Abstract

1. Introduction

Quorum sensing (QS) is the term coined to describe the process of cell-to-cell communication in bacteria. This intercellular communication enables the execution of coordinated behaviors in function of the bacterial population density. The process relies on the production, release and reception of signaling molecules that have been often classified into three main chemical categories: N-acyl homoserine lactones (AHLs), also referred as “autoinducer-1” (AI-1), which are characteristic of Gram-negative bacteria; autoinducing peptides (AIPs), employed by Gram-positive bacteria; and the so-called “autoinducer-2” (AI-2), furanones whose common precursor is (S)-4,5-dihydroxy-2,3-pentanedione (DPD) that have been proposed as a universal signal between Gram-positive and Gram-negative bacteria [1]. Nevertheless, other chemically different signaling molecules have also been identified over the last decades, such as the A-factor from Streptomyces (a γ-butyrolactone), 4-quinolones (Pseudomonas Quinolone Signal, PQS) or fatty acids (Diffusible Signal Factors, DSF) [2,3]. The most recent example are pyrones, recently identified as QS signals in Photorhabdus luminescens [4,5]. This likely represents only a small proportion of the extracellular metabolites involved in QS signaling. Similarly, the chemical nature of the molecules that are able to agonize or antagonize this phenomenon is equally diverse [6].

Once a threshold concentration of autoinducers is achieved in the extracellular milieu, bacterial gene expression is altered. These QS-regulated genes are involved in a variety of processes: production of virulence factors and secondary metabolites, sporulation, competence, or biofilm formation, among others [7]. For this reason, inhibition of QS constitutes a key target in the control of biofilm-related problems, including marine biofouling. Biofouling, the undesirable settlement of marine organisms on immersed substrata, begins with the adsorption of organic matter and the formation of bacterial biofilms, which in turn modulate the settlement of macroscopic foulers [8,9]. For example, Ulva zoospores are able to recognize AHLs from bacterial biofilms as a chemoattractive cue for settlement [10,11]. Spore release in the epiphytic alga Acrochaetium is also induced by bacterial AHLs [12]. Recently, a direct correlation between AHL concentration and cyprid settlement has been found in Balanus improvises [11]. As (ideally) QS blockers do not target bacterial growth, they do not exert a selective pressure on bacterial populations. In fact, this strategy is widely employed by nature to interfere with bacterial colonization, either by mimicking the bacterial autoinducers (e.g., brominated furanones from Delisea pulchra) [13,14], or by “quenching” bacterial signals through enzymatic degradation (e.g., lactonases from Bacillus, Pseudomonas or Shewanella species) [15].

Fungi are a renowned source of products with an array of bioactivities, from antibacterial to antiviral, cytotoxic, antiinflammatory, antifeeding, antifungal or antioxidant, among many others [16,17,18]. In recent years, research on fungi associated with marine invertebrates and algae has revealed the presence of antifouling secondary metabolites [19,20]. Indeed, there is increasing evidence that many of the bioactive metabolites produced by sponges or algae as chemical defenses to avoid epibiosis are not truly produced by these organisms themselves, but by microbes—mainly bacteria and fungi—living in association with them [21,22]. Although bacterial-fungal interactions are largely documented [23,24,25], there are relatively few reports on QS inhibition by fungal metabolites. For instance, Rasmussen and co-workers identified patulin and penicillic acid from Penicillium species as QS inhibitors (QSIs) in P. aeruginosa [26]. Conversely, the fungal QS molecule farnesol from Candida albicans has been reported to inhibit the production of PQS in P. aeruginosa, which in turn prevents the yeast from entering in the filamentous phase through the production of the QS inducer 3-oxo-C12 homoserine lactone [27,28].

As part of our search for novel antifouling agents, a collection of 75 strains of fungi were isolated from aquatic habitats in the states of Veracruz and Puebla, Mexico, cultured at laboratory scale and screened for the presence of QSIs with the reporter strain Chromobacterium violaceum CVO26. QSI production was screened in extracts from both the fungal biomass and its culture medium. For the most active isolates, a phylogenetic analysis by amplification of the ITS region (ITS1, 5.8S and ITS2) was conducted to assess a genetic identification. In order to investigate the chemical nature of the fungal metabolites involved in the observed bioactivity, LC-HRMS profiles of the most active extracts were analyzed.

