The INT-407 human epithelial cell-line (ATCC CCL-6) was purchased from the American Type Culture Collection (Manassas, VA, USA), and maintained at 37°C under 5% CO₂ in Minimum Essential Medium (MEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (10 U/ml penicillin G and 10 μg/ml streptomycin) (growth medium). Psammaplin A is a natural marine product that was isolated from two sponges, Jaspis sp. and Poecillastra wondoensis (17). The other compounds were isolated from a sponge-derived fungus Acremonium sp. and their configurations were determined by CD spectroscopic data, along with comparison of ¹H and ¹³C spectroscopic data (25). All chemicals used in the study were a gift from Professor Jung (College of Pharmacy, Pusan National University, Busan, Korea). The natural marine products were dissolved in anhydrous ethanol to make a 10 mg/ml stock solution. Subsequent dilutions were made in Dulbecco's modified Eagle's medium.

V. vulnificus strain MO6-24/O used in the present study was isolated from patients (9,10) and provided by Professor Sang Ho Choi (Seoul National University, Seoul, Korea). The V. vulnificus bacteria were grown to log phase at 30°C in Luria-Bertani medium (produced in the laboratory) supplemented with 2.0% NaCl LBS medium, after which they were diluted to 6×10⁸ CFU/ml in LBS medium, and then centrifuged for 3 min at 2,500 × g and resuspended in antibiotic-free MEM medium prior to infection of INT-407 cells. The concentration of bacteria was confirmed via viable colony counting conducted on LBS agar.

The V. vulnificus inoculum size was 6×10⁸ CFU/ml. Variable concentrations of natural pure compounds 1, 4, 6, 8 and 10 (1, 5, 10, 12.5, 20, 25, 40, 50, 75 and 100 μg/ml) were solubilized in 20 ml of growth medium (2% NaCl LBS) and then tested for their ability to alter bacterial growth by spectrometry (OD₅₄₀). This was conducted by culturing V. vulnificus for 0–13 h in the presence of 50 μg/ml psammaplin A or 0–100 μg/ml psammaplin A for 13 h at 37℃ in 2% NaCl LB medium, and bacterial growth was evaluated by measuring the optical density at 540 nm (OD₅₄₀). The V. vulnificus cultures were then incubated with aeration at 150 rpm using a gyratory shaker for 5 h at 37°C.

INT-407 human epithelial cells were infected with V. vulnificus as previously described (9,10). Briefly, INT-407 cells were grown in growth medium at 37°C in a 5% CO₂ incubator. Next, the cells were seeded onto 6-well (8×10⁵ cells/well) and 96-well (2×10⁴ cells/well) culture plates and then cultured for 24 h in antibiotic-free growth medium. Prior to infection, the bacteria were centrifuged for 3 min at 2,500 × g, resuspended and adjusted to 6×10⁸ CFU/ml in antibiotic-free MEM medium. The bacterial suspensions were then added to psammaplin A-treated or untreated-epithelial cells at various multiplicities of infection (MOI; the ratio of the number of bacteria to the number of epithelial cells), after which the infected cells were incubated for 1–4 h in antibiotic-free growth medium at 37°C under 5% CO₂.

The bacteria-infected INT-407 cell cultures were aliquoted into a 96-well tissue culture plate (Nunc, Roskilde, Denmark) as previously described (9,10). The cytotoxicity was then determined by measuring the activity of lactate hydrogenase (LDH) in the supernatant using a cytotoxicity detection kit (Roche, Mannheim, Germany). The cytotoxic level was expressed as a percentage relative to the total LDH activity of cells that were completely lysed by 1% Triton X-100 (9,10).

INT-407 (8×10⁵ cells/well) cells were incubated with bacteria in a 6-well plate for 3 h at an MOI of 10, after which the cells were washed with phosphate-buffered saline (PBS). The cells were then fixed with 4% para-formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature, washed and completely dried. Next, the cells were stained with Giemsa solution (Molecular Probes, Thermo Fisher Scientific, Inc.) for 1 h at room temperature. The cells were then washed twice with distilled water and dried, after which the images of the specimens were acquired using a microscope (Olympus IX 71, Tokyo, Japan).

A total of 35 female ICR mice (Samtaco Bio Korea, Gyounggi-do, Korea; age, 8 weeks; weight, 20–22 g) that were housed under specific-pathogen free conditions were used for all experiments. They were maintained at 24°C with a relative humidity of 50%, under a 12-h light/dark cycle. The mice had access to food and water ad libitum. The present study was approved by Korea University (Seoul, Korea). The mice were intraperitoneally infected with 0.1 ml of 250 μg iron dextran (Sigma-Aldrich) 30 min prior to injection with V. vulnificus. Next, the mice were intraperitoneally injected with 1×10³ CFU/0.1 ml V. vulnificus. The use of iron dextran produces a useful model to investigate systemic disease that results from V. vulnificus infection. The mice were administered 0.2 ml psammaplin A (DCM 1-9-1) solution (5, 10, 25 or 50 μg per mouse) or a PBS intraperitoneally (control), after which their survival status was assessed every hour for 24 h.

