Abstract
Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM) of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D) environment mainly composed of Collagen I (COL I). This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited “freeze and run” migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular “home”—macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model.
Highlights
The promastigote form of Leishmania amazonensis, causative agent of American cutaneous leishmaniasis (Barral et al, 1991; Murray et al, 2005), is transmitted through the bite of infected sand flies from the genus Lutzomyia (Kaye & Scott, 2011; Neuber, 2008)
L. amazonensis promastigotes extensively interact with and remodel 3D Collagen I (COL I) matrix fibers Scanning electron microscopy (SEM) of promastigotes cultivated in a 3D COL I matrix showed a huge number of promastigotes extensively adhered to COL I fibers (Fig. 1)
Leishmania amazonensis is an intracellular parasite that mainly proliferates inside macrophages
Summary
The promastigote form of Leishmania amazonensis, causative agent of American cutaneous leishmaniasis (Barral et al, 1991; Murray et al, 2005), is transmitted through the bite of infected sand flies from the genus Lutzomyia (Kaye & Scott, 2011; Neuber, 2008). Because L. amazonensis is an intracellular parasite that only proliferates inside a cellular host in the mammalian host, its interaction with the host ECM has been neglected. In order to establish an intracellular infection, the promastigote form must overcome the obstacles presented by the dermis ECM (Chang & McGwire, 2002). Except for broad ECM alterations during experimental Leishmania infection (Abreu-Silva et al, 2004; Melo et al, 2009; Silva-Almeida et al, 2012a) little is known about the interaction of L. amazonensis with the host ECM (Kulkarni et al, 2008; McGwire, Chang & Engman, 2003). Long viewed only as a supportive structure, the ECM is an essential part of the cell’s milieu that, through direct or indirect means, regulates almost all cellular behavior (Hynes, 2009), including inflammatory signaling (Larsen et al, 2006)
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