanol (1:1 v/v). This mixture was incubated at 48 • C for 30 min (Candau et al., 1992) and then exposed to an UV lamp. Samples showing green fluorescence were considered positive for gibberellin production.

Quantitative Determination of Gibberellins by HPLC
Seven-day old mycelium of each strain was inoculated in a flask with 100 ml of Czapek broth (1% glucose, 1% peptone) and incubated at 30 • C in the presence of light (as described by Bahalla et al., 2010). After 10 days, the culture was filtered and 50 ml of the filtrate was recovered and the pH adjusted to 3 by adding HCl (1N). The extraction was performed three times using ethyl acetate (1:1). The organic phase was separated and passed through anhydrous sodium sulfate. The solvent was eliminated in a rotary evaporator at 40 • C and 10 rpm. The residue was dissolved in 50 µl of HPLC grade methanol and used for reverse phase HPLC analysis. The analysis was done in triplicate in a Hewlett Packard HPLC instrument (series 1100) with operation and data analysis using Chemstation software running on a HP Vectra 486 computer (Hewlett Packard Co.). Gibberellins were chromatographed using LiChrospher on a RP-18 column (250 × 4 mm i.d.). Acetonitrile and acidic water (0.01% H 3 PO 4 ) in a 70:30 ratio was used as mobile phase with a flow rate of 0.6 ml/min for 15 min. One microliter of each sample was injected and gibberellins were identified at 206 nm (Barendse et al., 1980;Bahalla et al., 2010). Gibberellin quantification was performed as previously reported (Bahalla et al., 2010). Full spectra were obtained for commercial GA 3 and GA 4 (1 µg/µl, Sigma) that were used as standard.

RESULTS
In this work, a total of 891 isolates were recovered from six plants of S. tigrina collected from the botanical garden Xoxoctic in Puebla, Mexico (Figure 1). Out of the 891 fungal isolates recovered from the six S. tigrina plants, 257 isolates did not show filamentous characteristics, but were confirmed as yeast according to Gram and Lactophenol Cotton Blue staining.
Moreover, their ability to produce gibberellins was also tested but any of the yeast showed the ability to produce this metabolite under the conditions assayed (data not shown).
For the 634 fungal isolates that presented filamentous growth, morphological identification was carried out according to colony and hyphal morphology of the macroscopic culture and characteristics of the spores under microculture conditions. According to these characteristics, the 634 fungal isolates were classified into 139 distinct morphotypes, according to the morphological characteristics observed (Supplementary Table 1). Insights into the distribution and diversity of the fungal isolates recovered from S. tigrina is further presented.

Distribution of Fungal Isolates in S. tigrina
In addition to the morphological characterization, molecular analyses were carried out to confirm the identity of the different morphogroups (Wang et al., 2005;Jin et al., 2013). Using this combinatory approach, we identified 134 morphotypes that comprised 63 genera, while 10 isolates grouped in 5 morphotypes could not be identified.
From the total culturable population recovered, 80% of the isolates were epiphytes, while 20% of them were endophytes. Considering tissue distribution, the greatest number of isolates were recovered from pseudobulb (270 isolates), FIGURE 1 | Location map of the study site. Sampling was performed in the Botanical Garden Xoxoctic (Cuetzalan del Progreso, Puebla, México), a natural area located to the Sierra Norte (Puebla) devoted to preserve species characteristic of this region. followed by the leaf and root with 226 and 197 isolates, respectively. A total of 153 isolates were found in the rhizosphere and 45 in the flower (Figure 2). In general, the proportion of epiphytes than endophytes isolated was similar for the different tissues studied. Surprisingly, the flower resulted a very rich tissue if we keep in mind that we only recovered one flower during the sampling process. Considering that the flowers of this orchids only last a few days, the appearance of the flower at the sampling time was timely.
Concerning the genera identified, a total of 63 genera was established, being Trichoderma the most recurrent group identified showing 124 isolates, while the second most frequently recovered was Penicillium with 103 isolates (Figure 3). These were the dominant genera retrieved from S. tigrina, followed by Fusarium and Aspergillus with 40 and 33 isolates, respectively. The infrequent groups that presented only one isolate included Alternaria, Cladophialophora, Cochliobolus, Colletotrichum, Fusicolla, Gibberella, Hypocrea, Phialemonium, Phoma, Pseudobotrytis, Schizophyllum, and Talaromyces. Considering the provenance of the isolate, we found that the epiphytic population comprised 57 genera, while 32 genera constituted the endophytic population. On the other hand, we identified 33 genera in the rhizospheric community (Figure 3). Even when a total of 25 genera were found both in the endophytic and epiphytic populations, we also noticed differences in the composition of the two groups. In this regard, 32 genera were found only in the epiphytic population, while the following fungal taxa were recovered only as endophytes: Alternaria, Annulohypoxylon, Anthostomella, Colletotrichum, Letendraea, and Phaeosphaeriopsis (Figure 3). Interestingly, Trichoderma was frequently isolated both as epiphyte and from the rhizosphere.
We next analyzed the genera distribution for the different plants (Figure 4) and we found that plant 4, which was grown on substratum showed the highest number of associated epiphytic genera, while plant three that grew as litophyte presented the lowest number of fungal genera isolated ( Figure 4A). Concerning the endophytic community, plant 1 showed the greatest diversity of genera ( Figure 4B).

