Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding
To determine the specificity of the microbial proteases in triggering the respiratory proteins to produce bactericidal ROS, we compared the level of bacterial clearance of protease-producing and non-producing strains of typical Gram-negative and Gram-positive bacteria. For Gram-negative bacteria, we chose Pseudomonas aeruginosa strain PAO-Iglewski which produces PAE (the major extracellular protease virulence factor), and the PAE-knockout mutant PAO-B1A1 which does not produce PAE (1). For Gram-positive bacteria, we cultured Staphylococcus aureus PC1839 strain which produces active V8-protease, and AK3 strain which is a V8 protease-mutant (2). The genes controlling PAE in P. aeruginosa and the V8-protease in S. aureus are under the control of quorum-sensing signals. Therefore the culture conditions which facilitate the production of active extracellular proteases are utilized.
- Inoculate single colony of P. aeruginosa and S. aureus into 10 ml each of Luria-Bertani (LB) broth and Tryptone Soy Broth (TSB), respectively, and shake the culture at 200 rpm for 16 h at 37 ºC.
- For P. aeruginosa, in order to induce the production of PAE, dilute the overnight cultures of PAO-Iglewski and PAO-B1A1 with LB broth to 103 cfu/ml, and incubate as standing cultures at 37 ºC for 48 h (1).
- For S. aureus, dilute the overnight cultures of each strain in TSB until OD600nm reaches 1.0, and shake the culture at 220 rpm, 37 ºC for 4 h.
- For the naturally occurring Bacillus species, dilute the overnight culture with marine broth until OD600nm reaches 0.5, and then shake at 220 rpm, 37 ºC for 2 h.
- Collect the bacterial cultures by centrifugation at 8000 x g for 10 min.
- To evaluate the extracellular protease production, filter the supernatant through 0.22 μm pore-sized membrane-filter and measure the soluble protease activity in the filtered bacterium-free culture medium using the Azocoll protease assay (see protocol #12).
- For P. aeruginosa, the typical readout of the extracellular protease activity is: A550=0.4-0.7 for PAO-Iglewski, and A550<0.05 for PAO-B1A1.
- For S. aureus PC1839 and ATCC49775, the typical readout of the extracellular protease activity assay is A550= 0.2-0.3, whereas for AK3 strain and the clinical MRSA strains, the typical readout is A550 <0.05.
- For the naturally occurring Bacillus strains, the typical readout of the protease-producing strains is A550=0.3-0.5, while that of the protease non-producing strains is A550< 0.05.
- Toder, D. S., Ferrell, S. J., Nezezon, J. L., Rust, L. & Iglewski, B. H. lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity. Infect Immun. 62, 1320-1327 (1994).
- Karlsson, A., Saravia-Otten, P., Tegmark, K., Morfeldt, E. & Arvidson, S. Decreased amounts of cell wall-associated protein A and fibronectin-binding proteins in Staphylococcus aureus sarA mutants due to up-regulation of extracellular proteases. Infect Immun. 69, 4742-4748 (2001).
Respiratory protein–generated reactive oxygen species as an antimicrobial strategy, Naxin Jiang, Nguan Soon Tan, Bow Ho, and Jeak Ling Ding, Nature Immunology 8 (10) 1114 - 1122 26/08/2007 doi:10.1038/ni1501
Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding, National University of Singapore
Source: Protocol Exchange (2007) doi:10.1038/nprot.2007.469. Originally published online 31 October 2007.