Pseudopeptides of Marine Vibrio spp. from Taiwan and Their Combined Treatment Effects with Commercial Antibiotics

Abstract

Vibrio strains, identified by 16S rDNA, were isolated from the marine environment surrounding Taiwan, revealing diverse bioactive effects, such as iron-chelating and antimicrobial activities. Notably, the hierarchical clustering dendrogram of mass spectrum profiles of the Vibrio strains using matrix-assisted laser desorption ionization time-of-flight, in contrast to the phylogenetic tree based on 16S rDNA sequencing analysis, exhibited a strong correlation with their observed bioactivities. Within this set, global natural products social molecular network analysis by LC-HRMS/MS highlighted that three strains, Vibrio tubiashii DJW05 – 1, Vibrio japonicus DJW05 – 8, and Vibrio fortis DJW21 – 4, shared similar bioactive pseudopeptides in the same cluster. Subsequent chromatographical isolation and purification yielded an unprecedented unsaturated diketopiperazine, (Z)-3-(2-methylpropylidene)-2,3-dihydropyrrolo[1,2-a]pyrazine-1,4-dione (1), along with a series of diketopiperazines, and a potential new annotated pseudopeptide (2), as well as three pseudopeptides, including andrimid (10), moiramide B (11), and moiramide C (12), and several alkaloids from V. tubiashii DJW05 – 1. Further investigation into the combined applications of the major antimicrobial compound and commercial antibiotics revealed that andrimid (10) displayed significant inhibitory effects against gram-positive Staphylococcus aureus, and gram-negative Escherichia coli, Salmonella typhimurium, and Acinetobacter baumannii, but not Pseudomonas aeruginosa. Nevertheless, the potential for synergistic and additive effects of andrimid (10) with certain antibiotics remains, presenting valuable prospects for medicinal applications.

Keywords

Vibrio spp. - Biotyper - molecular network analysis - pseudopeptides - andrimid - combination treatments - antibiotics - synergism - antagonism

Introduction

Vibrio, a gram-negative bacterial genus commonly found in marine environments, is known for its ability to produce toxins, such as tetrodotoxin [1], [2], and various types of bioactive secondary metabolites [3], [4], [5], [6], such as vibrindole A [4], [5], an indole derivative against Staphylococcus aureus, vitroprocines [7], [8], a series of amino alcohols against Acinetobacter baumannii, andrimid [9], a pseudopeptide with wide antimicrobial activity, and amphibactins [10], lipopeptides with an iron-chelating effect. Therefore, it is a worthwhile consideration to explore potential Vibrio species and efficiently uncover their bioactive components.

Mass spectroscopies stand out as one of the most sensitive detectors in biological and biochemical research. For example, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers the capability to detect macromolecules without the need for an extraction process [11], which were further used to establish the metabolite profile of microorganisms (MALDI mass spectrum profiles) for Biotyper analysis [12], [13]. HRMS/MS serves as a highly sensitive detector tandem with liquid chromatography for tracing minor metabolites in a crude extract. In addition, the fragmentation information of the molecules by MS/MS could be submitted to the global natural products social molecular networking (GNPS) website (https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp) for molecular network analysis, in which the cosine similarity of all compounds in the extracts, compared to each other using a specific mathematic algorithm could be constructed and visualized in the software platform Cytoscape [14]. As a result, the GNPS, emerging as a mass spectrometry-based platform, offers an overview of the molecular features of the metabolomics of an extract. It has proven to be a potent tool for rapidly identifying known compounds prior to isolation work, and even for targeting previously undescribed compounds [14], [15].

By combining the various contemporary techniques mentioned above (Fig. 1S, Supporting Information), we would like to explore bioactive ingredients from marine microorganisms. Fourteen Vibrio strains isolated from a marine environment surrounding Taiwan ( Table 1S and Fig. 2S , Supporting Information) were identified by 16S rDNA analysis in our lab. Some of them exhibited varying degrees of antimicrobial effects ( Fig. 3S , Supporting Information). Additionally, a constructed MALDI-TOF MS Biotyper dendrogram of them by utilizing modern mass techniques ( Figs. 4S-8S , Supporting Information) seems to correlate with their bioactivities much better than the traditional phylogenetic analysis by 16S rDNA sequencing analysis ( Fig. 9S , Supporting Information). Noteworthy is that Vibrio tubiashii DJW05 – 1, Vibrio japonicus DJW05 – 8, and Vibrio fortis DJW21 – 4 exhibited the most potential antimicrobial activity and also shared a closer relationship in the Biotyper dendrogram compared to the 16S rDNA phylogenetic tree ( Table 2S and Figs. 3S and 9S , Supporting Information).

Furthermore, we tried to perform GNPS analysis of these bioactive Vibrio strains. The clusters in the GNPS analysis revealed the plausible presence of pseudopeptides, diketopiperazines, vitroprocines, and others ([Fig. 1]). Following scale-up cultivation of these Vibrio strains, these targeted compounds were isolated and their structures elucidated as a series of diketopiperazines, including one unprecedented compound (1), along with seven known cyclodipeptides (3 – 9), and four pseudopeptides, including one potential new annotated compound (2), as well as three known ones (10 – 12). The structures of all isolates were identified by 1D and 2D NMR and MS spectroscopic data. Among them, andrimid (10) was found to be the major antimicrobial compound from these three Vibrio strains (DJW05 – 1, DJW05 – 8, and DJW21 – 4).

