New antimicrobial compounds produced by Seltsamia galinsogisoli sp. nov., isolated from Galinsoga parviflora as potential inhibitors of FtsZ

A total amount of 116 fungal strains, belonging to 30 genera, were acquired from the rhizosphere soil and plant of Galinsoga parviflora. A strain SYPF 7336, isolated from the rhizospheric soil, was identified as Seltsamia galinsogisoli sp. nov., by morphological and molecular analyses, which displayed high antibacterial activity. In order to study the secondary metabolites of Seltsamia galinsogisoli sp. nov., nine compounds were successfully seperated from the strain fermentation broth, including two new compounds and seven known compounds. Their structures were elucidated based on spectral analysis including 1D and 2D NMR. All the seperated compounds were evaluated for their antimicrobial activities. Compounds 2, 5 and 1 displayed antimicrobial activities against Staphylococcus aureus with MIC values of 25, 32 and 75 μg/mL, respectively. Moreover, morphological observation showed the coccoid cells of S. aureus to be swollen to a volume of 1.4 to 1.7-fold after treatment with compounds 1, 2 and 5, respectively. Molecular docking was carried out to investigate interactions of filamentous temperature-sensitive protein Z (FtsZ) with compounds 1, 2 and 5.

Secondary metabolites coming from microorganism represent a large number of diverse components which have been treated as potential candidates for drugs [1][2][3][4][5] . Currently, due to the increase of antibiotic resistance, there is an urgent need for novel classes of lead compounds and novel mechanisms to confront the antibiotic crises 6,7 .
Microbial filamentous temperature-sensitive protein Z (FtsZ) is a novel target for drug discovery, which plays a key role in cell division 8,9 . The inhibitors of FtsZ prevent the cellular fission of bacteria, which lead to apoptosis of bacteria [10][11][12] . Therefore, morphological observation of microbial fission and molecular docking between the lead compound and FtsZ were accomplished to explore the possible mechanism 13,14 . Up to now, the discovered FtsZ inhibitors are divided into natural products (sanguinarine, berberine, totarol, curcumin and cinnamaldehyde) and synthetic small molecules (PC190723, UCM53, CCR-11) [15][16][17][18][19] . At present, PC190723 has been the candidate drugs to enter the clinical trials, which inspires scientists to make more effort to find potential FtsZ inhibitors as antibacterial agents [20][21][22] .
The endophytic fungi are known as a source of abundant secondary metabolites for functional bioactive substances. As we know, only a small amount of microbes have been studied so far, and a vast number of new taxa waiting for discovery, especially those separated from medicinal plants. This leads us to isolate and evaluate the potential pharmacological activity of bioactive compounds produced by endophytic fungi.
In the present study, the endophytic fungi diversity of the traditional Chinese herb, Galinsoga parviflora was surveyed and 116 fungal strains were isolated from the whole plant (stems, leaves and roots) which attributed to 30 genera. Forty-three percent of the strains revealed antimicrobial abilities against at least one kind of human pathogenic microorganisms. Strain SYPF 7336, presented the strongest antibacterial activity, could not be affiliated to any known taxon, so it was identified as a novel species of genus Seltsamia by phylogenetic analyses, given the name  The spectra of 1 were in part very similar to those of alter lactone except for the absence of a methylene group 24 . The HMBC spectra indicated the presence of long-range correlations ( Fig. 6) from H-1′ with C-2, C-3, and C-4, from H-5 with C-4a, C-7 and C-11b, from H-9 with C-7a and C-11, combined with the HSQC and HRESIMS data, the structure of compound 1 was verified as shown in Fig. 5 and it was given the common name Pyrenochaetayu.
Galinsogisoliyu (  2), one oxygenated methylene proton carbon (δ C 58.9), three methylene carbons (δ C 46.4, 32.4 and 23.5). The 1 H and 13 C NMR spectra of 2 were similar to those of 4-methyl-5,6-dihydro-2H-pyran-2-one except for the absence of two side chains 25,26 . The planar structure of 2 ( Fig. 6) was established by the 2D NMR data. The 2D NMR spectra of compound 2 ( Fig. 6) indicated the presence of long-range correlations from H-2 with C-1, C-3 and C-4, H-5  www.nature.com/scientificreports www.nature.com/scientificreports/ with C-1, C-14 and C-15, H-9 with C-11 and C-13 and H-15 with C-5 and C-14. According to the above evidence, the structure of 2 was verified as shown in Fig. 5 and it was given the common name Galinsogisoliyu.
The antimicrobial results of compounds 1-9. All the seperated metabolites were tested for antimicrobial effects against five common pathogenic bacteria, S. aureus, B. subtilis, P. aeruginosa, K. pneumonia and Bacillus cereus. The results were shown in Table 3.

