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Naxin Jiang

Nguan Soon Tan

Bow Ho

Jeak Ling Ding

Figure 1

_In vitro_ antimicrobial assay using chemically reconstituted system with hemocyanin or hemoglobin

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.

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 &#x3BC;g purified HMC plus 100 nmol 4-methylcatechol \(4ME) in 100 &#x3BC;l PBS at 37 &#xB0;C for 1 h. 


\(b) then enumerate the remnant bacterial population in the reaction mixture by plating 100 &#x3BC;l of serially diluted samples on nutrient agar plates and incubating at 37 &#xB0;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 &#x3BC;g HMC/PPO and 100 nmol 4ME in 100 &#x3BC;l PBS. 


\(b) apply one &#x3BC;l of the mixture to fluorescence microscopy and examine it at magnification of 63 x 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 &#x3BC;g metHb and 1.6 &#x3BC;mol H2O2 in 200 &#x3BC;l PBS at 37 &#xB0;C for 1 h. 


\(b) then enumerate the remnant bacterial population in the reaction mixture by plating 100 &#x3BC;l of serially diluted samples on nutrient agar plates and incubating at 37 &#xB0;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 &#x3BC;mol 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

Biological techniques

Microbiology

Chemical biology

Drug discovery

Method Article

Naxin Jiang, Nguan Soon Tan, Bow Ho, Jeak Ling Ding

Naxin Jiang

National University of Singapore

Nguan Soon Tan

National University of Singapore

Bow Ho

National University of Singapore

Jeak Ling Ding

National University of Singapore

DOI:

10.1038/nprot.2007.466

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Respiratory protein–generated reactive oxygen species as an antimicrobial strategy

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

Nature Immunology (13 June, 2007)

Method Article

Naxin Jiang, Nguan Soon Tan, Bow Ho, Jeak Ling Ding

Naxin Jiang

National University of Singapore

Nguan Soon Tan

National University of Singapore

Bow Ho

National University of Singapore

Jeak Ling Ding

National University of Singapore

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DOI:

10.1038/nprot.2007.466

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Respiratory protein–generated reactive oxygen species as an antimicrobial strategy

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

Nature Immunology (13 June, 2007)

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