In vivo antimicrobial assay of the PPO system using horseshoe crab as the model animal

doi:10.1038/nprot.2007.482

Method Article

In vivo antimicrobial assay of the PPO system using horseshoe crab as the model animal

https://doi.org/10.1038/nprot.2007.482

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Biological techniques

Microbiology

Chemical biology

Drug discovery

horseshoe crab

prophenoloxidase (PPO)

antimicrobial

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, PTU¹ or kojic acid². 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.

Culture the Gram-positive bacterial strains under pyrogen-free condition as described in protocols #4 and #5.
Adjust the bacterial population to 10⁶-10⁷ cfu/ml with pyrogen free 3% NaCl (isotonic to the horseshoe crab hemolymph).
Inject the horseshoe crab intracardially with 10⁵-10⁶ cfu bacteria /100 gram body, using #23 needle.
At 30 min post injection, collect hemolymph from the horseshoe crab by cardiac puncture using #18 needle.
Immediately after collection, remove the hemocytes by centrifuging the hemolymph at 150 x g for 10 min at room temperature.
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.
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.

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 10⁴ 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.

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).
Dowd, P. F. Relative inhibition of insect phenoloxidase by cyclic fungal metabolites from insect and plant pathogens. Natural Toxins, 7 (6), 337-341 (1999)

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In vivo antimicrobial assay of the PPO system using horseshoe crab as the model animal

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In vivo antimicrobial assay of the PPO system using horseshoe crab as the model animal

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