Materials and Reagents

Bacterial strains
1. Wild type (Vibrio vulnificus MO6-24/O, Clinical isolate: biosafety level 2) with pRK415 plasmid (vector control)
2. fur mutant with pRK415
3. fur mutant with pRK415-fur
1% human red blood cells (RBCs) (from healthy person who is a volunteer and diluted with PBS buffer to make a 1% RBC solution)
Tetracycline (3 mg/ml) (Sigma-Aldrich)
PBS buffer (Sigma-Aldrich, catalog number: P5368 )
0.02% Triton X-100 (Triton^TM X-100 for molecular biology) (Sigma-Aldrich, catalog number: T8787 )
LB broth (see Recipes)

Equipment

Shaking incubator
Centrifuge (14,000 rpm or 21,000 x g)
Spectrophotometry (540 and 600 nm)
0.22 micron filter

Procedure

Grow 5 ml of bacterial cells at 30 °C shaking incubator, in modified Luria–Bertani (LB) [addition of NaCl to LB at a final concentration of 2.5% (w/v): Vibrio vulnificus is a marine bacterium, so 2.5% NaCl makes cell happy to grow.] medium supplemented with antibiotics (tetracycline 3 mg/ml).
Take bacterial samples and measure cell density by spectrophotometry at 600 nm.
Collect the supernatants of cultures by centrifugation (14,000 rpm, 5 min, RT) and filtration (use with syringe filter: pore size 0.22 μm).
Prepare cell-free supernatants from each culture and serial dilute them with PBS by 1/2, 1/4 and 1/8 (final volume: 1 ml).
Incubate 1% RBC solution with the equal volume of the diluted supernatants at 37 °C for 1 h (no agitation).
After 1-h incubation, centrifuge of each samples to remove unlysed RBCs (14,000 rpm, 5 min, RT).
To make the fully lysis of 1% RBC solution, use equal volume of 0.02% Triton X-100 at 37 °C for 1 h (no agitation).
After centrifuge of each sample, take the upper part of samples. Measure the lysed RBCs samples by spectrophotometry at 540 nm, as described by Shinoda et al. (1985) (for the blank at 540 nm, you can use culture broth without bacteria cells).
Calculate Haemolytic activity (HU) (HU was expressed as the reciprocal of the dilution factor showing 50% haemolysis) (Shinoda et al., 1985).

Representative data

Figure 1. Haemolytic activities are decreased by fur mutation or iron deprivation. Haemolytic activities in the supernatants of wild type (pRK415), Δfur strain (pRK415) and Δfur strain (pRK415-fur) cultures. V. vulnificus strains were grown in LBS supplemented with 3 μg/ml tetracycline for 2 h, and each culture was treated for 4 h with 2,2′-dipyridyl at a concentration of 0.1 mM (+ chelator) or 0 mM (no chelator). OD₆₀₀ of the cultures of wild type (pRK415) and Δfur strain (pRK415-fur) were 2.0-2.5, and OD₆₀₀ of the Δfur (pRK415) cultures were 1.0-1.5. Serial dilutions of cell-free supernatants were added to 1% RBC solution, and lysed RBCs were measured by spectrophotometry. Activity was expressed as haemolytic units (HU), the reciprocal of the dilution factor showing 50% haemolysis (Lee et al., 2013).

Recipes

LB broth
10 g of Peptone
10 g of Yeast extract
5 g Sodium Cloride per one liter

Acknowledgments

This protocol has been adapted and modified from Shinoda et al. (1985) and Lee et al. (2013). This work was supported by the Mid-Career Researcher Program through a National Research Foundation grant funded by the Ministry of Education, Science and Technology, Korea (No. 2009-0092822 to K.-H.L.) and by the Marine and Extreme Genome Research Center program of the Ministry of Land, Transport and Maritime Affair, Korea (to K.-H.L.).

References

Lee, H. J., Kim, J. A., Lee, M. A., Park, S. J. and Lee, K. H. (2013). Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases. Mol Microbiol 88(4): 813-826.
Shinoda, S., Miyoshi, S., Yamanaka, H. and Miyoshi-Nakahara, N. (1985). Some properties of Vibrio vulnificus hemolysin. Microbiol Immunol 29(7): 583-590.

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