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Marine sponges are among the oldest animals to have emerged on Earth. They are metazoan holobionts that host diverse microbial symbionts, which constitute more than 40% of their biomass. Despite their morphological simplicity, sponges exhibit complex genetic architecture, unquestionably encoding ribosomally synthesised and post-translationally modified peptides (RiPPs) and proteins, essential for their biological functions. In addition to host-derived compounds, the associated microbiota also produce RiPPs, introducing further complexity in distinguishing the origin of these molecules. To date, marine sponge RiPPs research is confined to species within the class Demospongiae, with peptidomic, transcriptomic and genomic approaches employed for their discovery. This review provides a comprehensive account of current research on ribosomal peptides in marine sponges and associated microsymbionts, emphasising the need for expanded discovery efforts. Unravelling the genetic basis and biosynthetic pathways of these peptides will deepen our understanding of sponge biology and open new opportunities for peptide-based drug discovery.

With antibiotic resistance being a major global concern, there is a huge need of new treatment options to fight bacterial infections. In this study, we highlight the antibacterial and host-protective roles of a novel synthetic antimicrobial peptide in uropathogenic Escherichia coli–infected uroepithelial cells. This peptide, designed from a fragment of human cathelicidin LL-37 and named LD4-PP, was found to be highly potent against clinical isolates of E. coli as well as ESBL-producing and multi-drug resistant E. coli. Additionally, LD4-PP inhibited the formation of new biofilm, damaging both the bacterial surface and the bacterial genome. LD4-PP also modulated the host cell lipid vacuole, caveolin-1, and Rho GTPase B affecting bacterial survival. Furthermore, LD4-PP exerts immunomodulatory effects by modulating free radical formation, expression of antioxidants, and inflammasome-mediated cell death. Pronounced uroepithelial cell death was observed after E. coli infection which was significantly inhibited by LD4-PP without affecting the cellular toxicity. Overall, the peptide LD4-PP is shown to be a strong candidate for future clinical applications, particularly to prevent and treat urinary tract infections.

Glioblastoma (GBM) is the most aggressive cancer originating in the brain, but unfortunately combination treatments with resection, radiation, and chemotherapy are relatively ineffective. Therefore, novel methods of adjuvant therapy are critically needed. Cyclotides are plant-derived circular peptides that chemosensitize drug-resistant breast cancer to doxorubicin. We analyzed naturally occurring and synthetic cyclotides (Cycloviolacin O3, Cycloviolacin O19, natural Kalata B1, synthetic Kalata B1, and Vitri E) alone and in co-exposure treatments with the drug temozolomide (TMZ) in human glioblastoma cells. The cyclotides were identified by UPLC-PDA and HPLC-UV. The synthetic Kalata B1 sequence was verified with orbitrap LC-MS, and structural confirmation was provided by NMR spectroscopy. The cyclotides displayed dose-dependent cytotoxicity (IC50 values 2.4-21.1 μM) both alone and as chemosensitizers of U-87 MG and T 98 cells to TMZ. In fact, a 16-fold lower concentration of TMZ (100 μM) was needed for significant cytotoxicity in U-87 MG cells co-exposed to synthetic Kalata B (0.5 μM). Similarly, a 15-fold lower concentration of TMZ (75 μM) was required for a significant reduction in cell viability in T 98 cells co-exposed to synthetic Kalata B1 (0.25 μM). Kalata B1 remained stable in human serum stability assays. The data support the assertion that cyclotides may chemosensitize glioblastoma cells to TMZ.

Sri Lanka is a biodiversity hotspot and one of the richest geographical locations of marine sponges in the Indian ocean. However, the most extensive taxonomical study on Sri Lankan sponge biodiversity dates back similar to 100 years and only a limited number of studies have been conducted on sponge natural products. In the current study, 35 marine sponge specimens (collected from 16 sponge habitats around Sri Lanka) were identified, microfractionated and evaluated for antibacterial and anticancer assays. In total, 30 species were characterized, of which 19 species gave extracts with antibacterial and/or cytotoxic activities. Microfractionated organic extract of Aciculites orientalis gave the most potent antibacterial activity against Staphylococcus aureus and strongest lymphoma cell toxicity was exhibited by the organic extract of Acanthella sp. Guided by the molecular ion peaks in the bioactive fractions, large-scale extraction of Stylissa massa led to the isolation of three bromopyrrole alkaloids, sceptrin, hymenin and manzacidin A/C. Of these, sceptrin exhibited broad spectrum antibacterial activity against both Escherichia coli and S. aureus (MIC of 62.5 mu M against both species). Based on natural product literature, seven promising species were identified as understudied. Their further exploration may lead to the discovery of structurally novel compounds.