Tissue Distribution of the Fungal Isolates
In this study, we recovered fungal isolates from the following tissues in S. tigrina: leaf, pseudobulb, root, and flower. In the roots, the presence of mycorrhizal fungi was sought, however, the presence of pelotons was not observed and no isolates belonging to this type of fungi was obtained. The composition and abundance of the isolated genera varied according to the host tissues investigated. The tissue with the greatest number of associated genera was the leaf with 40, followed by pseudobulb with 32 and 27 from the root, while only 10 isolates were recovered from the flower (Figure 2). For all the tissues studied, epiphytes were retrieved from the surface, while endophytes were obtained from macerated disinfected tissue. Fungal isolates were also recovered from the associated rhizosphere. Out of the 45 genera recovered from the leaf, 12 were found in both the endophytic and epiphytic populations, while 30 genera were recovered only as epiphytes. In the case of the pseudobulb, we found 11 genera in both populations, whereas 19 genera were associated to the epiphyte population and five genera were exclusively endophytic. In the root, 12 genera were ubiquitous while 10 and 8 genera were found only as epiphytic or endophytic, respectively. Finally, in the flower we identified 9 epiphytic genera and only Paecilomyces was encountered as endophyte ( Table 2).
Regarding tissue distribution of epiphytes, the leaf was the tissue with the greatest diversity with 38 genera associated, followed 29 in pseudobulb, 21 in root and only 9 in flower, while 33 genera were recovered from the rhizosphere (Figure 3). The most frequent fungal genera were Trichoderma, Penicillium, Fusarium, Mucor, and Aspergillus, which were found in all tissues ( Figure 3A). However, we found tissue specificity for some genera. This was the case for Cochliobolus, Diplodia, Elaphocordyceps, Eutypa, Fusicolla, Gibberella, Hypocrea, Nigrograna, Phoma, Thyridaria that were recovered only from the leaf. On the other hand, Phialemonium, Pseudobotrytis, Roussoella, and Umbelopsis were found only in pseudobulb while Bionectria, Cladophialophora, Microsporum, Phialemoniopsis, Pyrenochaeta, and Talaromyces seemed exclusive from rhizosphere.
On the other hand, for the endophytic population we found a more balanced abundance of fungal isolates. In this case, out of the 32 genera identified, 20 were found in the root, 16 in pseudobulb, 15 in leaves and only one in flower ( Table 1). Regarding the identity of the isolated genera, only Paecilomyces was identified in all tissues. Consistent with our observations of the epiphytic population, we discovered the following tissuespecificity: Chaetomium, Eutypella, Geotrichum, Hypoxylon, Mucor, Pestalotiopsis, Phaeosphaeriopsis were identified only in the leaf, Alternaria, Bionectria, Daldinia, Macrophoma, Phialemonium, and Pyrenochaeta were found only in the root, while Cladosporium, Colletotrichum, Letendraea, Nectria, and Phialemoniopsis were retrieved only from the pseudobulb ( Figure 3B).

Fungal Distribution According to S. tigrina Growth Habit
Considering the ability of S. tigrina to grow in different substrates, in this study we recovered fungal isolates from one lithophyte plant, two epiphyte specimens and three orchids that grew on substrate. When we compared the abundance of genera according to these growth conditions, we realized that there were 65 genera recovered from plants that grew as epiphytes and 58 from orchids grown on substrate. The similar abundance of genera observed in orchids grown as epiphytes or on the substrate was noticed for both the epiphytic and endophytic fungal populations, albeit their composition was different. On the other hand, we recovered only 28 genera from the lithophyte orchid. Insights into the special composition of the epiphytic and endophytic fungal populations discovered are further presented (Figure 5).