Morphological observation and molecular docking. The cells of S. aureus treated with compounds
were observed carefully (Fig. 7). Interestingly, the coccoid cells of S. aureus were swelled to larger volume after treatment with compound 1 (1.4 fold), 2 (1.7 fold) and 5 (1.6 fold), respectively. In order to explain the possible mechanism, FtsZ, key protein of cell division 35 , was explored for molecular docking study.
The docking simulation of active compounds 1, 2 and 5 to FtsZ from S. aureus (PDB:ID 3VOB) (Fig. 8) resulted in the binding energies of −109, −125, and −113 kcal/mol, respectively (Table 4). Thus, compound 2 had the best binding energies with FtsZ. Furthermore, the binding patterns were also different. Compound 2 displayed four hydrogen bonds and one more hydrophobic bond to relevant residues comparing with compound 5 (Table 4). Compound 1 showed two hydrogen bonds and five hydrophobic bonds to relevant residues. However, an unfavorable bump LEU261 was observed for compound 1 (Table 4). Five hydrophobic bonds for compound 1 were observed and considered to make major contribution to the combinations. The interactions between FtsZ with compounds 1-2 and 5 are displayed in Table 4.

Discussion
All the endophytic fungi isolated from the rhizosphere of G. parviflora were fermented and the crude extracts of each strain were tested for the microbial activities (Table S1). Forty-three percent of the strains showed antimicrobial activities against at least one kind of human pathogenic microorganisms. These results provide references for further study of the strains.
Among the stains, SYPF 7336, showed the best antibacterial activity. The strain SYPF 7336 was carefully studied and identified as Seltsamia galinsogisoli sp. nov. by morphology and molecular analyses, and only pycnidia was observed whereas no perfect stage available. This is the first-found of pycnidia in the genus Seltsamia for only perfect stage was recorded whereas no asexual information in the publication in S. ulmi, which was isolated from Hapalocystis bicaudata on corticated Ulmus glabra in Norway in 2018 23 . Though differences of the reproductive body between the two species could not be compared, they are remarkably different species based on the Compd.  Table 3. Antibacterial effects of compounds 1-9. a As positive control. "+": antibacterial rate 0-30%, "+": 30-60%, "+++", 60-80%, "++++", 80-100%, "−" no antibacterial activities.   www.nature.com/scientificreports www.nature.com/scientificreports/ phylogenetic analyses (Fig. 2). Moreover, the difference between the two species are that Seltsamia galinsogisoli sp. nov. produces dark grey colonies with annular and radical grooves on MEA 23 . Seltsamia is a newly introduced genus, the finding of Seltsamia galinsogisoli sp. nov. expands the host range of this genus.

