(B) Kinin-releasing activity of recombinant cruzipain-1 or cruzipain-2

(B) Kinin-releasing activity of recombinant cruzipain-1 or cruzipain-2. cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas’ disease. (trypomastigotes) rapidly enter the bloodstream, from where they disseminate the infection to multiple tissues. After invading macrophages, muscle, and other nucleated cells, the trypomastigotes escape from endocytic vacuoles and migrate into the cytoplasm where they transform into round-shaped amastigotes, the replicating forms. Within 5C6 d, RG7713 the host cells rupture, releasing large numbers of trypomastigotes and amastigotes into interstitial spaces. Acute pathology and parasite tissue load subside with the onset of immunity, but the pathogen is not eradicated. After years of asymptomatic infection, 10C24% of the patients develop a severe chronic cardiomyopathy characterized by myocarditis, fibrosis, microcirculatory lesions, cardiomegaly, and conduction system abnormalities 1 2 3. At the cellular level, trypomastigotes invade nonphagocytic cells by a unique mechanism distinct from phagocytosis 4 5. Penetration by tissue culture trypomastigotes (TCTs) is preceded by energy-dependent adhesive interactions RG7713 6 involving the parasites’ surface glycoproteins RG7713 7 8 and negatively charged host surface molecules 9. Depending on the host cellCparasite combination studied, invasion requires activation of the TGF- signaling pathway 10 or stimulation of host cell receptors coupled to heterotrimeric G proteins 11 12. Efforts to characterize the hitherto unknown Ca2+-signaling agonist pointed to a crucial role of a cytosolic parasitic serine protease of 80 kD, oligopeptidase B 13. Although null mutants generated by targeted deletion of the oligopeptidase B gene were poorly infective 14, purified or recombinant oligopeptidase B alone failed to induce intracellular free calcium ([Ca2+]i) transients in the mammalian cells 13. Because addition of recombinant oligopeptidase B to null parasite extracts reconstituted [Ca2+]i signaling, it was suggested that the agonistic activity was generated by oligopeptidase BCmediated processing of a cytoplasmic precursor molecule 14. Mouse monoclonal to MYST1 Other RG7713 clues to understand the role of proteases in host cell invasion emerged from in vitro assays performed with synthetic inhibitors of cruzipain 15, the parasite’s major cysteine proteinase 16 17 18. Encoded by multiple polymorphic genes 19 20, this cathepsin LClike proteinase is the most extensively characterized isoform expressed by replicating forms of the parasite 16 17 18 21. Given the broad pH range of the activity profile RG7713 and the high stability of cruzipain 17, the finding of antigen deposits of this molecule in foci of myocardial inflammation 22 suggested that this proteinase may contribute to pathology. Our findings that the substrate specificity of cruzipain resembles that of tissue kallikrein and that cruzipain releases the bradykinin (BK)-like vasoactive peptide lysyl-bradykinin (kallidin) from its large precursor forms, high (H-) and low (L-) molecular weight kininogens 23, suggested that may directly trigger the kinin system through the activity of this cysteine proteinase. Here we demonstrate that the short-lived kinin peptides and their cognate G proteinCcoupled cellular receptors 24 are engaged in the signaling mechanisms leading to invasion. We also show that invasion of cells that overexpress the constitutive B2 subtype of BK receptor is critically modulated by the kinin-degrading activity of host kininase II, also known as the angiotensin ICconverting enzyme (ACE). The finding that activation of the proinflammatory kinin cascade by trypomastigotes potentiates invasion may shed light on the molecular basis of.