Capability of Some Fungal Isolates to Produce Gibberellins
As part of our ongoing efforts toward finding novel alternatives to revert the threatened state of S. tigrina, we investigated the ability of these fungal isolates to produce secondary metabolites. It is also important to identify these potentially bioactive fungi for a better understanding of the characteristics of the relevant fungal community associated to the orchid. In the search of fungal isolates from S. tigrina that could have a positive impact on plant development, we performed a general screening to identify gibberellin-producing isolates using a qualitative method described previously (Candau et al., 1992). Using this approach, we tested all the 891 isolates and found that 21 of them were positive for gibberellin production using the qualitative test. For these 21 isolates, the production of gibberellins was quantitatively determined by HPLC, using commercial GA 3 (Sigma G7645) and GA 4 (Sigma G7276) as standards (Supplementary Material). This analysis showed the presence of GA 3 in the 21 isolates, being STF062 the isolate with the highest production of GA 3 (4.1 µg), followed by STF624 that produced 2.0 µg of GA 3 . Other gibberellin producers were isolates STF282, STF524, STF526, STF538, STF599, STF637, STF657, STF756, and STF757, all with gibberellin production around 1 µg (Table 3). Even when the assay was designed to identify both GA 3 and GA 4, in most isolates only the production of GA 3 was detected and the production of both gibberellins was observed only for isolate STF723, with a predominant production of GA 4 (2.1 µg) over GA 3 (0.1 µg).

Molecular Identification of Gibberellin Producing Isolates
In addition to the morphological characterization, molecular analyses were carried out to confirm the identity of the 21 isolates recovered from S. tigrina that were determined as gibberellin producers and the results are included in Table 4. The ITS1-5.8S-ITS2 sequences of these isolates were compared to the sequences annotated in the GenBank and using this approach, we found that isolates STF62, STF147, STF149, STF538, STF591, STF599, STF654, STF723, and STF859 were associated to various reported sequences for the genus Penicillium. Isolates STF664, STF673, and STF860 showed the highest similarity with the Xylariales order. The ITS sequence for the isolate STF526 did not show significant homology to any known cultured fungal species. The other isolates were associated to the following fungal species: Arthrinium marii (STF757), Bionectria cf Ochroleuca (STF624), Macrophoma (STF282), Nectria pseudotrichia (STF753), Neopestalotiopsis sp. (STF524), Talaromyces funiculosus (STF343), Trichoderma sp. (STF864), and Diplodia quercivora (STF756). The sequences used for the molecular identification were handled in order to construct the phylogenetic tree shown in Figure 6. The phylogenetic analysis of the sequences divided the strains into two main clades. The first clade comprising 12 isolates includes two classes: Eurotiales and Botryosphaeriales. The Eurotiales clade contains several wellsupported branches and represents basically two genera: Penicillium and Talaromyces. The largest group shows a bootstrap support of 96% and includes different known strains for the genus Penicillium. Isolates STF062, STF654, STF599, STF859, and STF599 were all grouped with Penicillium oxalicum and  in a different branch STF723 was also associated to this group. Isolate STF149 and Penicillium charlesii formed a group with a bootstrap of 86%. In a different clade, isolate STF538 was grouped with Penicillium pinophilum, while isolate STF343 clustered together with Talaromyces funiculosus with a bootstrap support of 100%. Phylogenetic analysis suggests that isolates STF062, STF149, STF538, STF591, STF599, STF654, STF723, and STF859 belong to the genus Fusarium, while STF343 belong to the genus Talaromyces. The Botryosphaeriales clade was supported for a bootstrap of 100% and groups Macrophoma theicola with the isolate STF282 in one clade and isolate STF756 with Diplodia quercivora in a separate clade (Figure 6), associating the isolates with the corresponding genus. Within the Xylariales clade, STF864 formed a group with Trichoderma asperellum and STF624 clustered together with Bionectria cf. ochroleuca, both with a bootstrap support of 100%. STF664 and STF673 were grouped with Xylaria cf. heliscus with a bootstrap value of 93%, while STF860 was grouped within the Xylariaceae family. Therefore, phylogenetic analysis suggests that STF864 belong to the genus Trichoderma, STF624 relates to the genus Bionectria, and STF664, STF673 match the genus Xylaria, while STF860 associates with the Xylariaceae family.
The final clade groups two isolates and two genera corresponding to Hypocreales. On one hand, we have isolate STF524 grouped with Neopestalotiopsis sp. with a strong bootstrap of 100% and in separate clades STF757 releted to Arthrinium sp. with a bootstrap value of 87%. Given that all the isolates identified as gibberellin producers, four sequences for reported basidiomycetes were used as outgroup.
Interestingly, we report here for the first time several genera as fungal partners for orchids, including the following: Aplosporella, Microsporum, Mucor, Trichophyton, Diplodia, Fonsecae, and Scedosporium, indicating that the composition of the fungal communities associated to different orchid species should be further analyzed. Even when these genera correspond to endophytes, pathogens, or latent pathogens in other hosts (Meletiadis et al., 2002;Becker, 2003;Burgess et al., 2004;Prusky et al., 2004;Cortez et al., 2008;Lazzizera et al., 2008;Leelasuphakul et al., 2008;Slippers et al., 2009;Taylor et al., 2009;Twizeyimana et al., 2013;Dreaden et al., 2014;Dou et al., 2017), their biotechnological potential as secondary metabolite producers (Malonek et al., 2005;Bi et al., 2013)  should also be considered when designing future applications to rescue this endangered orchid. In this regard, fungal isolates reported here could have a strong impact on the health of S. tigrina by maintaining a balance in the composition of the associated microbiome (Omacini et al., 2001), serving as a defense against different pathogens or helping to deal with biotic stress (Brundrett, 2006). Thus, a better knowledge of the fungal community associated with S. tigrina could help to preserve endangered species like this orchid from further destruction.