2. Results

2.1. Sampling and Isolation

Three different ecological niches were selected for the isolation of fungal strains: endophytes from reef organisms (corals, sponges, and algae), mangrove rhizosphere soils, and saline lakes. These are all aquatic ecosystems with marked differences that are likely to host a rich microbial diversity. Indeed, the sampling resulted in 75 isolates belonging to 21 genera (Table 1). The marine sources were particularly prolific, since more than half the isolates (34) were endophytes, followed by those associated with mangrove roots (28). Altogether, these two groups accounted for 83% of the isolates (Figure 1).

Table 1

Strains of fungi isolated from Mexican aquatic habitats.

Strain ID.	Fungus Genus	Biological Source	Location
LAEE 01	Cladosporium sp.	Amphimedon compressa	Arrecifes Blancas
LAEE 02	Aspergillus sp.	Amphimedon compressa	Arrecifes Blancas
LAEE 03	Curvularia sp.	Amphimedon compressa	Arrecifes Blancas
LAEE 04	Fusarium sp.	Agelas sp.	Arrecifes Blancas
LAEE 05	Aspergillus sp.	Agelas sp.	Arrecifes Blancas
LAEE 06	Sarocladium sp.	Agelas sp.	Arrecifes Blancas
LAEE 07	Fusarium sp.	Aplysina sp.	Arrecifes Blancas
LAEE 08	Acremonium sp.	Chondrilla sp.	Arrecifes Blancas
LAEE 09	Fusarium sp.	Acropora palmata	Arrecifes Blancas
LAEE 10	Acremonium sp.	Acropora palmata	Arrecifes Blancas
LAEE 11	Acremonium sp.	Acropora palmata	Arrecifes Blancas
LAEE 12	Fusarium sp.	Diploria clivosa	Arrecifes Blancas
LAEE 13	Fusarium sp.	Diploria strigosa	Arrecifes Blancas
LAEE 14	Epicoccum sp.	Diploria strigosa	Arrecifes Blancas
LAEE 15	Trichoderma sp.	Diploria strigosa	Arrecifes Blancas
LAEE 16	Aspergillus sp.	Diploria strigosa	Arrecifes Blancas
LAEE 17	Fusarium sp.	Diploria strigosa	Arrecifes Blancas
LAEE 18	Aspergillus sp.	Montastrea cavernosa	Arrecifes Blancas
LAEE 19	Aspergillus sp.	Montastrea cavernosa	Arrecifes Blancas
LAEE 20	Aspergillus sp.	Montastrea cavernosa	Arrecifes Blancas
LAEE 21	Khuskia sp.	Plexaura flexuosa	Arrecifes Blancas
LAEE 22	Cladosporium sp.	Plexaura flexuosa	Arrecifes Blancas
LAEE 23	Trichoderma sp.	Plexaura flexuosa	Arrecifes Blancas
LAEE 24	Monilia sp.	Plexaura flexuosa	Arrecifes Blancas
LAEE 25	Acremonium sp.	Pseudoplexaura porosa	Arrecifes Blancas
LAEE 26	Acremonium sp.	Pseudoterogorgia americana	Isla de Sacrificios
LAEE 27	Aspergillus sp.	Pseudoterogorgia americana	Isla de Sacrificios
LAEE 28	Alternaria sp.	Siderastrea siderea	Arrecifes Blancas
LAEE 29	Aspergillus sp.	Siderastrea siderea	Arrecifes Blancas
LAEE 30	Aspergillus sp.	Siderastrea siderea	Arrecifes Blancas
LAEE 31	Fusarium sp.	Siderastrea siderea	Arrecifes Blancas
LAEE 32	Aspergillus sp.	Siderastrea siderea	Arrecifes Blancas
LAEE 33	Fusarium sp.	Zoanthus sp.	Isla de Sacrificios
LAEE 34	Aspergillus sp.	Hypnea cervicornis	Isla de Sacrificios
LAEE 35	Monilia sp.	Sample from littoral zone	Laguna de Atexcac
