Cell Culture Model Organisms Isolation Purification and Separation

scientificprotocols authored about 3 years ago

Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding 

Introduction

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.

Procedure

  1. 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.
  2. 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).
  3. 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.
  4. 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.
  5. Collect the bacterial cultures by centrifugation at 8000 x g for 10 min.
  6. 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).

Anticipated Results

  1. 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.
  2. 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.
  3. 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.

References

  1. 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).
  2. 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).

Associated Publications

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

Author information

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.

Average rating 0 ratings