Fig. 1 Molecular network generated by Cytoscape software (https://cytoscape.org/) of six extracts from marine Vibrio strains with antimicrobial or iron-chelating activities. MN consists of the ions with m/z between 150 and 1500. Boxes A – D are the clusters of nodes in the MN. The relative quantification of each ion from different extracts in the node is displayed as a pie chart diagram. The proportions of the pie chart indicate that the areas of the corresponding extracts were recorded from the area of the mass chromatogram. ^aThe structures were determined by HR-ESI-MS/MS fragmentation patterns; ^bthe compounds were isolated from Vibrio spp.; ^cthe compounds have been published in pervious study [17].)

On the other hand, there is a pressing issue of antimicrobial resistance in clinical treatment. The combined treatment of antibiotics with other antimicrobial agents has gained significance. Considering this, we would like to evaluate the combined effects (synergistic, additive, or antagonistic) of the major antibacterial compound andrimid (3) as a cotreatment with commercial antibiotics in the quest for new medicinal applications of this pseudopeptide, which was proposed as a potential inhibitor of acetyl-CoA carboxylase against gram-positive and gram-negative bacteria [16].

Results and Discussion

To streamline the search for bioactive metabolites from these Vibrio strains, high-resolution liquid chromatography tandem mass spectrometry (LC-HRMS/MS) data of these Vibrio strains were used to generate a mass spectral molecular network (MN) ([Fig. 1]). The LC-HRMS/MS data of the six MeOH extracts from Vibrio strains H100-10, DJW05 – 1, DJW05 – 8, DJW21 – 4, and DJW23 – 6, each associated with different colors, were utilized to construct an extensive MN, while the strain QWI-06 was used as a positive control ([Fig. 1]). A series of amino alcohols, vitroprocines, were identified previously in our lab [17]. Through a search for known compounds and analogues in the MS/MS database of the GNPS website, we were able to annotate a series of diketopiperazines and vitroprocines in two isolated clusters ([Fig. 1 a, b]). After scaling up the cultivation of bioactive Vibrio sp. DJW05 – 1, an unprecedented unsaturated diketopiperazine, (Z)-3-(2-methylpropylidene)-2,3-dihydropyrrolo[1,2-a] pyrazine-1,4-dione (1), was isolated and identified, along with seven known diketopiperazines, including cyclo-(L-leucine-L-proline) (3) [18], [19], cyclo-(glycine–L-proline) (4) [20], cyclo-(D-alanin-4-trans-hydroxy-D-proline) (5) [21], cyclo-(L-proline-L-phenylalanine) (6) [18], [20], [22], cyclo-(D-proline-L-phenylalanine) (7) [23], cyclo-(4-trans-hydroxyl-L-proline-L-phenylalanine) (8) [23], and cyclo-(L-leucine-L-valine) (9) [24], [25]. In addition, a distinct MN cluster ([Fig. 1 d]) was observed, exclusively associated with Vibrio strains DJW05 – 1, DJW05 – 8, and DJW21 – 4, demonstrating dissimilarity from other Vibrio strains. Despite no credible matches being found after extensively searching the GNPS database, certain antimicrobial pseudopeptides, andrimid (10), moiramides B (11), and C (12), were recommended by the subsequent exploration of molecular weights in the Reaxys database. Furthermore, we isolated a potential new annotated pseudopeptide (2) as well as andrimid (10), moiramides B (11), and C (12) from the scaled-up cultivations of Vibrio sp. DJW05 – 1. Moreover, the additional five known compounds, uracil (13) [26], thymine (14) [27], [28], indole-3-aldehyde (15) [29], trisindoline (16) [30], [31], and indole-3-glyoxylamide (17) [32], [33], were isolated from the strain DJW23 – 6 and their structures confirmed with comparison of their spectroscopic data (UV, IR, ¹H and ¹³C NMR, and MS) with those in the literature.

Compound 1 was isolated as a yellow amorphous solid. Its molecular formula, C₁₁H₁₂ N₂O₂, was established through HRESIMS, which showed a sodium adduct ion [M + Na]⁺ at m/z 227.0792 (calcd. for C₁₁H₁₂ N₂NaO₂ ⁺, m/z 227.0791 [M + Na]⁺), conducting seven indexes of hydrogen deficiency (IHD). The infrared (IR) spectrum showed the presence of a carbonyl at 1667 cm⁻¹. ¹³C NMR and HSQC spectra showed 11 carbon signals, including two carbonyl carbons (δ _C 155.1 and 154.9), four olefinic carbons (δ _C 132.7, 120.8, 117.8, and 114.8), two quaternary carbons (δ _C 125.6 and 124.9), one methine carbon (δ _C 25.5), and two methyl carbons (δ _C 20.8 for both) ([Table 1]). The ¹H NMR spectrum of 1 showed four aromatic signals at δ _H 7.70 (1H, dd, J = 3.2, 1.5 Hz), 7.13 (1H, dd, J = 3.6, 1.5 Hz), 6.61 (1H, dd, J = 3.6, 3.2 Hz), and δ _H 6.31 (1H, d, J = 10.5) as well as a set of isopropyl signals at δ _H 2.93 (1H, m) and δ _H 1.12 (6H, d, J = 6.4 Hz) ([Table 1]). The ¹H-¹H COSY correlations between δ _H 7.70 (H-7), 6.61 (H-8), and 7.14 (H-9) and between δ _H 6.32 (H-10), 2.94 (H-11), and 1.12 (H-11a, 11b) indicated the presence of a pyrrole group and the isobutyl moiety in 1. The HMBC correlations between δ _H 6.31 (H-10)/δ _C 155.1 (C-2) and δ _H 2.93 (H-11)/δ _C 125.6 (C-3) supported that the isobutyl moiety was attached at C-3 and linked with C-2, which was identified as the unsaturated leucine subunit. In addition, for two carbonyls, one pyrrole, and an unsaturated double bond in leucine, the remaining one IHD suggested the cyclization of the unsaturated leucine and proline moieties ([Fig. 2]). Thus, 1 was deduced as (Z)-3-(2-methylpropylidene)-2,3-dihydropyrrolo[1,2-a] pyrazine-1,4-dione, an unprecedented unsaturated diketopiperazine.