LAEE 36	Ulocladium sp.	Sample from littoral zone	Laguna de Atexcac
LAEE 37	Stachybotrys sp.	Sample from littoral zone	Laguna de Atexcac
LAEE 38	Fusarium sp.	Sample from littoral zone	Laguna de Atexcac
LAEE 39	Fusarium sp.	Sample from littoral zone	Laguna de Atexcac
LAEE 40	Absidia sp.	Potamogeton sp.	Laguna de Atexcac
LAEE 41	Paecilomyces sp.	Sample from littoral zone	Laguna de Atexcac
LAEE 42	Chaetomium sp.	Sample from littoral zone	Laguna de Atexcac
LAEE 43	Stemphylium sp.	Juncus sp.	Laguna de Atexcac
LAEE 44	Penicillium sp.	Cladophora sp.	Laguna de Atexcac
LAEE 45	Acremonium sp.	Sample from littoral zone	Laguna de Atexcac
LAEE 46	Aspergillus sp.	Sample from littoral zone	Laguna de Atexcac
LAEE 47	Aspergillus sp.	Cladophora sp.	Laguna de Atexcac
LAEE 48	Fusarium sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 49	Alternaria sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 50	Fusarium sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 51	Trichocladium sp.	Rhizosphereof Rhizophora mangle	Manglar de Tuxpan
LAEE 52	Aspergillus sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 53	Penicillium sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 54	Aspergillus sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 55	Fusarium sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 56	Aspergillus sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 57	Mucor sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 58	Cladosporium sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 59	Fusarium sp.	Rhizosphere of Rhizophora mangle	Manglar de Tuxpan
LAEE 60	Aspergillus sp.	Rhizosphere of Avicennia germinans	Manglar de Tuxpan
LAEE 61	Blastomyces sp.	Rhizosphere of Avicennia germinans	Manglar de Tuxpan
LAEE 62	Aspergillus sp.	Rhizosphere of Avicennia germinans	Manglar de Tuxpan
LAEE 63	Fusarium sp.	Rhizosphere of Avicennia germinans	Manglar de Tuxpan
LAEE 64	Fusarium sp.	Rhizosphere of Avicennia germinans	Manglar de Tuxpan
LAEE 65	Penicillium sp.	Rhizosphere of Avicennia germinans	Manglar de Tuxpan
LAEE 66	Acremonium sp.	Rhizosphere of Avicennia germinans	Manglar de Tuxpan
LAEE 67	Aspergillus sp.	Rhizosphere of Laguncularia racemosa	Manglar de Tuxpan
LAEE 68	Aspergillus sp.	Rhizosphere of Laguncularia racemosa	Manglar de Tuxpan
LAEE 69	Penicillium sp.	Rhizosphere of Laguncularia racemosa	Manglar de Tuxpan
LAEE 70	Acremonium sp.	Rhizosphere of Laguncularia racemosa	Manglar de Tuxpan
LAEE 71	Aspergillus sp.	Rhizosphere of Laguncularia racemosa	Manglar de Tuxpan
LAEE 72	Aspergillus sp.	Rhizosphere of Laguncularia racemosa	Manglar de Tuxpan
LAEE 73	Fusarium sp.	Rhizosphere of Laguncularia racemosa	Manglar de Tuxpan
LAEE 74	Paecilomyces sp.	Rhizosphere of Laguncularia racemosa	Manglar de Tuxpan
LAEE 75	Paecilomyces sp.	Rhizosphere of Laguncularia racemosa	Manglar de Tuxpan

