Pharmacology Viruses Model Organisms

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


To demonstrate the ability of microbial factor-activated PPO activity in clearing the invading pathogen in vivo, we infected horseshoe crabs in the presence or absence of PO-specific inhibitor, PTU (1) or kojic acid (2). A comparison of the remnant bacterial load under these conditions should help to clarify the specific contribution of PO, if any, to the antimicrobial activity. Previously, it was reported that HMC/PPO is activated by host intracellular factors released through LPS-dependent degranulation of hemocytes. To avoid provocation of PPO by such cellular components and to unequivocally demonstrate that the microbial factor-activated PPO contributes to the antimicrobial defense, Gram-positive bacteria lacking LPS were used to avoid LPS-induced hemocyte lysis. To this end, the S. aureus laboratory strains, PC1839 (V8 protease-producing) and AK3 (V8 protease inactive mutant), were injected into the animals.


  1. Culture the Gram-positive bacterial strains under pyrogen-free condition as described in protocols #4 and #5.
  2. Adjust the bacterial population to 10e6-10e7 cfu/ml with pyrogen free 3% NaCl (isotonic to the horseshoe crab hemolymph).
  3. Inject the horseshoe crab intracardially with 10e5-10e6 cfu bacteria /100 gram body, using #23 needle.
  4. At 30 min post injection, collect hemolymph from the horseshoe crab by cardiac puncture using #18 needle.
  5. Immediately after collection, remove the hemocytes by centrifuging the hemolymph at 150 x g for 10 min at room temperature.
  6. Quantify the remnant bacterial load in the extracellular milieu by applying 100 μl of the cell-free hemolymph (from step 5) to the nutrient agar plate and incubate at 37 °C overnight.
  7. In order to confirm the contribution of PO activity in the bacterial clearance, include 5 mM PTU or 5 mM kojic acid in the bacterial injection to block in vivo PO activity, if any.

Anticipated Results

As shown in Figure 7, at 30 min post-injection, the remnant bacterial load in the cell free hemolymph (injected without PTU or kojic acid) is less than 10e4 cfu/ml. Co-injection of PTU or kojic acid with the bacteria results in significantly higher bacterial load of the extracellular-protease positive strains such as S. aureus PC1839. In contrast, the clearance of the extracellular-protease-negative strains such as AK3, is unaffected by PTU or kojic acid.


  1. Nellaiappan, K. & Sugumaran, M. On the presence of prophenoloxidase in the hemolymph of the horseshoe crab, Limulus. Comp Biochem Physiol B Biochem Mol Biol. 113, 163-168 (1996).
  2. Dowd, P. F. Relative inhibition of insect phenoloxidase by cyclic fungal metabolites from insect and plant pathogens. Natural Toxins, 7 (6), 337-341 (1999)


Figure 1: The PO triggered by the microbial protease contributes to in vivo antimicrobial activity.

Fig 1

Injection of the S. aureus laboratory strains, PC1839 and AK3, which are active V8 protease-positive and -negative, respectively into the horseshoe crab at 105 cfu/100 gram body weight, in the presence or absence of 5 mM PTU. At 30 min post injection, the remaining bacterial load in the hemolymph was measured. The protease-positive strain which specifically evoked the ROS-production by HMC/PPO, is in turn killed effectively. However, co-injection with PTU inhibited the HMC/PPO activity, and allowed the bacteria to remain viable in the host. On the other hand, the clearance of the V8-inactive strain was unaffected by PTU. This is probably due to the antimicrobial effects of parallel PO-independent mechanisms (see main text for further explanations).

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.2007.482. Originally published online 31 October 2007.

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