Table 1 ¹H (400 MHz) and ¹³C (100 MHz) NMR data of 1 in CD₃OD [δ (ppm), J (Hz)].

	δ H	δ C	COSY	HMBC
2		155.1 qC
3		125.6 qC
5		154.9 qC
6		124.9 qC
7	7.70, dd (3.2, 1.5)	117.8 CH	8	6
8	6.61, dd (3.6, 3.2)	114.8 CH	7, 9
9	7.13, dd (3.6, 1.5)	120.8 CH	8
10	6.31, d (10.5)	132.7 CH	11	2, 11a, 11b
11	2.93, m	25.5 CH	10, 11a, 11b	3, 10
11a, 11b	1.12, d (6.4)	20.8 CH3	11	10, 11

Fig. 2 ¹H-¹H COSY and HMBC correlations of compound 1.

Compound 2 was isolated by RP-HPLC at t _R 38 min (MeCN-H₂O, 50 : 50, flow rate: 1 mL/min) from the same fraction with the known pseudopeptides 10 – 12. They all exhibited the same MS² fragmentation pattern and NMR spectral characteristics. To accelerate the structure identification of the pseudopeptides, we briefly outlined the spectral characteristics of compounds 10 – 12 before delving into the structure elucidation of compound 2. Compounds 10 – 12 were obtained as brown amorphous powders by RP-HPLC at t _R 29.0, 34.0, and 44.0 min, respectively (MeCN-H₂O 50 : 50, 1 mL/min). The HR-ESIMS ion peaks of these compounds at 502.2314 [M + Na]⁺, 476.2155 [M + Na]⁺, and 518.2260 [M + Na]⁺ led to the suspected molecular formulas of C₂₇H₃₃ N₃O₅, C₂₅H₃₁ N₃O₅, and C₂₇H₃₃ N₃O₆, respectively. The characteristic ¹H signals of the benzyl group (δ _H 7.39 – 7.18 in 10 – 12), olefinic protons (δ _H 6.96, 6.50, 6.22, 6.14, 5.97, and 5.86 in 10; δ _H 7.07, 6.18, 6.09 and 5.91 in 11; δ _H 7.12, 6.50, 6.21, 6.18, 5.97, and 5.91 in 12), alpha-protons of amino acids (δ _H 5.43 and 4.73 in 10; δ _H 5.42 and 4.73 in 11; δ _H 5.25 and 5.18 in 12), methyl groups (δ _H 1.78, 0.86, and 0.77 in 10; δ _H 1.80, 0.86, and 0.77 in 11; δ _H 1.79, 0.81, and 0.66 in 12), and the methyl on the succinimide group (δ _H 1.14 in 10; δ _H 1.13 in 11; δ _H 0.87 in 12) were matched to the ¹H signals of andrimid and moiramides B and C ([Table 2]). Combined with the molecular formula and ¹³C spectral signals, 10 was identified to andrimid [9], 11 to moiramide B [9], and 12 to moiramide C [9]. In addition, the MS² fragmentation of these compounds showed a specific and robust pattern as follows: the HR-ESI-MS/MS fragments at m/z 343.1643 (C₁₉H₂₃ N₂O₄ ⁺, fragment A), 268.1323 (C₁₇H₁₈NO₂ ⁺, fragment B), and 226.1218 (C₁₅H₁₆ NO⁺, fragment C) in 10, and at m/z 343.1643 (C₁₉H₂₃ N₂O₄ ⁺), 242.1187 (C₁₅H₁₆NO₂ ⁺, lost a C₂H₂ compared to fragment B in 10), and 200.1066 (C₁₃H₁₄ NO⁺, lost a C₂H₂ compared to fragment C in 10) in 11, and at m/z 359.1630 (C₁₉H₂₃ N₂O₅ ⁺, one more oxygen than fragment A in 10), 268.1323 (C₁₇H₁₈NO₂ ⁺), and 226.1218 (C₁₅H₁₆ NO⁺) in 12 (Table 5S, Supporting Information), respectively, indicated the cleavages between C-13 and the amino group of C-20, and between C-13 and C-12, and C-11 in the amide group breaking ([Fig. 3]), which further confirmed the structures of these known pseudopeptides.

Table 2 ¹H (400 MHz) NMR data of pseudopeptides 2 – 5 in CD₃OD [δ (ppm), J (Hz)].

