In vitro antimicrobial assay using chemically reconstituted system with hemocyanin or hemoglobin
Lab/Group: Jeak Ling DING’s lab (National University of Singapore)
Related Journal & Article Information
Journal: Nature Immunology
Introduction
Here we describe the chemically-reconstituted system for evaluating the antimicrobial activity of the microbial protease-mediated ROS production from the hemocyanin-prophenoloxidase (HMC/PPO)or hemoglobin. The antimicrobial activity is examined by endpoint measurement of the remnant microbial population or by real-time observation of the bactericidal process. To demonstrate the specificity of PO-induced ROS-production which causes the antimicrobial activity, PTU is used here as a specific inhibitor of PO activity.
Materials
Reagents
Equipment
Procedure
1. Prepare the bacterial culture as described in protocol #4, and then adjust the bacterial population to 106 - 107 cfu/ml.
2. For the end-point measurement of HMC/PPO:
(a) incubate the bacteria under test at 106 cfu/ml with 60 μg purified HMC plus 100 nmol 4-methylcatechol (4ME) in 100 μl PBS at 37 °C for 1 h.
(b) then enumerate the remnant bacterial population in the reaction mixture by plating 100 μl of serially diluted samples on nutrient agar plates and incubating at 37 °C overnight. Perform the colony count and calculate the bacterial cfu/ml.
(c) add 10 nmol of phenylthiourea (PTU) to the reactions in order to evaluate the impact of PO activity.
(d) also set up controls by incubating bacteria with HMC/PPO or 4ME or PTU separately, or in combinations.
3. For real-time imaging of the bacterial clearance elicited by HMC/PPO:
(a) mix each bacterial strain at 107 cfu/ml with 60 μg HMC/PPO and 100 nmol 4ME in 100 μl PBS.
(b) apply one μl of the mixture to fluorescence microscopy and examine it at magnification of 63 × 1.6, with time-elapse method.
(c) capture images at intervals of 30 s for 1 h, and make a movie.
4. For the end-point measurement of metHb:
(a) incubate the bacteria under test at 106 cfu/ml with 36 μg metHb and 1.6 μmol H2O2 in 200 μl PBS at 37 °C for 1 h.
(b) then enumerate the remnant bacterial population in the reaction mixture by plating 100 μl of serially diluted samples on nutrient agar plates and incubating at 37 °C overnight. Perform the colony count and calculate the bacterial cfu/ml.
(c) to further prove that the antibacterial activity was indeed attributable to ROS, apply 2 μmol GSH to the incubation mixture to quench the superoxide ions.
(d) set up controls by incubating bacteria with metHb, H2O2 or GSH separately or in combinations.
Troubleshooting
Critical Steps
Anticipated Results
In the presence of the HMC/PPO and its substrate, more of the protease-producing bacteria are cleared compared to those of the protease-inactive strains. This difference can be reduced when ROS quenchers (eg: GSH for both quinone and superoxide anion or SOD for superoxide anion) are applied. When supplemented with exogenous proteases, the antimicrobial effect of the protease-inactive strains is increased dose-dependently. (Refer to the example shown in figure 5 as a typical result obtained using HMC).
References
Acknowledgements
Keywords
in vitro; antimicrobial assay; PPO; ROS
Figure 1
The in vitro antimicrobial action of the bacterial component-activated PO: the end-point measurement and the real-time observation.
(a) The end-point antimicrobial activity assay using PAE-producing and PAE non-producing strains of Pseudomonas aeruginosa. (b) Supplementation of exogenous PAE increased the antimicrobial activity against the PAE non-producing strain. (c) The interception of the microscopy images at 0, 30, 30 min during the real-time observation; (d) the bacterial counts at each time-point of observation.