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Figure 1

Distribution of the strains isolated in this study in function of their origin (central circle); proportion of active and inactive strains in each case (blue circles); and origin (biomass or culture medium) of the active extracts in each case (green circles).

2.2. Quorum Sensing Assays

Extracts from the fungal biomasses and their liquid culture media were screened for the presence of QSIs with the reporter strain C. violaceum CVO26. Thus, two extracts (biomass and spent medium) were prepared for each fungal isolate. Two screening rounds were conducted. In the first-round screening, extracts were evaluated at 100 μg·mL⁻¹. Extracts inhibiting violacein production above 40% with respect to the control were considered active samples. 14 of the 34 marine endophytic strains, 8 of the 28 mangrove root-associated fungi and 10 of the 13 saline lake-derived isolates exhibited activity, either from their broth extracts or their biomasses (Figure 1). 26 of these active samples (58%) belonged to fungal biomasses, suggesting that most of the bioactive molecules were not delivered to the extracellular milieu under the laboratory culture conditions.

The active samples were subjected to a second-round screening in which serial dilutions from 500 to 50 μg·mL⁻¹ were evaluated (Figure 2). Four samples stood out for their high activity (inhibitions above 70% in violacein production): LAEE06C (strain LAEE06, culture medium extract), LAEE13B (strain LAEE13, biomass extract), LAEE14B (strain LAEE14, biomass extract) and LAEE21C (strain LAEE21, culture medium extract). These samples inhibited violacein production dose-dependently (Figure 2), all belonging to marine endophytic fungi. Interestingly, two of these isolates, LAEE13 and LAEE17, were isolated from the same coral species, Diploria strigosa (Table 1). Although statistically significant differences were quantified for bacterial growth in the presence of the fungal extracts (One-way ANOVA, Dunnett’s post-hoc test, p < 0.05), a dose-dependent trend on violacein production was observed (Figure 2). As evidenced in Figure 2a,c,d, this dose-dependent inhibition of violacein synthesis is not correlated with higher toxicities (i.e., growth inhibition) on the reporter strain. In effect, extracts LAEE06C, LAEE14B and LAEE21C seem to exert a basal effect on bacterial growth that does not change significantly in the range of tested concentrations. This is in marked contrast with the effect observed on violacein production, which is clearly inhibited by increasing concentrations of the fungal extracts. By contrast, extract LAEE13B exerted an evident antibacterial activity that explains by itself the dose-dependent phenotypic evidence (Figure 2b).

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Figure 2

Growth inhibition (white bars) and violacein inhibition (black bars) caused by different concentration of the fungal extracts: (a) LAEE06C; (b) LAEE13B; (c) LAEE14B and (d) LAEE21C. Data represent the mean ± SD. Asterisks indicate significant differences respect the untreated control (one-way ANOVA, Dunnett’s multiple comparison test, p < 0.05).

2.3. ITS Phylogeny and Taxonomic Identification of the Isolates

ML and NJ tree reconstructions were used in the phylogenetic analysis, however, as both showed the same topology, only the ML tree is included in the text (Figure 3, Supplementary Figure S1). The four isolates were placed, with high statistical support (99% and 100% bootstrap values), on four different branches corresponding to the genera Sarocladium (LAEE06), Fusarium (LAEE13), Epicoccum (LAEE14), and Khuskia (LAEE21).

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Figure 3

Maximum-likelihood (General Time Reversible, with gamma distribution) phylogenetic tree based on ITS sequences (552 nt) of isolates of this study and reference strains. Accession numbers are given in parentheses. Numbers at the nodes are bootstrap support values for 500 replicates. The scale bar indicates the number of substitutions per site.

The isolate LAEE06, from genus Sarocladium, shared 99.8% sequence similarity with the species S. strictum, thus, this isolate could be considered a new strain of this species. Within the Fusarium cluster, LAEE13 occupied an intermediate position between two closely related lineages, the F. incarnantum-F. equiseti species complex (FIESC) and the F. chlamydosporum species complex (FCSC). So, with the current data, isolate LAEE13 cannot be assigned to a specific species, as the ITS sequences were not discriminative enough to resolve at the species level for this clade [29]. The closest relative of isolate LAEE14 is the species Epicoccum nigrum, with which it shares 99.4% sequence similarity, and it could be a new strain of E. nigrum or a novel sister species. In the Khuskia/Nigrospora cluster, Nigrospora (anamorph of Khuskia) sphaerica was the closest species of isolate LAEE21, with 98.8% sequence similarity. The ITS sequence of LAEE21, interestingly, was almost identical (99.8% similar) to another unclassified endophytic fungal isolate.

2.4. Metabolic Profiles

In order to investigate the chemical nature of the observed inhibitions, LC-HRMS profiles of the four samples, LAEE06C, LAEE13B, LAEE14B and LAEE21C, were obtained. The identified metabolites are summarized in Table 2 and the most relevant chemical structures are included in Figure 4.

Table 2

Annotated peaks observed in the chromatograms of the extracts LAEE06C, LAEE13B, LAEE14B and LAEE21C.