Epiphytic and Endophytic Fungal Communities
In this work, a total of 891 fungal isolates was recovered using a culture-dependent approach to identify both epiphytes and endophytes, revealing the richness of the fungal community present in S. tigrina plants. This is the first report about culturable fungi isolated from this endangered orchid. Endophytic fungi have been reported as mutualists that could show entomopathogenic activity or produce biologically active secondary metabolites (Stone et al., 2000). In orchids, endophytic fungi could exhibit strong antibacterial or antifungal activities as well as plant-growth promoting effect (Chen et al., 2010;Xing et al., 2011). These findings support the notion that endophytic fungi isolated from S. tigrina could have potentially diverse applications. On the other hand, it has been shown that epiphytic fungi are determinant for plant health, plant protection (Andrews and Harris, 2000;Santamaría and Bayman, 2005;Rodriguez et al., 2009), and microbial biodiversity maintenance (Huang et al., 2008;Kharwar et al., 2010). However, the relationship between fungal epiphytes and orchids is practically non-existent. In this study, we obtained a higher number of epiphytic isolates than endophytes in all tissues investigated. This behavior has been reported for other plants including Coffea arabica leaves and Eucaluptus citriodora (Santamaría and Bayman, 2005;Osono, 2008;Kharwar et al., 2010). It would be relevant to examine both the endophytic and epiphytic fungal populations associated with orchids to fully uncover the ecological potential associated to these plants as a valuable source of biotechnological applications.
Current studies of orchid-associated fungi are mainly focused on mycorrhizae (Kristiansen et al., 2001;Yamato et al., 2005;Huang et al., 2014;Oliveira et al., 2014) and endophytic fungi associated with roots and leaves (Chen et al., 2010;Chutima et al., 2011;Tan et al., 2012), leaving aside the study of fungi present in other tissues. In this work, we examined both the endophytic and epiphytic fungal populations in the orchid to fully consider the great microbiological richness associated to this plant. We found that the leaf was the tissue with the greatest diversity of epiphytes, while the root exhibited the highest diversity of endophytic genera, in accordance with the significant microbial interactions that occur in these plant tissues and with previous reports (Vendramin et al., 2010;Sudheep and Sridhar, 2012;Bunch et al., 2013;Chen et al., 2013). However, we also found that other tissues like the pseudobulb showed a considerable diversity for both epiphytic and endophytic isolates, supporting the notion that other tissues should also be considered in this type of studies. Moreover, this capability of S. tigrina to relate with many fungal partners could represent a biological and survival advantage for the orchid (Otero et al., 2004).
Concerning genera distribution that conform the epiphytic or endophytic communities isolated from this orchid, we found Trichoderma and Fusarium as the most prevalent genera for the epiphytic and endophytic communities, respectively. It has been reported that Trichoderma produces enzymes involved in cell plant degradation and could be applied for biocontrol (Naher et al., 2014;Schmoll et al., 2016), while Fusarium produces secondary metabolites such as gibberellins (O'Donnell et al., 1998;Malonek et al., 2005) and protect plants from pathogenic isolates (Minerdi et al., 2011). Besides these prevalent fungi, we identified 25 genera as part of both the epiphytic and endophytic communities in S. tigrina, where the most widely distributed genera were Paecilomyces, Penicillium, and Scedosporium. These fungal isolates could be applied to benefit the orchid by inducing growth promotion through hormone secretion, by generating plant resistance due to the activation of multiple defense signals or they could also show antagonistic activity against pathogens, as reported for other hosts (Kiewnick and Sikora, 2006;Hossain et al., 2007;Khan et al., 2011a;Radhakrishnan et al., 2013).
Even when several genera showed ubiquitous distribution, some appeared to have a preference. This was the case for Alternaria, Annulohypoxylon, Colletotrichum, and Phaeosphaeriopsis which were recovered only as part of the endophytic community and their natural ability to inhabit the orchid could be advantageous for future biotechnological applications. For example, Annulohypoxylon (previously referred as Hypoxylon) presents an endophytic stage as part of its life cycle and produces several secondary metabolites with antibiotic and antiparasitic activities (Piettre et al., 2002;Bills et al., 2012). On the other hand, we found that 31 genera were exclusively epiphytic, which could be related mainly to the general richness observed for this community.