	Compound 2	Andrimid (10)	Moiramide B (11)	Moiramide C (12)
*The signal was suspected of overlapping with CD3OD
2	4.22, bs	4.59, bs	4.59, bs	–
3	3.26, q (7.4)	3.11, q (7.4)	3.11, q (7.4)	3.33, m*
5	1.14, d (7.4)	1.14, d (7.4)	1.13, d (7.4)	0.87, d (7.0)
7	4.73, d (4.6)	4.73, d (4.9)	4.73, d (4.9)	5.23, bs
8	2.39, m	2.38, m	2.38, m	2.59, m
9	0.77, d (6.9)	0.77, d (6.9)	0.77, d (6.7)	0.66, d (6.9)
10	0.86, d (6.8)	0.86, d (6.8)	0.86, d (6.9)	0.81, d (7.1)
12	2.77, m 2.86, m	2.76, m 2.84, m	2.81, m 2.84, m	2.81, m 2.84, m
13	5.43, t (7.2)	5.43, t (7.2)	5.42, t (7.2)	5.39, t (7.4)
15 – 19	7.39 – 7.18, m	7.39 – 7.18, m	7.39 – 7.18, m	7.39 – 7.18, m
21	5.94, d (15.2)	5.98, d (15.1)	5.91, d (15.1)	5,97, d (15.2)
22	7.09, dd (15.2, 9.6)	7.12, dd (15.1, 11.2)	7.07, dd (15.1,10.4)	7.12, dd (15.2, 11.9)
23	6.18, m	6.21, m	6.18, m	6.21, m
24	6.16, m	6.50, dd (14.6, 10.4)	6.09, m	6.50, dd (14.3, 10.4)
25	2.18, m	6.18, m	1.80, d (6.6)	6.18, m
26	1.06, t (7.5)	5.92, m		5.91, m
27		1.78, d (6.8)		1.79, d (6.8)

Fig. 3 An MN cluster of the bioactive fractions from the three antimicrobial strains, DJW05 – 1, DJW05 – 8, and DJW21 – 4, pertinent to pseudopeptide-type metabolites. MNs 1 – 6 are predicted as pseudopeptide analogues with similar HR-ESI-MS/MS fragmentation patterns (see detailed spectral data in Table 5S and 6S, Supporting Information).

Compound 2 was obtained as a colorless powder with a molecular formula of C₂₆H₃₃ N₃O₅ based on a sodium adduct ion [M + Na]⁺ at m/z 490.2315 (calcd. for C₂₆H₃₃ N₃O₅Na, 490.2312). ¹H NMR spectrum of compound 2 revealed the presence of a benzyl group at δ _H 7.39 – 7.18 (m), olefinic protons at δ _H 7.09 (dd), 6.18 (m), 6.16 (m), and 5.94 (d), five methine groups at δ _H 5.43 (t), 4.73 (d), 4.22 (bs), 3.26 (q) and 2.39 (m), two methylene at δ _H 2.79/2.59 (m) and 2.18 (m), and four methyl groups at δ _H 1.14 (d), 1.06 (t), 0.92 (d), and 0.83 (d) ([Table 2]). While the available quantity of compound 2 was insufficient for ¹³C NMR analysis, we utilized 2D spectra, including HSQC, COSY, and TOCSY, to gather further structural information (Fig. 22S-24S, Supporting Information). Additionally, we estimated the ¹³C-NMR chemical shifts based on the HSQC spectrum, as detailed in Table 3S, Supporting Information. Key correlations observed in ¹H-¹H COSY (Fig. 23S, Supporting Information) and TOCSY (Fig. 24S, Supporting Information) spectra, such as those between δ _H 7.09 (H-22), 6.18 (H-23), 6.16 (H-24), 2.18 (m), and 1.06 (H-26), confirmed the presence of a short unsaturated aliphatic chain with a terminal ethyl group, which was also supported by the evidence of HR-ESI-MS/MS fragments at m/z 343.1643 (C₁₉H₂₃ N₂O₄ ⁺), 256.1332 (C₁₆H₁₈NO₂ ⁺, lost one carbon compared to fragment B in 10), and 214.1208 (C₁₄H₁₆ NO⁺), following the previous empirical MS/MS fragmentation rules (Table 5S, Supporting Information). To further confirm the structure of 2, we compared the predicted spectrum generated using MetFrag [34] with our HRLC-MS/MS data. As a result, compound 2 achieved a final score of 3.0, providing additional evidence to support its structure (Table 7S, Supporting Information). Thus, compound 2 was annotated as (2E,4E)-N-(3-((3-methyl-1-(4-methyl-2,5-dioxopyrrolidin-3-yl)-1-oxobutan-2-yl)amino)-3-oxo-1-phenylpropyl)hepta-2,4-dienamide. Further NMR analysis is therefore required to confirm its structure.

Andrimid (10) has been previously identified as a significant antibacterial compound effective against gram-positive S. aureus and gram-negative Escherichia coli [16], and was recognized as a carboxylase inhibitor [35]. Following this, we assessed the inhibitory effect of andrimid (10) against four bacterial indicators, E. coli, S. aureus, Salmonella typhimurium, and A. baumannii. However, 10 did not exhibit any inhibitory effect against Pseudomonas aeruginosa. The escalating issue of antimicrobial resistance is a growing concern. Addressing this challenge necessitates exploring strategies, such as combining antibiotics with antimicrobial compounds to mitigate the situation until the discovery of next-generation antibiotics. To this end, we plan to evaluate the combined treatment effect of andrimid (10) with several commercial antibiotics. Specifically, we selected six commercial antibiotics, including ampicillin (a penicillin-type antibiotic for inhibiting cell wall synthesis), kanamycin (an aminoglycoside-type antibiotic for inhibiting ribosome protein synthesis), erythromycin (a macrolide-type antibiotic for inhibiting ribosome protein synthesis), tetracycline (a polyketide antibiotic for inhibiting ribosome protein synthesis), trimethoprim (a diaminopyrimidine-type antibiotic as an inhibitor of folic acid synthesis), and levofloxacin (a fluoroquinolone antibiotic to inhibit the DNA synthesis) based on their diverse mechanisms of antimicrobial actions.