Sample	RT (min)	Suggested MF	FM Database ID	Fungal Metabolites with This MF Included in the DNP
LAEE06C	4.36	C27H49O12P		Lysofungin (3)
	4.76	C33H43NO4		Antibiotic GKK 1032A1 (2)
	5.04	C33H45NO4		Sespendole (1)
	5.39	C18H30O2		Linolenic acid
	6.64	C18H35NO2		Not found in the DNP
LAEE13B	0.90	C10H13NO2	Fusaric acid (4)
	2.74	C9H13NO		Not found in the DNP
	3.26	C16H21NO4		N-Acetyl-O-prenyltyrosine (7)
	4.02	C13H16O3		5 coincidences in the DNP
	4.11	C18H30O3		11-Oxo-9,12-octadecadienoic acid (8)
	5.41	C21H29NO4	Trichosetin (5)
	6.39	C44H55N3O9		Beauvericin D (9)
	6.56	C45H57N3O9	Beauvericin (6)
LAEE14B	3.29	C20H22N4O2		Phenylahistin (12)
	3.48	C24H29N3O4		Variecolorin N (13)
	3.76	C23H32O7		(E,E)-6-(6′,7′-dihydroxy-2′,4′-octadienoyl)-strobilactone A
	5.00	C31H55N5O7		Emericellamide A (10)
	5.74	C15H20O3		>50 coincidences in the DNP
	6.15	C25H34O		Not found in the DNP
	6.53	C18H32O2		Linoleic acid
LAEE21C	2.38	C10H16O4		13 coincidences in the DNP
	3.08	C15H22O4		>50 coincidences in the DNP
	3.59	C18H34O5		Penicitide B (15)
	3.79	C23H32O7		(E,E)-6-(6′,7′-dihydroxy-2′,4′-octadienoyl)-strobilactone A
	4.06	C17H14N2S		Not found in the DNP
	4.58	C27H32O9	Verrucarin B (14)
	5.04	C31H55N5O7		Emericellamide A (10)
	6.61	C18H32O2		Linoleic acid

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Figure 4

Electrospray ionization positive mode base peak chromatograms of the QS active samples. The numbers above the peaks identify the metabolites listed in Table 2.

3. Discussion

Among the natural sources of bioactive compounds, microbes are more likely to support a sustainable, cost-effective production through large-scale cultivation [30]. In spite of the wide array of bioactivities found in marine and marine-derived microbes, the role of fungi from aquatic habitats—including those associated with marine organisms—as a source of antifouling substances, has been largely underestimated [31]. It was only in recent years that several antifouling compounds have been isolated from marine-derived fungi [32,33,34,35,36] and even patented as additives for antifouling coatings [37].

In this study, several aquatic ecosystems were screened for the presence of filamentous fungi (Table 1). A total of 75 isolates were recovered and identified on the basis of their microscopic and macroscopic features. Up to 21 different genera were isolated, thus representing a wide ecological diversity. This richness is particularly evident in the mangroves: 12 isolates were recovered from the sediments from Rhizophora mangle, whereas the rhizosphere of Laguncularia racemosa and Avicennia germinans yielded nine and seven isolates, respectively. Among the endophytes, the corals Diploria strigosa and Siderastrea siderea were particularly prolific, with five different strains being isolated from each specimen (Table 1). All the isolates were facultative and not marine obligates, that is, they are also found in terrestrial environments [38]. This does not mean that these strains are not ‘truly’ marine; indeed, they are considered as a highly specialized group of microbes of major ecological importance with particular physiological adaptations [39]. Batch cultures of the isolates reached the deceleration/stationary phase within 7 days, with biomass yields in the range 8–16 g·L⁻¹ that coincide well with the values reported for other filamentous fungi isolated from aquatic environments [40] and highlight their potential for being cultured at a larger scale.

It is well known that filamentous fungi secrete secondary metabolites to the surrounding medium that are involved in diverse processes, including chemical defense towards microorganisms [41,42]. However, the secretion, or even the production of these bioactive molecules must often be triggered by or is subjected to environmental factors, for example, by a limiting nutrient availability, under different conditions of pH, temperature and/or salinity, or by inducing a competitive pressure by co-cultivation with other microorganisms [43,44]. This may explain the apparent lack of difference between the number of active extracts from the spent media and those from the fungal biomass.

Remarkably, the most active samples were identified among endophytic fungi from reef organisms. It is interesting to note that three of these isolates were obtained from corals (LAEE13, LAEE14, and LAEE21), two of them from the same species, Diploria strigosa (Table 1). Corals are indeed the paradigm of a holobiont, the term coined by Lynn Margulis in the 1990s to design an organism and its associated microbial symbionts. Thus, corals are hosts of a plethora of microorganisms that live in a mutualistic association [45]. Although previous studies have focused on the ability of whole-organism extracts [46,47], coral-associated bacteria [48,49,50] or bacteria associated to endosymbiotic dinoflagellates [51] to interfere with QS-regulated phenotypes, this is, to the best of our knowledge, the first evidence of this kind of behavior in marine endophytic fungi. It is tempting to hypothesize a possible ecological role of these fungal symbionts on the antifouling protection of their hosts.

