Upregulation and Identification of Antibiotic Activity of a Marine-Derived Streptomyces sp. via Co-Cultures with Human Pathogens

Abstract

Marine natural product drug discovery has begun to play an important role in the treatment of disease, with several recently approved drugs. In addition, numerous microbial natural products have been discovered from members of the order Actinomycetales, particularly in the genus Streptomyces, due to their metabolic diversity for production of biologically active secondary metabolites. However, many secondary metabolites cannot be produced under laboratory conditions because growth conditions in flask culture differ from conditions in the natural environment. Various experimental conditions (e.g., mixed fermentation) have been attempted to increase yields of previously described metabolites, cause production of previously undetected metabolites, and increase antibiotic activity. Adult ascidians-also known as tunicates-are sessile marine invertebrates, making them vulnerable to predation and therefore are hypothesized to use host-associated bacteria that produce biologically active secondary metabolites for chemical defense. A marine-derived Streptomyces sp. strain PTY087I2 was isolated from a Panamanian tunicate and subsequently co-cultured with human pathogens including Bacillus subtilis, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa, followed by extraction. Co-culture of Streptomyces sp. PTY087I2 with each of these human pathogens resulted in increased production of three antibiotics: granaticin, granatomycin D, and dihydrogranaticin B, as well as several analogues seen via molecular networking. In addition, co-cultures resulted in strongly enhanced biological activity against the Gram positive human pathogens used in these experiments. Expanded utilization of co-culture experiments to allow for competitive interactions may enhance metabolite production and further our understanding of these microbial interactions.

Keywords: antibiotic secondary metabolites; co-culture with human pathogens; microbial natural product drug discovery; mixed fermentation.

Figure 1

Fold increase of peak area for peaks A–G from Streptomyces sp. PTY087I2 monoculture and co-culture with human pathogens (BS = Bacillus subtilis; MSSA = methicillin-sensitive Staphylococcus aureus; MRSA = methicillin-resistant Staphylococcus aureus; PA = Pseudomonas aeruginosa).

Figure 2

Mass spectral identification of peaks C–E as granatomycin D, granaticin, and dihydrogranaticin B, respectively. (A) Peak C eluted at retention time (t_R) 11.4 min, with [M + H]⁺ of 447.2, consistent with a molecular formula of C₂₂H₂₃O₁₀, confirming production of granatomycin D (446.4 g/mol); (B) Peak D eluted at t_R 13.2 min, with [M + H]⁺ of 445.2, consistent with a molecular formula of C₂₂H₂₁O₁₀, confirming production of granaticin (444.4 g/mol); (C) Peak E eluted at t_R 17.0 min, with [M + Na]⁺ of 583.1, consistent with a molecular formula of C₂₈H₃₂O₁₂Na, confirming production of dihydrogranaticin B (560.5 g/mol).

Figure 3

Comparison of granaticin biosynthetic gene clusters from Streptomyces sp. PTY087I2 and S. violaceoruber TÜ22. The granaticin biosynthetic gene cluster from Streptomyces sp. PTY087I2 was compared to that from S. violaceoruber TÜ22 and showed 83% similarity. Figure adapted from antiSMASH output [28].

Figure 4

Utilization of molecular networks to identify naphthoquinone derivatives from extracts of Streptomyces sp. PTY087I2 monoculture (red), PTY087I2 co-cultured with MRSA (yellow), and PTY087I2 co-cultured with MSSA (blue). Highlighted area includes nodes for granatomycin D, granaticin, and dihydrogranaticin B, as well as indicating presence of several derivatives of these compounds in one or more extracts.