Before combining commercial antibiotics with andrimid (10), we measured the minimum inhibitory concentration (MIC) values of the selected antibiotics toward five bacterial indicators using the broth microdilution method ([Table 3]). The fractional inhibitory concentration (FIC) index [36], determined through the checkerboard method, was introduced to assess the antimicrobial effects of the combination treatment ([Table 3]). Based on the results, andrimid (10) showed a synergistic effect in combination with erythromycin A and levofloxacin toward E. coli, with ΣFIC values of 0.28 and 0.50, respectively. For S. aureus, andrimid (10) demonstrated additive and synergistic effects with ampicillin, kanamycin, erythromycin A, tetracycline, and levofloxacin, with ΣFIC values of 0.63, 0.75, 0.31, 0.25, and 0.26, respectively. Against S. typhimurium, andrimid (10) showed a synergistic effect with ampicillin, kanamycin, and levofloxacin, with ΣFIC values of 0.16, 0.49, and 0.13, respectively. However, for A. baumannii, andrimid (10) only exhibited a minor additive effect with kanamycin. Conversely, andrimid (10) displayed apparent antagonistic effects (ΣFIC values > 2) with trimethoprim toward E. coli, S. aureus, and A. baumannii. Similar antagonistic effects were observed against E. coli and A. baumannii when combined with tetracycline. Toward S. typhimurium, andrimid (10) only showed an antagonistic effect with erythromycin A ([Table 3]).

Table 3 The MIC and FIC values of andrimid (10) and clinical antibiotics toward pathogenic indicators.

Indicators	E. coli		S. aureus		S. typhimurium		A. baumannii
	MICa	FIC	MICa	FIC	MICa	FIC	MICa	FIC
aMIC was recorded by µg/mL; ND indicates no detection. Since the commercial antibiotics did not show any inhibitory effect against pathogenic indicators at concentrations below 1024 µg/mL, we were unable to confirm the FIC indexes. Active means that the commercial antibiotic was too potent to calculate the FIC index. *Independent effect; **additive effect; ***synergistic effect; Δantagonistic effect
MICa of Andrimid (10)	10		40		25		25
Ampicillin	> 32	ND	2	0.63**	32	0.16***	> 32	ND
Kanamycin	> 32	ND	4	0.75**	32	0.49***	32	0.75**
Erythromycin A	1	0.28***	2	0.31***	16	2.12 Δ	32	0.90**
Tetracycline	8	> 2.5Δ	8	0.25***	2	1.00*	8	2.4 Δ
Trimethoprim	4	> 2.5Δ	8	> 2.5Δ	2	0.13***	32	2.5 Δ
Levofloxacin	0.5	0.50***	4	0.26***	< 0.13	Active	0.5	1.01*

Although andrimid (10) was initially identified as an inhibitor of acetyl-CoA carboxylase, the observed antagonistic effects in combined treatments with tetracycline, trimethoprim, and erythromycin A suggest that andrimid (10) might hinder the antibacterial actions of these antibiotics or engage in interactions with them. Nevertheless, the potential for synergistic and additive effects of andrimid (10) with other antibiotics could still offer valuable applications in clinical treatment.

In the black box of exploring undiscovered natural products, it poses a significant challenge to isolate chemically unstable natural products during multicomponent extractions and purification processes. It is often difficult to determine whether these isolates are natural products or artifacts. Modern advanced mass techniques and data processing tools, such as molecular networking that computes the similarity of fragmentation patterns of secondary metabolites, offer promising solutions to these issues. In this study, we isolated and identified the structures of the antimicrobial pseudopeptides, as well as a series of diketopiperazines and alkaloids, from three Vibrio strains guided by GNPS analysis. This approach also provides a viable solution for identifying chemically unstable compounds during the experimental process. Additionally, the major antimicrobial pseudopeptide, andrimid (10), confirmed the synergistic effect in combination with commercial antibiotics, highlighting its potential for further medicinal application.

Material and Methods

General experimental procedures

Optical rotations were determined on a Jasco P-2000 digital polarimeter. UV spectra were measured on a Jasco V-650 spectrometer. IR spectra were recorded on a Jasco FT/IR-4100 spectrometer. 1D and 2D NMR spectra were obtained by Varian Unity 400 FT NMR spectrometers and a Bruker UltraShield 600 MHz NMR spectrometer with a TXO 5-mm CryoProbe. ESIMS was performed with a Bruker AmaZon SL, and HRESIMS was performed on a Thermo Fusion Lumos Tribrid. Reversed-phase medium-pressure liquid chromatography (MPLC) was performed on Teledyne Combiflash Rf⁺ flash chromatography system equipped with C18 reverse-phase columns (80 g, 35 – 45 µm). HPLC was performed on a Hitachi 5110 equipped with a Hitachi 5430 diode array detector. Discovery HS C18 (250 × 4.6 mm, i. d., 5 µm) and Ascentis C18 (250 × 4.6 mm, i. d., 5 µm) were used for analytical purposes. Discovery HS semipreparative C18 (250 × 10 mm, i. d., 5 µm), preparative C18 (250 × 21.2 mm, i. d., 5 µm), and Ascentis semipreparative C18 (250 × 10 mm, i. d., 5 µm) were used for preparative purposes.

