Shrimp alpha-2-macroglobulin prevents the bacterial escape by inhibiting fibrinolysis of blood clots.
Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis.
Department of Biochemistry, Faculty of Science, Center of Excellence for Molecular Biology and Genomics of Shrimp, Chulalongkorn University, Bangkok, Thailand.
Vorrapon
V
Chaikeeratisak
Kunlaya
K
Somboonwiwat
Anchalee
A
Tassanakajon
D000818:Animals; D005342:Fibrinolysis; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006434:Hemocytes / Q000378:metabolism / Q000821:virology; D006458:Hemolymph / Q000378:metabolism / Q000821:virology; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D033561:Penaeidae / Q000378:metabolism* / Q000821:virology*; D011485:Protein Binding; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014733:Vibrio / Q000502:physiology*; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D000511:alpha-Macroglobulins; D049452:Green Fluorescent Proteins; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins