In vitro antimicrobial assay using chemically reconstituted system with hemocyanin or hemoglobin

doi:10.1038/nprot.2007.466

Method Article

In vitro antimicrobial assay using chemically reconstituted system with hemocyanin or hemoglobin

https://doi.org/10.1038/nprot.2007.466

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Biological techniques

Microbiology

Chemical biology

Drug discovery

in vitro antimicrobial assay

PPO

ROS

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.

Prepare the bacterial culture as described in protocol #4, and then adjust the bacterial population to 10⁶ - 10⁷ cfu/ml.
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.

(d) also set up controls by incubating bacteria with HMC/PPO or 4ME or PTU separately, or in combinations.

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 x 1.6, with time-elapse method.

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.

(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.

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).

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In vitro antimicrobial assay using chemically reconstituted system with hemocyanin or hemoglobin

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