Material

The marine invertebrates Agelas nemoechinata DJW05, Sinularia exilis DJW21, and Cladiella tuberculosa DJW-23 were collected from the Dongjiyu, which belongs to the South Penghu Marine National Park for scuba diving. V. tubiashii DJW05 – 1 and V. japonicus DJW05 – 8 were isolated from A. nemoechinata. Vibrio xuii DJW21 – 3 and V. fortis DJW21 – 4 were isolated from S. exilis DJW21. Vibrio hyugaensis DJW23 – 4, Vibrio hepatarius DJW23 – 5, and V. japonicus DJW23 – 6 were isolated from C. tuberculosa DJW-23. Vibrio alginolyticus (red-1and green-1), Vibrio caribbeanicus (H100 – 3, H100 – 5, and H100 – 6), and Vibrio harveyi (H100-10) were isolated from the seawater in the Qi-jin coastal area, Kaohsiung County, Taiwan. The accession numbers are shown in the Supporting Information (Table 1S, Supporting Information). All the strains were grown in Difco marine agar medium (peptone 5 g/L, yeast 1 g/L, NaCl 19.45 g/L, MgSO₄ 3.24 g/L, CaCl₂ 1.8 g/L, MgCl₂ 5.9 g/L, NH₄NO₃ 1.6 mg/L, ferric citrate 0.1 g/L, KCl 0.55 g/L, NaHCO₃ 0.16 g/L, KBr 0.08 g/L, SrCl₂ 34.0 mg/L, H₃BO₃ 22.0 mg/L, NaF 2.4 mg/L, Na₂SiO₃ 4.0 mg/L, Na₂HPO₄ 8.0 mg/L, agar 15 g/L, and pH 7.6) in petri dishes (90 mm × 15 mm) at 30 °C.

Extraction

For the upscaling of cultivation, H100-10 was cultured in GGP broth (30 mL glycerol, 10 g peptone, 0.5 g K₂HPO₄, 0.5 g MgSO₄, and 30 g sea salt). After a 3-day culture, the culture filtrate (5 L) was applied to an Amberlite XAD-16 column (500 mL) and eluted with acetone to obtain the organic extract (7.59 g). On the other hand, DJW05 – 1 and DJW23 – 6 were cultured in solid mediums of marine agar (600 plates per batch). After the 3-day cultures, solid mediums with cultured Vibrio strains DJW05 – 1 and DJW23 – 6 were cut into small pieces and further macerated in EtOAc for 1 day at room temperature 3 times. After the solvent was removed, the EtOAc extract (4.0 – 4.5 g per batch) was partitioned successively into MeOH (2.0 – 2.5 g per batch) and n-hexane layers (1.0 – 1.3 g per batch).

Acquisition of UHPLC-HRMS/MS data for molecular networking analysis.

For quantitation analysis, the mass data were acquired in triplicate using UHPLC-HRMS/MS (Thermo Fusion Lumos Tribrid system). The MeOH layers of Vibrio spp. (0.1 mg/mL) were dissolved in LC-grade MeOH, of which 5 µL were injected and separated by ACQUITY UPLC BEH C18 (100 × 2.1 mm, i. d., 1.7 µm) with the a gradient [MeCN-H₂O (0.1% formic acid), 5 : 95 to 100 : 0 for 6 min; 100 : 0 for two min, 100 : 0 to 5 : 95 for 0.2 min, and 5 : 95 for 1.2 min; flow rate: 0.4 mL/min]. The mass data were acquired in the profile mode, with positive mode ion detection between m/z 150 – 1500 with 60 000 resolutions. The top five intense ions from each full mass scan were selected for collision-induced dissociation (CID) fragmentation. For CID, the isolation width was 1.2 Da and the selected ions were fragmented with a normalized collision energy of 35, activation Q of 0.250, activation time of 10.0, and 15 000 resolutions. The raw LC-MS/MS data (.RAW files) were converted to an. mzXML format by MSConvert software [37] and subjected to GNPS (https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp) to generate molecular networking. The parameter settings on the GNPS website were as follows: parent ion tolerance, 2.0 Da; ion tolerance, 0.5 Da; minimum pair cosine score, 0.5; minimum matched peaks, 6. The generated molecular network was downloaded as Cytoscape data (.graphml files) and edited in Cytoscape (https://cytoscape.org/).

