Study On The Pathogenic Mechanism Of Thermolysin To Bombyx Mori Plasma

Thermolysin, a metallopeptidase secreted by pathogenic microbes, that needs one zincion for enzyme activity and four calcium ions for structural stability. Thermolysin isclassified as the M4family of metallopeptidase, and this family of enzyme is found inanimal pathogenic bacteria and fungi.Thermolysin is an important virulence factor due toits ability to cleave host proteins. To date, studies assessing thermolysin virulence havefocused on the ability of this enzyme to cleave purified proteins in vitro. Using Bombyxmori as a model system, we for the first time tested the effect of thermolysin on plasmaprotein profiles in an organismal context. The results are as follows:1. Lethality of injected thermolysin to the silkworm (Bombyx mori) larvae. Silkwormlarvae on day1of5th larval stage (V-1) were injected with thermolysin, or trypsindissolved in sterilized water. After injection, the silkworm larvae were put on ice for20min and then transferred to tissue papers. The larvae could not stop bleeding likely due todestruction of the coagulation system when thermolysin was injected. In the meanwhile,the hemolymph was melanized. Trypsin injection did not affect the plasma coagulationsystem and the plasma was not melanized.2. Thermolysin cleaves recombinant PPO into an inactive form.Purified recombinantDrosophila melanogaster rPPO1was used to determine whether thermolysin and/ortrypsin activate PPO directly. Absorbance readings after incubation of rPPO1with eitherthermolysin or trypsin failed to detect obvious rPO1activity, showing thatthermolysin-mediated cleavage in vitro does not activate PPO directly. When thermolysinwas injected into the hemocoel of na ve silkworm larvae, the plasma became melanized.To test whether thermolysin induces B. mori plasma PPO activation in vitro, plasma fromlarvae that had been injected with Micrococcus was incubated with thermolysin, trypsin, orwithout either protease. Results confirmed that plasma from Micrococcus injected larvaehas negligible PO activity, and showed that while trypsin treatment does not activate PO,thermolysin treatment induces high levels of plasma PO activity. Together, these data showthat, in vitro, thermolysin induces the activation of PPO in whole plasma, but not theactivation of recombinant PPO in the absence of other plasma proteins. This suggests thatthermolysin-induced PPO activation occurs through the increased activity of plasma serineproteases that cleave PPO into its bioactive form. 3. Serine protease, is not activated by thermolysin. Treatment of α-chymotrypsinogenA as well as incubation with α-chymotrypsin with trypsin increased α-chymotrypsinactivity. However, thermolysin incubation with α-chymotrypsinogen A as well asincubation with α-chymotrypsin showed that this enzyme cannot processα-chymotrypsinogen A into its active form. This result suggesting that thermolysin cannotcleave this protein. This result was more pronounced when non-reducing SDS-PAGE wasperformed: most α-chymotrypsinogen A remained intact after treatment with thermolysinwhile it disappeared after treatment with trypsin. Together, these data show thatthermolysin cannot cleave α-chymotrypsinogen A. This suggests that thermolysin cannotdirectly activate serine proteases.4. Cleavage on mammalian plasma proteins by addition of thermolysin. Thermolysincan cleave mammalian plasma proteins at37℃and42℃. Plasmas from the followingmammals were assayed: rabbit antiserum, human AB serum, fetal bovine serum, andBALB/C mouse plasma. Higher levels of plasma protein degradation were observed ateach time point when the temperature was increased to42℃. When trypsin was used at thesame enzyme activity or at the same amount as thermolysin, plasma protein degradationwas not significantly observed. Therefore, thermolysin-induced plasma protein degradationis a universal phenomenon.5. Proposed model for thermolysin action.Normally, there are many serpin proteinsthat can regulate different protease activity in animal plasmas. Plasma serpins inhibit theactive serine protease like trypsin activity. However, thermolysin can specifically andquickly cleave serpin, by which to liberate the active proteases that might be activated bythermolysin directly or indirectly. Therefore, the cleavage on serpins and activation onvarious proteases since the addition of thermolysin are the reasons to boost plasma proteindegradation.