Chronic infection by Leishmania amazonensis mediated through MAPK ERK mechanisms

Abstract

Leishmania amazonensis is an intracellular protozoan parasite responsible for chronic cutaneous leishmaniasis (CL). CL is a neglected tropical disease responsible for infecting millions of people worldwide. L. amazonensis promotes alteration of various signaling pathways that are essential for host cell survival. Specifically, through parasite-mediated phosphorylation of extracellular signal regulated kinase (ERK), L. amazonensis inhibits cell-mediated parasite killing and promotes its own survival by co-opting multiple host cell functions. In this review, we highlight Leishmania-host cell signaling alterations focusing on those specific to (1) motor proteins, (2) prevention of NADPH subunit phosphorylation impairing reactive oxygen species production, and (3) localized endosomal signaling to up-regulate ERK phosphorylation. This review will focus upon mechanisms and possible explanations as to how Leishmania spp. evades the various layers of defense employed by the host immune response.

Figure 1. Life Cycle of Leishmania spp.

Parasite entry into the human host occurs upon an infected sandfly ingesting a blood-meal and thereby injecting a plug of infectious promastigotes into the host. Within the dermis, the parasite is phagocytized and transforms into the amastigote form. Amastigotes divide and thrive within macrophages. Once a naïve sandfly takes a blood-meal from the infected host, it will ingest infected phagocytes. These parasites will be released from the cell and the parasite replicates within the sandfly midgut as promastigotes. Image from the CDC. www.cdc.gov/parasites/leishmaniasis/biology.html

Figure 2. Murine fibroblast infected with Leishmania amazonensis

Morphology of infectious Leishmania amazonensis promastigote. There is not one, but rather several parasites in the process of infecting one single fibroblast. The flagella is clearly visible as transformation into the amastigote stage begins after engulfment and entrance into the parasitphorous vacuole.

Figure 3. MAPK classic and unconventional pathways

MAPKs are a family of pathways united by a three-tiered module that starts with activating stimuli that leads to the activation of the MAPKKK, followed by MAPKK, and to MAPK. Conventional MAPKs consist of ERK5, ERK1/2, p38, and JNK1/2/3. Atypical MAPKs are ERK3/4, ERK7/8 and NLK. Once MAPKs are activated they further phosphorylate and activate a plethora of substrates that located in the cytoplasm, nucleus, membrane and cytoskeleton. Adapted from Cargnello et al. [44].

Figure 4. NADPH phox subunits in basal and activated cells

A.) During a resting state, the cytoplasmic subunits of p47phox, p67phox and p40phox are found free in the cytoplasm. Membrane subunit is flavocytochrome (p22phox and gp91phox/NOX2). B.) Upon activation, Rac will activate cytoplasmic subunits. Once p47phox is activated through phosphorylation, it will travel to the membrane along with p67phox and p40phox. It is only upon interaction with the membrane subunits of the NADPH system that the complex is activated and can produce ROS including to eliminate intracellular pathogens including L. amazonensis.

Figure 5. NADPH complex of bone marrow derived macrophages activated with Zymosan

A.) BMM not exposed to any stimulant, labeled with anti-p47phox (AF488) and gp91phox (AF568). No activation NADPH oxidase activation, cells have basal levels of these proteins with no visualization of vesicles. B.) BMMs activated with Zymosan for 1 hour. Zymosan is a strong activator of NADPH oxidase complex formation. Cell has delineated vesicles around engulfed particles of Zymosan.

Figure 6. Model. Leishmania amazonensis mediated ERK1/2 activation promotes parasite intracellular survival

Leishmania amazonensis is able to take of many pathways upon infection of host macrophages. A.) after a generic stimulus, the MAPK pathway is activated, or there can be activation and engagement of the PI3K pathway, which leads to Akt phosphorylation, and further phosphorylation of p47phox via Akt. p47phox translocation to the membrane with the rest of the cytosolic subunits will bind with b558 and start the RB. B.) 1. MP1-MEK scaffolding complex is needed for parasite induced ERK1/2 phosphorylation. 2. ERK1/2 phosphorylates intermediate chain of dynein essential for motor protein movement, thought to facilitate the PV access to the host nucleus. 3. ERK1/2 activating a downstream phosphatase. This keeps Akt in a state of inactivation – and p47phox would not be activated, and therefore no activation of the NADPH oxidase complex or production of ROS, leading to intracellular parasite survival.