Isolation

The MeOH layer of DJW05 – 1 was subjected to reversed-phase MPLC using a C18 column (40 µm irregular; 120 g) with a gradient (MeOH-H₂O, 5 : 95 to 100 : 0 for 90 min, 100 : 0 for 10 min, 100 : 0 to 5 : 95 for one min, and 5 : 95 for 19 min; flow rate: 10 mL/min) to obtain six fractions (Fr. 1–Fr. 6) detected by a UV-Vis detector. Fr. 2 (289.0 mg) was further subjected to reversed-phase MPLC using a C18 column (40 µm irregular; 120 g) with a gradient (MeOH-H₂O, 5 : 95 to 100 : 0 for 90 min, 100 : 0 for 10 min, 100 : 0 to 5 : 95 for one min, and 5 : 95 for 19 min; flow rate: 10 mL/min) to yield five subfractions (Fr. 2 – 1–Fr. 2 – 5) detected by a UV-Vis detector. Subfraction Fr. 2 – 2 (15.0 mg, MeOH, 100%) was separated by reversed-phase HPLC (MeOH-H₂O, 5 : 95; flow rate: 1 mL/min) using Discovery HS C18 (250 × 10 mm, i. d., 5 µm) to obtain uracil (13, 1.1 mg, 0.05%, t _R = 17.2 min) and thymine (14, 2.6 mg, 0.12%, t _R = 25.0 min) by a UV-Vis detector at 220 nm. Subfraction Fr. 2 – 3 (11.3 mg, MeOH, 100%) was separated using reversed-phase HPLC (MeOH–H₂O, 5 : 95; flow rate: 1 mL/min) using Discovery HS C18 (250 × 10 mm, i. d., 5 µm) to obtain cyclo-(glycine–L-proline) (4, 1.5 mg, 0.07%, t _R = 32.0 min) and cyclo-(D-alanin-4-trans-hydroxy-D-proine) (5, 1.7 mg, 0.08%, t _R = 22.6 min) by a UV-Vis detector at 220 nm. Fr. 3 (110.0 mg) subjected to reversed-phase MPLC using a C18 column (40 µm irregular; 80 g) with a gradient (MeCN-H₂O, 5 : 95 to 30 : 70 for 50 min, 30 : 70 for 10 min, 30 : 70 to 50 : 50 for 50 min, 50 : 50 for 10 min, 50 : 50 to 100 : 0 for 10 min, 100 : 0 for 10 min; flow rate: 10 mL/min) to gain eight subfractions (Fr.3 – 1–Fr. 3 – 8) detected by a UV-Vis detector. Subfraction Fr. 3 – 6 (24.3 mg, MeOH, 100%) was separated by reversed-phase HPLC (MeOH-H₂O, 50 : 50; flow rate: 1 mL/min) using Discovery HS C18 (250 × 10 mm, i. d., 5 µm) to yield (Z)-3-(2-Methylpropylidene)-2,3-dihydropyrrolo[1,2-a] pyrazine-1,4-dione (1, 2.1 mg, 0.1%, R _t = 42.3 min) by a UV-Vis detector at 275 nm. Fr. 4 (135.3 mg, MeOH, 100%) was separated by reversed-phase HPLC (MeCN-H₂O, 15 : 85; flow rate: 1 mL/min) using Discovery HS C18 (250 × 10 mm, i. d., 5 µm) to yield cyclo-(L-leucine-L-proline) (3, 12.0 mg, 0.55%, t _R = 59.5 min), cyclo-(L-proline-L-phenylalanine) (6, 10.0 mg, 0.45%, t _R = 85.0 min), cyclo-(D-proline-L-phenylalanine) (7, 2.2 mg, 0.1%, t _R = 70.0 min), cyclo-(4-trans-hydroxyl-L-proline-L-phenylalanine) (8, 26.5 mg, 1.2%, t _R = 68.0 min), and cyclo-(L-leucine-L-valine) (9, 0.9 mg, 0.04%, t _R = 50.0 min) by a UV-Vis detector at 220 and 280 nm. Fr. 5 (30.2 mg, MeOH, 100%) was purified by reversed-phase HPLC (MeCN-H₂O, 50 : 50; flow rate: 1 mL/min) using Discovery HS C18 (250 × 10 mm, i. d., 5 µm) to yield andrimid (10, 3.5 mg, 0.16%, t _R = 44.0 min), moiramide B (11, 1.3 mg, 4.3%, t _R = 29.0 min), moiramide C (12, 0.5 mg, 0.02%, t _R = 34.0 min), compound 2 (0.2 mg, 0.01%, t _R = 38.0 min), and indole-3-aldehyde (15, 0.7 mg, 0.03%, t _R = 24.5 min) by a UV-Vis detector at 280 nm.

The MeOH layer of DJW23 – 6 was subjected to reversed-phase MPLC using a C18 column (40 µm irregular; 120 g) with a gradient (MeOH-H₂O, 5 : 95 to 100 : 0 for 90 min, 100 : 0 for 10 min, 100 : 0 to 5 : 95 for one min, and 5 : 95 for 19 min; flow rate: 10 mL/min) to obtain six subfractions (Fr. 1–Fr. 6) detected by a UV-Vis detector. Subfraction Fr. 3 (94.9 mg) was subjected to reversed-phase MPLC (MeOH-H₂O, 30 : 70; flow rate: 10 mL/min) using a C18 column (40 µm irregular; 80 g) to yield seven subfractions (Fr. 3 – 1–Fr. 3 – 7) detected by a UV-Vis detector. Subfraction Fr. 3 – 5 (28.2 mg, MeOH, 100%) was separated by reversed-phase HPLC [MeCN-H₂O (0.1% TFA), 25 : 75; flow rate: 2 mL/min] using Discovery HS C18 (250 × 10 mm, i. d., 5 µm) to yield indole-3-glyoxylamide (17, 0.8 mg, 0.04%, t _R = 25.0 min) by a UV-Vis detector at 254 nm. Fr. 4 (32.4 mg, MeOH, 100%) was separated by reversed-phase HPLC using Discovery HS C18 (250 × 21.2 mm, i. d., 5 µm) with a gradient (MeOH–H₂O, 5 : 95 to 100 : 0 for 30 min, 100 : 0 for 10 min, 100 : 0 to 5 : 95 for one min, and 5 : 95 for 19 min; flow rate: 1 mL/min) to yield three subfractions (Fr. 4 – 1–Fr. 4 – 3) detected by a UV-Vis detector. Subfraction Fr. 4 – 3 (10.2 mg, MeOH, 100%) was separated by reversed-phase HPLC [MeOH-H₂O (0.1% TFA), 55 : 45; flow rate: 2 mL/min] using Discovery HS C18 (250 × 10 mm, i. d., 5 µm) to yield trisindoline (16, 1.0 mg, 0.05%, t _R = 44.0 min) by a UV-Vis detector at 254 nm.

MetFrag application

The MetFrag application features a user-friendly web interface, https://msbi.ipb-halle.de/MetFrag/. The required input includes the tandem MS peak list with intensities (Table 7S, Supporting Information), and the selection of the upstream compound database and respective search parameters. Alternatively, a list of database IDs can be provided explicitly. This allows for selecting the candidates based on their occurrence in specific pathways. A feedback form allows for storing all input data, user ratings of the hypotheses, and further comments. This helps to collect user-provided test and training data. Spectra will not be saved unless explicitly granted [38].