The classical fungal taxonomy has been mainly based on macro-morphological and microscopical features, and this approach has not always given a reliable classification of closely related organisms. Over the last decades due to improvements in molecular techniques, this classification has been remodeled, and we have seen a revolution in many aspects of the fungal taxonomy by using genes for a molecular identification, especially functionally conserved genes as the ribosomal genes, giving a more reliable and objective classification of the organisms. The ITS regions are part of the ribosomal DNA operon. They are located between the 18S and 28S rRNA genes and divided into two segments, the ITS1 and ITS2, by the rRNA 5.8S. As part of the ribosomal operon, they are highly represented with multiple copies in the genome. However, the different ITS copies in the genome of a particular organism tend to be similar. The conserved regions of the flanking genes, 18S and 28S, facilitate the design of broad-range primers for PCR amplification across a broad range of fungal lineages and the more variable regions of the ITS allow their identification. Thus, ITS regions have been confirmed as important molecular markers for taxonomic studies and proposed as a universal barcode for fungal identification [52,53].

In this work, using a phylogenetic analysis based on the sequences of the ITS region, we have identified four marine endophytic fungal isolates as belonging to four genera: Sarocladium (LAEE06), Fusarium (LAEE13), Epicoccum (LAEE14) and Khuskia (LAEE21). One isolate, LAEE06, belonging to genus Sarocladium, could be assigned to the previously described species, S. strictum [54]. Isolate LAEE21 is likely to represent a novel species within the Khuskia clade, and interestingly another endophytic fungus from Hevea brasiliensis (Khuskia sp. PP96, Figure 3) could also belong to the same species [55]. However, the precise taxonomic position of the Epicocum isolate, LAEE14, could not be determined. Either the current data from the ITS sequences could resolve the Fusarium clade. Though the ITS sequences has, in general, a high resolution power to discriminate among fungal species, it is known that it shows some limitations to resolve large genera, like Aspergillus or Fusarium, which include closely related species, that form a “species complex” [56,57].

In recent years, some extra markers have been used, as the β-tubulin or the Elongation Factor-1α genes [54,57]. Thus, in order to precisely resolve the taxonomic position of some of our fungal endophytes, further analysis will be necessary using protein-coding markers.

LC-HRMS analysis of these bioactive extracts led to the identification (in-house database)/tentative identification (Dictionary of Natural Products, DNP) of several major components whose QS-inhibitory properties have not been reported so far (Figure 4 and Figure 5, Table 2). The structures of some of these main constituents are presented in Figure 5.

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Figure 5

Chemical structures of representative secondary metabolites identified by LC-HRMS in the bioactive extracts.

Strain LAEE06, extract LAEE06C, produced linoleic acid and three nitrogenated metabolites, two of them with the same MF and UV spectra as the previously reported fungal metabolites sespendole (1) and antibiotic GKK 1032A₁ (2). A phosphorus-containing compound whose Molecular Formulae (MF) and UV spectra matched those of lysofungin (3) was also identified. The sixth molecule had a MF of C₁₈H₃₅NO₂, not described in the DNP for any fungal metabolite, and therefore it may represent a new natural product.

Fusaric acid (4), trichosetin (5) and beauvericin (6) were identified as the main components of LAEE13B. Several other compounds whose MF and UV spectra were coincident with those of N-acetyl-O-prenyltyrosine (7), 11-oxo-9,12-octadecadienoic acid (8) and beauvericin D (9) compound with MF C₁₃H₁₆O₃ and UV-Vis λ_max of 226; 275 nm matched five coincidences in the DNP, whereas a nitrogenated compound with MF C₉H₁₃NO and UV-Vis λ_max of 230(sh); 276; 301 nm did not match any previously reported fungal metabolite.

Strain LAEE14 (extract LAEE14B) also produced linoleic acid, emericellamide A (10) and (E,E)-6-(6′,7′-dihydroxy-2′,4′-octadienoyl)-strobilactone A (11). Two components with MF C₂₀H₂₂N₄O₂ and C₂₄H₂₉N₃O₄ were tentatively classified as two diketopiperazines (DKPs), namely phenylahistin (12) and variecolorin N (13). DKPs are not only a widely recognized group of QSIs [58] but also a family of autoinducer signals in bacterial intercellular communication [59], being able to act, consequently, as QS agonists or antagonists. A compound with MF C₁₅H₂₀O₃ and UV-Vis λ_max of 307 nm matched more than 50 compounds in the DNP and could not be identified, whereas another compound with MF C₂₅H₃₄O and UV-Vis λ_max of 236; 300 nm did not yield any match in the DNP.