Geophila repens (L.) I.M. Johnst (Rubiaceae) is a traditional medicinal plant used in Sri Lanka for the treatment of bacterial infections. Due to its rich endophytic fungi content, it was postulated that endophytically-produced specialized metabolites may be responsible for its purported antibacterial effects. To test this hypothesis, eight pure endophytic fungal cultures were isolated from G. repens then extracted and screened for antibacterial activity in a disc diffusion assay against Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. Large scale culturing, extraction, and purification of the most active fungal extract, obtained from Xylaria feejeensis, led to the isolation of 6′,7′-didehydrointegric acid (1), 13-carboxyintegric acid (2), and four known compounds including integric acid (3). Compound 3 was isolated as the key antibacterial component (MIC = 16 μg/mL against Bacillus subtilis, 64 μg/mL against Methicillin-Resistant S. aureus). Compound 3 and its analogues were devoid of hemolytic activity up to the highest tested concentration of 45 μg/mL. This study demonstrates that specialized metabolites produced by endophytic fungi may contribute to the biological activity of some medicinal plants. Endophytic fungi should be evaluated as a potential source of antibiotics, especially from unexplored medicinal plants traditionally used for the treatment of bacterial infections.

The SARS-CoV-2 virus that causes COVID-19 is a global health issue. The spread of the virus has resulted in seven million deaths to date. The emergence of new viral strains highlights the importance of continuous surveillance of the SARS-CoV-2 virus by using timely and accurate diagnostic tools. Here, we used a stable cyclic peptide scaffolds to present antigenic sequences derived from the spike protein that are reactive to SARS-CoV-2 antibodies. Using peptide sequences from different domains of SARS-CoV-2 spike proteins, we grafted epitopes on the peptide scaffold sunflower trypsin inhibitor 1 (SFTI-1). These scaffold peptides were then used to develop an ELISA to detect SARS-CoV-2 antibodies in serum. We show that displaying epitopes on the scaffold improves reactivity overall. One of the scaffold peptides (S2_1146-1161_c) has reactivity equal to that of commercial assays, and shows diagnostic potential.

The subtilisin-like macrocyclase PatGmac is produced by the marine cyanobacterium Prochloron didemni. This enzyme is involved in the last step of the biosynthesis of patellamides, a cyanobactin type of ribosomally expressed and post-translationally modified cyclic peptides. PatGmac recognizes, cleaves, and cyclizes precursor peptides after a specific recognition motif comprised of a C-terminal tail with the sequence motif -AYDG. The result is the native macrocyclic patellamide, which has eight amino acid residues. Macrocyclase activity can be exploited by incorporating that motif in other short linear peptide precursors, which then are formed into head-to-tail cyclized peptides. Here, we explore the possibility of using PatGmac in the cyclization of peptides larger than the patellamides, namely, the PawS-derived peptide sunflower trypsin inhibitor-1 (SFTI-1) and the cyclotide kalata B1. These peptides fall under two distinct families of disulfide constrained macrocyclic plant peptides. They are both implicated as scaffolds for drug design due to their structures and unusual stability. We show that PatGmac can be used to efficiently cyclize the 14 amino acid residue long SFTI-1, but less so the 29 amino acid residue long kalata B1.

Cyclotides are an intriguing class of structurally stable circular miniproteins of plant origin with numerous potential pharmaceutical and agricultural applications. To investigate the occurrence of cyclotides in Sri Lankan flora, 50 medicinal plants were screened, leading to the identification of a suite of new cyclotides from Geophila repens of the family Rubiaceae. Cycloviolacin O2-like (cyO2-like) gere 1 and the known cyclotide kalata B7 (kB7) were among the cyclotides characterized at the peptide and/or transcript level together with several putative enzymes, likely involved in cyclotide biosynthesis. Five of the most abundant cyclotides were isolated, sequenced, structurally characterized, and screened in antimicrobial and cytotoxicity assays. All gere cyclotides showed cytotoxicity (IC50 of 2.0-10.2 mu M), but only gere 1 inhibited standard microbial strains at a minimum inhibitory concentration of 4-16 mu M. As shown by immunohistochemistry, large quantities of the cyclotides were localized in the epidermis of the leaves and petioles of G. repens. Taken together with the cytotoxicity and membrane permeabilizing activities, this implicates gere cyclotides as potential plant defense molecules. The presence of cyO2-like gere 1 in a plant in the Rubiaceae supports the notion that phylogenetically distant plants may have coevolved to express similar cytotoxic cyclotides for a specific functional role, most likely involving host defense.

Is it possible to enhance structural stability and biological activity of KR-12, a truncated antimicrobial peptide derived from the human host defense peptide LL-37? Based on the mapping of essential residues in KR-12, we have designed backbone-cyclized dimers, cross-linked via a disulfide bond to improve peptide stability, while at the same time improving on-target activity. Circular dichroism showed that each of the dimers adopts a primarily alpha-helical conformation (55% helical content) when bound to lyso-phosphatidylglycerol micelles, indicating that the helical propensity of the parent peptide is maintained in the new cross-linked cyclic form. Compared to KR-12, one of the cross-linked dimers showed 16-fold more potent antimicrobial activity against human pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans and 8-fold increased activity against Escherichia coli. Furthermore, these peptides retained antimicrobial activity at physiologically relevant conditions, including in the presence of salts and in human serum, and with selective Gram-negative antibacterial activity in rich growth media. In addition to giving further insight into the structure-activity relationship of KR-12, the current work demonstrates that by combining peptide stabilization strategies (dimerization, backbone cyclization, and cross-linking via a disulfide bond), KR-12 can be engineered into a potent antimicrobial peptide drug lead with potential utility in a therapeutic context.