Trypanosoma cruziextracellular amastigotes trigger the protein kinase D1-cortactin-actin pathway during cell invasion

Abstract

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells.

Candida albicans: The Ability to Invade Epithelial Cells and Survive under Oxidative Stress Is Unlinked to Hyphal Length

“…Western blots were performed essentially as described by Bonfim-Melo et al (2015). HeLa cells were seeded with 10 8 C. albicans blastospores and incubated for various time intervals (ranging from 0 to 180 min) at 37°C in 5% CO 2 .…”

“…In addition, C. albicans exploits clathrin-dependent endocytosis, which requires dynamin and cortactin, to invade human epithelial cells (Moreno-Ruiz et al, 2009). Cortactin is involved in actin-related cellular processes ranging from lamellipodium protrusion and extracellular matrix degradation to the uptake of intracellular pathogens such as bacteria and parasites (Chen et al, 2003; Bonfim-Melo et al, 2015). Cortactin interacts with the Arp2/3 complex, newly formed actin filaments and a variety of actin-binding/regulation proteins (Daly, 2004); it is also regulated by phosphorylation (Martinez-Quiles et al, 2004).…”

“…Both events occur via an actin-dependent mechanism and involve cortactin (Moreno-Ruiz et al, 2009; Bonfim-Melo et al, 2015). In addition, T. cruzi recruited actin and cortactin to sites of adherence and invasion into HeLa cells and also selectively induced cortactin phosphorylation during cell invasion (Bonfim-Melo et al, 2015). However, possible cortactin phosphorylation in response to C. albicans invasion into non-phagocytic cells, such as HeLa cells, has never been assessed.…”

The Mitogen-Activated Protein Kinase (MAPK) Pathway: Role in Immune Evasion by Trypanosomatids

“…However, it is well established that upon cell invasion, T. cruzi begins to subvert signaling pathways and to use host molecules to favor its entry and survival inside host cells. For instance, T. cruzi extracellular amastigotes (EAs) recruit both host protein kinase D1 (PKD1) and cortactin to induce PKD1 autophosphorylation and cortactin activation by ERK, leading to the recruitment of host actin that allows parasite entry into HeLa cells ( Bonfim-Melo et al, 2015 ). T. cruzi , as well as Leishmania spp., is able to induce: (i) ERK1/2, but not p38 MAPK, activation in macrophages and dendritic cells ( Mukherjee et al, 2004 ); and (ii) increased IL-10 and decreased IL-12 production ( Poncini et al, 2008 ).…”

ERM Proteins Play Distinct Roles in Cell Invasion by Extracellular Amastigotes of Trypanosoma cruzi

“…Host cell invasion by these forms is mediated by complex cellular signaling events triggered by parasite surface proteins and secreted molecules (reviewed in Mortara et al, 2005 ; reviewed in Ferreira et al, 2012 , 2016 ) leading to actin filament reorganization, the main event in EA uptake. During EA internalization, the participation of actin cytoskeleton regulators, such as Rac1, gelsolin (Procópio et al, 1999 ; Fernandes and Mortara, 2004 ) and other proteins related to actin cytoskeleton regulation have been demonstrated to colocalize with actin at parasite invasion sites (Procópio et al, 1999 ; Bonfim-Melo et al, 2015 ). Additionally, host cell membrane components participate in EA invasion (Fernandes et al, 2007 , 2013 ).…”