Staphylococcus
Another aim of this study is to isolate antimicrobial secondary metabolites secreted by the novel strain, Seltsamia galinsogisoli sp. nov. It is the first time to report secondary metabolites from the genus Seltsamia, family Cucurbitariaceae. Two new compounds (1-2) and seven known compounds (3-9) (Fig. 5) were purified, identified and tested for their antimicrobial abilities against S. aureus, B. subtilis, P. aeruginosa, K. pneumonia, and E. coli. As results, compounds 2, 5 and 1 displayed well antibacterial activities toward S. aureus with MIC values of 25, 32 and 75 μg/mL, respectively. These results from the present work provide further information about the diversity and activities of compounds in the genus Seltsamai.
FtsZ is a pop target for drug discovery in recent years. The gene of FtsZ has the ability of high conservation and presented almost in all bacteria 9,36 . In bacterial cytokinesis, FtsZ protein is the earliest known step to build a contractile ring on the inner surface of the cytoplasmic membrane 9,36 . The inhibitors of FtsZ might prevent the cellular fission of bacteria, which lead to apoptosis of bacteria. Therefore, morphological observation and molecular docking were carried out to search the possible interactions between the active compounds and FtsZ.
Thus, the cells of S. aureus treated with active compounds 1, 2 and 5 were observed to further study the possible antibacterial mechanism. Interestingly, As Fig. 7 showed, the coccoid cells of S. aureus were swollen to 1.4 to 1.7-fold volume after treatment with compound 1 (1.4 fold), 2 (1.7 fold) and 5 (1.6 fold), respectively. In order to explain this interesting appearance, FtsZ, the key protein of cell division 8,9 , was explored to illustrate the mechanism of cells that became swollen. Thus, a molecular docking study was carried out to verify the deduction.
The docking results are shown in Fig. 8 and Table 4. The FtsZ from S. aureus (PDB:ID 3VOB) displayed the docking score of the ligand (−167 kcal/mol) was the lowest. Compound 5 (−113 kcal/mol) displayed three hydrophobic bonds with THR309, ASN263, LEU209 residues but no hydrogen bonds with neighbouring amino acid residues. Compound 2 (−125 kcal/mol) formed four hydrogen bonds with the ILE228*2, THR309, VAL297 residues and four hydrophobic bonds with the ASN263*2,THR309 and THR265 residues. Although compound 1 formed two hydrogen bonds and five hydrophobic bonds with the residues, one unfavorable interactions between compound 1 and the active region or intramolecular of FtsZ were existed. Thus, compound 1 showed a weak ability to combine with the docking score of −109 kcal/mol. The docking scores −125 kcal/mol (2) −113 kcal/mol (5) and −109 kcal/mol (1), indicated that compounds 2 and 5 might form lower potential energies and more stable binding sites with the target protein FtsZ compared to coumpound 1 which validated the observed antimicrobial activities. Based on the antimicrobial activities, phenotypic consequences and docking studies, compounds 2 and 5 were identified as promising antimicrobial lead molecules.

Methods
General experimental procedures. Optical rotations were recorded using a P-2000 Digital Polarimeter (JASCO, United Kingdom) 37 . IR spectra were measured on an Equinox55 spectrophotometer in KBr discs (Bruker Optik BmbH, Ettlingen, Germany). The 1D-and 2D-NMR spectra were recorded at 600 for 1 H and 150 MHz for 13 C (Bruker, Rheinstetten, Germany). HR-ESI-MS data were acquired on a Bruker Customer micrOTOF-Q 125 mass spectrometer (MA, Germany). Solvents were purchased from Tianjin Kemiou Chemical Reagent Company (Tianjin, China), MeOH and CH 3 CN for HPLC analysis were chromatographic grades (Merck, Darmstadt, Germany). Silica gel (200-300 mesh, Qingdao Marine Chemistry Ltd, Qingdao, China) were used for column chromatography.
sampling, fungal isolation, morphological study. Samples of soil and plant were collected from the field of a traditional Chinese medical herb G. parviflora in Huludao city, Liaoning province, northeast of China (40°82′26.5″N, 119°78′52.0″E). The samples were conducted as described previously 38,39 . All plates were incubated at 26 °C and examined daily. Single colonies were picked and transferred to freshly prepared PDA plates. The single spore-origin strain was stored at 4 °C and/or conidia suspension in 20% glycerol for further study.

