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.
- Culture the Gram-positive bacterial strains under pyrogen-free condition as described in protocols #4 and #5.
- Adjust the bacterial population to 10e6-10e7 cfu/ml with pyrogen free 3% NaCl (isotonic to the horseshoe crab hemolymph).
- Inject the horseshoe crab intracardially with 10e5-10e6 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 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.
- 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)
Figure 1: The PO triggered by the microbial protease contributes to in vivo antimicrobial activity.
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).
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
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.