Fungal Gibberellin Producers
It has been reported that GA 3 has the ability to alter orchid seedling morphology by elongating seedlings (Hadley and Harvais, 1968;Ávila-Díaz et al., 2009) and GAs are known to promote cell elongation and division in various plant tissues (Potter et al., 1993;Bianco et al., 1996;Ozga et al., 2002;Kazmierczak, 2003;Little and MacDonald, 2003;Bilkay et al., 2010). Orchid embryos typically form globular structures, expanding outward in all directions and germination could conceivably be enhanced by the action of phytohormones like gibberellins. Here, we identified several gibberellin-producing isolates in suitable yields according to concentrations that have shown biological activity in different plants (Hamayun et al., , 2010bKhan et al., 2011c;Waqas et al., 2012), suggesting that these isolates could be useful to improve propagation, adaptation, and conservation of S. tigrina, particularly at early stageS. When identified, these isolates were associated to 11 genera that comprise Penicillium and Arthrinium, which have already been reported as gibberellin-producers (Mitter et al., 2002;Kawaide, 2006;Khan et al., 2009;Bahalla et al., 2010;Leitão and Enguita, 2016) with the ability to enhance plant growth and to help the plants to resist abiotic stresses (Hamayun et al., 2010a;Khan et al., 2011bKhan et al., , 2015a.
Gibberellins play a relevant role in plant growth and development. However, many fungi that associate with plants or soil have not been tested for their ability to produce GAs. Here, we found that isolates with the ability to produce gibberellins displayed a high degree of similarity with the following genera, according to the fungal ITS regions: Bionectria, Diplodia, Macrophoma, Nectria, Neopestalotiopsis, Talaromyces, and Trichoderma. Even when we found a high similarity when identifying the isolates, further investigations will help to fully characterize the identity of these isolates and their ability to produce gibberellins and other useful metabolites. Finally, the physiological effects of this metabolite in orchids, particularly in S. tigrina remains to be elucidated.
Overall, this study shows novel information about the composition of the mycobiome present in S. tigrina, an endangered species of the rich endemic Mexican ecosystem. As found for other orchids, S. tigrina shows a complex fungal community that includes diverse groups, suggesting the probable relevance of its biodiversity in maintaining equilibrium with its environment. Moreover, interesting insights related to the composition of the epiphytic and endophytic communities and their distribution in the plant showed a great fungal richness for this orchid. Finally, we believe that the description of the natural mycobiome associated to S. tigrina and the capability of some of the isolates to produce secondary metabolites could be applied in different biotechnological strategies oriented to increase the population of S. tigrina in Mexico. This integrated approach could benefit delicate members of ecosystems, particularly for endangered species. Similar studies could be conducted to gain insights into the fungal microbiome associated to other plants, in order to identify microbial symbionts that may play a role in providing stability for the host in their particular ecosystem.

AUTHOR CONTRIBUTIONS
Overall approach of this study was designed by NM-M and RM-C. SS-C conducted the experiments. MC-L designed and supervised HPLC experiments. All authors were involved in experiment design, data analysis, and manuscript preparation.

ACKNOWLEDGMENTS
S. tigrina plants were identified by the Biologist Raúl Álvarez Mora (Xoxoctic Botanical Garden). PCR reactions were partially performed in the Functional Genomics Laboratory, at the University of Sherbrooke (Sherbrooke, Quebec, Canada) with the support of Dr. Benoit Chabot. SS-C received a PhD fellowship from CONACYT.