Conventional hydrogen bond
www.nature.com/scientificreports www.nature.com/scientificreports/ DNA isolation, PCR and sequencing. The mycelia grown in PDB at 26 °C for 7 days were prepared for DNA isolation. Total genomic DNA was extracted as described previously 40,41 . The internal transcribed spacer (ITS) region was amplified with primers ITS4 and ITS5 42 . The partial 28S ribosomal RNA (LSU) gene region was amplified with primers LROR and LR7 43,44 . The thermocycling conditions for amplifications had an initial denaturing step of 94 °C for 5 min, 32 cycles of 94 °C for 60 s, 55 °C for 30 s, 72 °C for 90 s, followed by a final elongation step at 72 °C for 7 min. A G1000 Thermal Cycler (BIOER, Hangzhou, China) was used for PCR amplification. Amplicons were verified with 1% agarose electrophoresis gel, and the expected bands were excised and purified with an AxyPrepTM gel purification kit (Axygen, Hangzhou, China). These fragments were cloned into the pEASY-T5 zero cloning kit (Transgen Biotech, Beijing, China) and followed by sequencing (Sangon Biotech, Shanghai, China). The nucleotide sequences of the genes have been deposited in GenBank (Table 1). phylogenetic analyses. All the sequences (Table 1) were aligned by Clustal X (Larkin, Blackshields et al. 2007) and Mega 7.0 45,46 . Datasets were analyzed using both maximum parsimony (MP) and Bayesian tree inference (BI). MP analyses were performed using PAUP* 4.0b10, a heuristic search option was chosen with random addition of sequences as 1,000 replications; gaps were treated as missing data 47 . BI analyses were run with MrBayes 3.2.4 using the GTR substitution model with gamma-distributed rate variation across sites and a proportion of invariable sites 48 . Two sets of four chains were executed until the standard deviation of split frequencies reached 0.01. Sample frequency was set at 100 and 25% of trees removed as burn-in.
Fermentation and extraction. This assay was performed according to our previous method 37 . Briefy, sterile water (53 ml) and rice (40 g) were mixed to an Erlenmeyer flask (250 ml), which were autoclaved at 121 °C for 30 min. The strain of SYPF 7336 (1 ml) was inoculated in each Erlenmeyer flask (250 ml × 120), which were cultivated at 28 °C for 30 days. The fermented material was extracted using ethyl acetate (12 L × 3) to give the crude extract (127 g). Then it was dissolved in 90% MeOH-H 2 O (1 L), and extracted by hexane (1 L × 3) to obtain the residue (62 g).
Minimal inhibitory concentration (MIC) of compounds 1, 2 and 5 were assessed against bacteria S. aureus (CMCC26003). The MIC values of the isolated compounds against human pathogenic bacteria were determined by the modified CLSI M38-A method 49,50 . Briefly, compounds 1, 2 and 5 were dissolved using DMSO with the final concentrations of 100, 50, 25, 12.5, and 6.25 ug/mL, respectivly for the MIC determination. The dosage-response curve was drawed according to different concentrations of compound to S. aureus cells growth inhibition rates. The MIC values were calculated from the dosage-response curves. DMSO and Ampicillin were used as the negative control and positive control, respectively. Molecular docking. This assay was performed according to our previous method 37 . Briefy, the crystal structure of protein obtained from RCSB Protein Data Bank (PDB Code 3VOB) were used for docking 51 . The 3D structures of the compounds 1-2 and 5 were prepared and Gasteiger-Hückel charges were added using Sybyl software (Tripos, America). The ligand, guanosine-5′-diphosphate, was subjected to energy minimization with Tripos force filed parameters 51 . Blind docking was carried out using Molegro Virtual Docker 4.0 (Molegro ApS, Aarhus, Denmark) program. The 3D docking grid was sufficiently large to cover the protein.

Morphological observation of bacterial fission.
To identify whether there are changes in morphology of S. aureus after treated with compounds 1-2 and 5, observations under a transmission electron microscope (TEM, HT7700, Japan) were performed. S. aureus cells were grown at 37 °C on an agar plate, then diluted by LB broth to an OD 600 of 0.2, and 25 μg/ml compounds 1-2 and 5 were added to the suspensions. Samples of treated cells and controls were further cultivated at 37 °C for 3 h. Then, the bacterial suspensions were dyed with 2% phosphotungstic acid (v/v = 1:1 pH 6.5) for 3-5 min, and transmission scan was performed as previously described 20 .