Fibronectin-Degrading Activity of Trypanosoma cruzi Cysteine Proteinase Plays a Role in Host Cell Invasion

Abstract

Trypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insectstage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-␤1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi metacyclic forms, through its fibronectin-degrading activity, is implicated in host cell invasion. E xtracellular matrix (ECM) proteins, which serve as substrates for diverse adhesion molecules and are involved in many important physiological processes, also may mediate cell attachment and/or invasion of pathogenic microorganisms. Among these molecules, fibronectin (FN) has been reported to play a role in adherence to and invasion of host cells by bacteria such as Staphylococcus aureus, Streptococcus pyogenes, and Campylobacter jejuni (1-6). The interaction of fibronectin with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, also has been described. T. cruzi infection is initiated by metacyclic trypomastigote (MT) from the insect vector. This parasite form is responsible for the initial T. cruzi-host cell interaction upon entering the mammalian host through the skin or by the oral route. Following MT internalization in a membrane-bound vacuole and escape to the cytoplasm, the parasite differentiates into amastigote form. After several rounds of replication, amastigotes transform into trypomastigotes that are released in the circulation upon host cell rupture. Experiments with tissue culture trypomastigote (TCT), which corresponds to the bloodstream trypomastigote, revealed the involvement of fibronectin in target cell adhesion/invasion. The treatment of 3T3 fibroblasts or rat peritoneal macrophages, or TCT, with human plasma FN increased parasite-cell association (7), and binding ...

Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part III: Protozoa (3): Kinetoplastids

“…It was concluded that CRZ proteolytic activity played an essential role in fibronectin degradation, facilitating entry of the metacyclic forms, i.e. host cell invasion [128] .…”

Biochemical, Cellular, and Immunologic Aspects during Early Interaction between Trypanosoma cruzi and Host Cell

“…However, this interaction inhibits cell invasion. The presence of the major cysteine proteinase cruzipain (TCC) helps to degrade these extracellular matrix proteins enabling cell invasion [20]. These surface glycoproteins are very polymorphic among T. cruzi strains resulting in different grades of virulence .…”

Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

“…Trypomastigotes and AMAs release EVs containing virulence factors involved in: (i) host cell invasion and intracellular parasite development, such as the TS and TS-like proteins ( Zingales et al, 1985 ; Gonçalves et al, 1991 ; Schenkman et al, 1991 ; Abuin et al, 1996a , b ; Torrecilhas et al, 2009 , 2012 ; Cánepa et al, 2012a ; Maeda et al, 2012 ; Bayer-Santos et al, 2013 ; Marcilla et al, 2014 ; Mattos et al, 2014 ), peptidyl prolyl cis-trans -isomerase ( Moro et al, 1995 ), oligopeptidases and proteases ( Meirelles et al, 1992 ; Caler et al, 1998 ; Scharfstein et al, 2000 ; Cuevas et al, 2003 ; Bastos et al, 2005 ; Doyle et al, 2011 ; Maeda et al, 2014 ), phospolipases A1 and C ( Andrews et al, 1988 ; Rosenberg et al, 1991 ; Furuya et al, 2000 ; Okura et al, 2005 ; Belaunzarán et al, 2007 , 2013 ; Castillo et al, 2013 ); mucins and mucin-like proteins ( Gazzinelli et al, 1991 ; de Diego et al, 1997 ; Kierszenbaum et al, 1999 ; Pereira-Chioccola et al, 2000 ; Kierszenbaum et al, 2002 ; Alcaide and Fresno, 2004 ; Cánepa et al, 2012b ), MASP ( de Pablos et al, 2011 ; Cánepa et al, 2012a ), SAP ( Baida et al, 2006 ; Zanforlin et al, 2013 ), P21 AMA specific proteins ( da Silva et al, 2009 ), surface membrane proteins (TcSMP; Martins et al, 2015 ); (ii) immune evasion ( Andrews et al, 1990 ; Norris et al, 1991 ; Norris and Schrimpf, 1994 ; Ouaissi et al, 1995 ; Reina-San-Martin et al, 2000 ; Martin et al, 2006 ; Mott et al, 2011 ; Kulkarni et al, 2013 ; Nogueira et al, 2015 ); and (iii) increased heart parasitism, inflammation, and arrhythmia that contrib...…”

“…Several studies using synthetic irreversible cysteine peptidase inhibitors have demonstrated that T. cruzi infectivity, host immune evasion, and intracellular growth depend on the activity of cruzipain ( Meirelles et al, 1992 ; Waghabi et al, 2005 ; McKerrow et al, 2008 ). To facilitate entry into non-phagocytic cells like endothelial cells and cardiomyocytes, cruzipain acts on a cell-bound kininogen to generate bradykinin, which upon recognition by the B2 bradykinin receptor, triggers the Ca 2+ mobilization required for parasite internalization ( Scharfstein et al, 2000 ; Guiñazú et al, 2007 ; Maeda et al, 2014 ).…”