Minimum inhibitory concentration assay

The pathogenic bacteria E. coli, A. baumannii ATCC 19 606, S. aureus, and S. typhimurium were provided by Prof. Sung-Pin Tseng from Kaohsiung Medical University. The MICs of antibiotics and compounds against clinical isolates of pathogenic bacteria were determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) recommendation as described previously [39]. Briefly, for bacterial indicators, after being grown overnight in BHI broth at 37 °C, the bacterial was diluted with Mueller hinton broth II to adjust the turbidity approximately to the standard McFarland 0.5 (2 × 10⁸ CFU/mL) [40], [41]. Then, the bacterial suspension was further diluted 200 times with MH II broth to achieve an initial loading of 1 × 10⁶ CFU/mL. Most of the antibiotics were prepared and series were diluted in H₂O at various concentrations, except for erythromycin A, trimethoprim, and levofloxacin. Erythromycin A, trimethoprim, and andrimid (10) were prepared in DMSO while levofloxacin was prepared in 0.1 M NaOH. The bacterial suspension (199 µL) was mixed with an equal volume (1 µL) of sample solution at various concentrations in each well of a 96-well plate and incubated for 24 h at 37 °C. To ensure that DMSO and 0.1 M NaOH would not influence the growth of the bacteria, an equal volume of buffer solution was also mixed with the bacterial suspension to monitor individually. The MIC was defined as the lowest concentration in which no visible turbidity was detected with unaided eyes and an ELISA reader (SpectraMax ABS Plus-MOLECULAR DEVICES) in OD₆₀₀. Broth with bacteria was only used as a negative control, and each MIC was measured and tested in triplicate.

Fractional inhibitory concentration index analysis [36]

All antibiotics were prepared in the same solution as the MIC assay. The initial concentrations that were used followed a half or one time of MIC values of the respective data. Of the diluted bacteria, 199 µL were plated into each well following a self-drug combination test. Andrimid (10) was serially diluted 2-fold in the vehicle 4 times, and the antibiotics were added to all wells in decreasing concentrations (Figs. 42S-45S, Supporting Information). After incubating statically for 24 h at 37 °C, the OD₆₀₀ readings were taken for FICs calculation. The sum of the FICs (ΣFIC) of the wells corresponding to an MIC (isoeffective combinations) were calculated using the following equation:

∑ F I C = F I C A + F I C B = C A M I C A + C B M I C B \sum FIC={FIC}_{A}{+}{FIC}_{B}=\left({CA\over{MICA}}\right){+}\left({CB\over{MICB}}\right)

Here, MIC_A and MIC_B mean the MICs of drugs A and B alone, respectively, and C_A and C_B are the concentrations of the drugs in combination, respectively. The ΣFICs calculated were for all isoeffective combinations used to capture synergistic, additive, and antagonistic interactions. An FIC index of ≤ 0.5 indicates synergism, > 0.5 – 1 as additive effects, > 1 to < 2 as indifference, and ≥ 2 as antagonism [42].

Biotyper

After a culture of 24 h at 37 °C, some quantity of biological material (from one single colony to 5 – 10 mg) was transferred to an Eppendorf tube with 300 µL water. The mixture further added 900 µL ethanol and then was pipetted to a homogeneous condition. Next, the mixture was kept centrifuging at 14 000 rpm for 2 min. After that, the supernatant was discarded, and the pellet was resuspended in 70% formic acid (50 µL) and mixed very well by pipetting and/or by vortexing. Further, pure MeCN (50 µL) was added to the mixture and mixed by pipetting again. After centrifugation at 14 000 rpm for 2 min, about 1 µL of the supernatant (between 0.5 and 2 µL) was placed onto a steel target plate and air-dried. After drying, each mixed sample was overlayed with 1 µL of matrix solution [250 µL basic organic solvent (500 µL MeCN, 475 µL H₂O, and 25 µL pure TFA)] directly with a Bruker HCCA portioned matrix (Art. #255 344).

Contributorsʼ Statement

Conception and design of the work, financial support assurance: C. C. Liaw and W. C. Cheng; compound isolation, bioactive assay, FIC index: M. X. Hong, W. T. Lin, and G. E. D. Jayalath; marine bacteria isolation and purification: M. X. Hong, B. W. Wang, and L. H. Lo; pathogenic bacteria culture and provision: S. P. Tseng and Y. H. Chen; critical review of paper content: Y. H. Chen and W. C. Tsai; data collection, interpretation of mass data, and molecular network analysis: M. X. Hong, Y. L. Yang, and C. C. Liaw; interpretation of all data and drafting of the paper: C. C. Liaw and M. X. Hong.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors appreciate Mrs. Hsiao-Ching Yu from the Center for High-Value Instrumentation at National Sun Yat-sen University and Dr. Sychyi Cheng from the Mass Spectrometry Core Facility, Agricultural Biotechnology Research Center at Tainan, Academia Sinica, for their assistance with HRMS analysis. The authors also thank Academia Sinica High-Field NMR Center (HFNMRC) for technical support and appreciate Dr. Chi-Fon Chang in the Genomics Research Center, Academia Sinica, for her assistance with 600 MHz NMR analysis. HFNMRC is funded by the Academia Sinica Core Facility and Innovative Instrument Project (AS-CFII-111 – 214).

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Publication History

Received: 27 June 2024

Accepted after revision: 10 February 2025

Accepted Manuscript online:
11 February 2025

Article published online:
14 March 2025