Finally, strain LAEE21 (extract LAEE21C) was a source of chemically diverse metabolites, including emericellamide A (10), compound 11, verrucarin B (14) and penicitide B (15). Two compounds with UV-Vis λ_max of 210 nm and MF C₁₀H₁₆O₄ and C₁₅H₂₂O₄, respectively, matched several coincidences in the DNP. Interestingly, a sulfur-containing compound with MF C₁₇H₁₄N₂S and UV-Vis λ_max of 223; 267 nm was also detected in this extract, which did not match any known natural product and could represent a novel chemical entity. Linoleic acid was also identified in this sample. Linoleic acid and other fatty acids have been previously reported as QSIs [60,61].

It is interesting to note that several metabolites highlighted in Figure 5 have recent literature precedents in marine-derived strains. Beauvericin (6), first isolated in Beauveria bassiana as an antibacterial and cytotoxic agent [62] was later identified in Fusarium species [63] and, last year, in a mangrove-endophytic Aspergillus terreus [64]. Emericellamide A (10) was recently isolated from the marine-derived fungus Emericella sp., an epiphyte of the green alga Halimeda sp. [65], and later from A. nidulans [66]. (E,E)-6-(6′,7′-dihydroxy-2′,4′-octadienoyl)-strobilactone A (11) has been reported in two marine-derived Aspergillus species: A. ustus and A. insuetus, isolated from sponges and algae [67,68,69]. Ocean sediments were the source of Penicillium griseofulvum, from which variecolorin N (13) was isolated for the first time [70]. Finally, the isolation of penicitide B (15) from P. chrysogenum, an endopytic strain of a Laurencia sp., was reported in 2011 [71].

The widespread distribution of the secondary metabolites reported in this work within different fungal genera complicates the identification of clear chemotaxonomic markers in the metabolic profiles. In spite of this fact, a clear ‘chemotaxonomic fingerprint’ was observed for LAEE13, clustered within the Fusarium genus (Figure 3). For this isolate, fusaric acid (4), trichosetin (5) and two beauvericins (6, 9) were identified (Figure 4, Table 2). These compounds are frequent in Fusarium [72,73,74]. Fusaric acid is particularly renowned and, in addition, its ability to interfere with bacterial QS has been previously documented [75,76].

4. Experimental Section

5. Conclusions

This study highlights the potential of fungal secondary metabolites to interfere with bacterial QS. Although fungi are a renowned source of bioactive compounds, their ability to thwart bacterial cell-to-cell communication is poorly characterized. Among the diversity of aquatic environments screened in this study, marine endophytes clearly stood out. With the exception of LAEE13B, the QS-disrupting activity exhibited by the extracts of these endophytic filamentous fungi was unrelated to toxic effects on the reporter strain Chromobacterium violaceum CVO26. LC-HRMS profiles of these extracts revealed the presence of a broad diversity of fungal metabolites (Figure 5), some of them of recent identification in marine-derived strains [62,63,64,65,66,67,68,69,70,71]. Interestingly, all these fungal extracts contained compounds that might represent new natural products whose identity and biological activity will be determined in further experiments. The identity of the compounds tentatively identified by HRMS and UV analysis and whether the presence of the abovementioned metabolites can explain the QS-inhibitory activity observed in the extracts or it is due to the presence of minor components, or even to the synergistic effect of combinations of these molecules are issues that remain to be determined. To the best of our knowledge, this is also the first report of QS inhibition in endophytic fungi isolated from marine organisms.

Acknowledgments

Supplementary Files

Author Contributions

A.J.M.-R. and J.J.F. conceived and designed the experiments; A.J.M.-R. performed the experiments; F.R. and J.M. conducted the LC-HRMS analyses; J.P-Y. and M.L.-B. conducted the phylogenetic identification; A.T. designed the sampling campaign. A.C. and C.E. conducted the isolation work and the taxonomical identification; J.J.F., A.T., V.M. and M.N. contributed reagents, materials and financial support; A.J.M.-R. and J.J.F. wrote the paper. All authors have read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References