The V. vulnificus-inoculated mice were sacrificed by cervical dislocation 7 h after infection. A ventral incision was made to observe the abdomen of the infected mice treated with or without psammaplin A (Nikon D60; Nikon Corporation, Tokyo, Japan), and the spleen, liver and small intestine lesions were then aseptically removed. The removed specimens were homogenized in 2 ml PBS using glass tissue homogenizers, after which the homogenates were diluted in PBS and plated on 2% NaCl HI agar. The samples were then incubated at 37°C for 12 h and bacterial colonies were counted.

The data were analyzed with Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Student's t-test and one-way analysis of variance followed by the Bonferroni method were employed to identify statistical differences between the values of the various experimental and control groups. P<0.05 was considered to indicate a statistically significant difference.

Twelve pure compounds were isolated from natural marine products, and their structures were characterized as previously described (17,25) (Fig. 1). The inhibitory effects of these compounds were determined on V. vulnificus-induced cytotoxicity (Fig. 2). INT-407 cells were infected with V. vulnificus at an MOI of 10 for 3 h in the presence or absence of the 12 marine product-derived compounds. Then, the cytotoxicities of the compounds were evaluated in cells using LDH assays. As shown in Fig. 2A, there was significantly decreased cytotoxicity in cells treated with compounds 1, 4, 6, 8, 9 and 10 compared with the untreated cells infected with V. vulnificus, indicating that these compounds have inhibitory effects on V. vulnificus-induced cytotoxicity. Treatment with these compounds significantly inhibited the cytotoxicity of V. vulnificus in a concentration- and time-dependent manner (Fig. 2B and C). Of these compounds, psammaplin A (compound 8) had the strongest inhibitory effect on the V. vulnificus-induced cytotoxicity.

To confirm the inhibitory effects of psammaplin A on the V. vulnificus-induced cytotoxicity of INT-407 cells, the size and morphology of nuclei were assessed using a microscope (Fig. 3). INT-407 cells infected with V. vulnificus at an MOI of 10 for 2–3 h showed typical phenotypic features of cell death, such as cytoplasmic loss and cellular damage, while treatment with psammaplin A reversed that phenotype. Psammaplin A ameliorated the significant cellular damage at 3 h after infection with V. vulnificus. These results suggest that psammaplin A inhibits the cytotoxicity against host cells induced by V. vulnificus infection.

To investigate whether psammaplin A prolonged survival, mice were infected with V. vulnificus and administered psammaplin A (0–50 μg per mouse). Mice inoculated intraperitoneally with 1×10³ CFU V. vulnificus all died within 16 h. However, psammaplin A treatment of V. vulnificus-infected mice increased the survival rate. Following psammaplin A treatment, four out of five mice infected with V. vulnificus (50 μg per mouse) survived for 24 h (Fig. 4A).

To investigate the effects of psammaplin A treatment on the growth of V. vulnificus in vivo, mice were intraperitoneally infected with 1×10³ CFU V. vulnificus and administered psammaplin A (0, 10, 25 and 50 μg per mouse). After 7 h, several tissue samples, including from the spleen, liver and small intestine were excised from the mice, and the number of V. vulnificus colonies in each tissue was evaluated. Fig. 4B shows that the number of V. vulnificus colonies was significantly reduced in all tissue samples isolated from psammaplin A-treated mice compared with the number of V. vulnificus colonies isolated from untreated controls. In addition, the necropsy results of V. vulnificus-infected mice at 7 h post infection showed edema, hemorrhage, vasodilation and necrosis in the intestines, livers and spleens isolated from the untreated mice. However, the tissue samples from the psammaplin A-treated mice did not show any of the symptoms observed in the tissues of untreated mice (Fig. 4C). These results suggest that psammaplin A significantly suppresses the growth of V. vulnificus and the associated pathology in vitro and in vivo.

To investigate the antibacterial activities of psammaplin A against V. vulnificus, V. vulnificus was incubated in the presence or absence of psammaplin A (0–100 μg/ml) for 0–13 h. As shown in Fig. 5, the bacterial numbers of V. vulnificus increased in an incubation time-dependent manner. However, psammaplin A treatment inhibited the growth of V. vulnificus in a concentration-dependent manner. These findings suggest that psammaplin A significantly inhibited the growth of V. vulnificus.