Actinobacteria Isolated From Laminaria ochroleuca: A Source of New Bioactive Compounds

Abstract

Nature is the major reservoir of biologically active molecules. The urgent need of finding novel molecules for pharmaceutical application is prompting the research of underexplored environments, such as marine ecosystems. Here, we investigated cultivable actinobacteria associated with the macroalgae Laminaria ochroleuca and assessed their potential to produce compounds with antimicrobial or anticancer activities. A specimen of L. ochroleuca was collected in a rocky shore in northern Portugal, and fragments of tissues from different parts of the macroalgae (holdfast, stipe, and blades) were surface sterilized and plated in three culture media selective for actinobacteria. A total of 90 actinobacterial strains were isolated, most of which affiliated with the genus Streptomyces. Isolates associated with the genera Isoptericola, Rhodococcus, Nonomuraeae, Nocardiopsis, Microbispora, and Microbacterium were also obtained. Organic extracts from the isolates were tested for their antimicrobial activity using the agar-based disk diffusion method, followed by determination of minimum inhibitory concentration (MIC) values. Forty-five isolates inhibited the growth of Candida albicans and/or Staphylococcus aureus, with MIC values ranging from <0.5 to 1000 μg mL^-1. The actinobacterial isolates were also tested for their anticancer potential on two human cancer cell lines. Twenty-eight extracts affected the viability of at least one human cancer cell line (breast carcinoma T-47D and neuroblastoma SH-SY5Y) and non-carcinogenic endothelial cell line (hCMEC/D3). Seven extracts affected the viability of cancer cells only. This study revealed that L. ochroleuca is a rich source of actinobacteria with promising antimicrobial and anticancer activities and suggests that macroalgae may be a valuable source of actinobacteria and, consequently, of new molecules with biotechnological importance.

Keywords: Laminaria ochroleuca; anticancer; antimicrobial; bioactivity; endophytic actinobacteria; kelp; macroalgae; marine actinobacteria.

FIGURE 1

Actinobacterial genera recovered from L. ochroleuca. (A) Number of actinobacterial strains affiliated with the different genera retrieved from L. ochroleuca, (B) genera distribution in the holdfast, stipe, and blades of the macroalgae and (C) genera distribution according to the selective culture media used for the isolation.

FIGURE 2

Morphological diversity of some actinobacterial strains isolated from L. ochroleuca: (A) strain KENS1, (B) strain KENR91, (C) strain KENR92, (D) strain KENR94, (E) strain KENB10, (F) strain KENR56, (G) strain KENR81, and (H) strain KENB8.

FIGURE 3

Phylogenetic tree (16S rRNA gene) obtained by Maximum Likelihood analysis of the 87 actinobacterial isolates recovered from L. ochroleuca, together with a selection of closely related strains with sequences deposited in GenBank. The tree was generated using 1281 bp and 1000 bootstraps. Numbers at nodes represent bootstrap values (%) when higher than 60%. Numbers in parenthesis correspond to GenBank accession numbers. Bacillus subtilis was used as an outgroup.

FIGURE 4

The effect of extracts from actinobacterial strains isolated from L. ochroleuca on cellular viability. The effects on the viability of (A) breast carcinoma T-47D and (B) neuroblastoma SH-SY5Y tumor cell lines, and (C) a non-tumor cell line (human brain capillary endothelial cells, hCMEC/D3) are shown after 24 and 48 h of exposure. Only the extracts with significant differences are shown on the graphs for SHSY-5Y and T47D cell lines. All of these extracts were re-tested on the non-cancer cell line (C) for general cytotoxicity. PC and SC indicate positive and solvent controls, respectively. Data are presented as mean ± standard deviation from two independent experiments performed in triplicates each, and significant differences compared to the solvent control are marked with asterisks in the graphs (^∗ = p < 0.05).

FIGURE 5

GNPS-based LC-HRESIMS/MS molecular network analysis of actinobacterial extracts likely to contain new bioactive compounds. The extracts obtained from three Streptomyces sp. strains showed molecular clusters unique to each strain (depicted by connected ellipses, labeled with the corresponding m/z value for the parent ion). The results of Dictionary of Natural Products (^∗) searches for the accurate masses of the most abundant species in each cluster (as estimated from the respective UV and extracted ion chromatograms, EICs, and shown in different color from the remaining nodes in the cluster) are presented for each m/z species (for two molecular clusters, no correspondence was found in this database).