Biology subjects

Barrie, U.

Publications and source records attributed to Barrie, U..

3 recordsLinked to original sources

bioRxiv2024

Using machine learning to dissect host kinases required for Leishmania internalization and development

The Leishmania life cycle alternates between promastigotes, found in the sandfly, and amastigotes, found in mammals. When an infected sandfly bites a host, promastigotes are engulfed by phagocytes (i.e., neutrophils, dendritic cells, and macrophages) to establish infection. When these phagocytes die or break down, amastigotes must be re-internalized to survive within the acidic phagolysosome and establish disease. To define host kinase regulators of Leishmania promastigote and amastigote uptake and survival within macrophages, we performed an image-based kinase regression screen using a panel of 38 kinase inhibitors with unique yet overlapping kinase targets. We also targeted inert beads to complement receptor 3 (CR3) or Fc{gamma} receptors (FcR) as controls by coating them with complement/C3bi or IgG respectively. Through this approach, we identified several putative host kinases that regulate receptor-mediated phagocytosis and/or the uptake of L. amazonensis. Findings included kinases previously implicated in Leishmania uptake (such as Src family kinases (SFK), Abl family kinases (ABL1/c-Abl, ABL2/Arg), and spleen tyrosine kinase (SYK)), but we also uncovered many novel kinases. Our methods also predicted host kinases necessary for promastigotes to convert to amastigotes or for amastigotes to survive within macrophages. Overall, our results suggest that the concerted action of multiple interconnected networks of host kinases are needed over the course of Leishmania infection, and that the kinases required for the parasites life cycle may differ substantially depending on which receptors are bound and the life cycle stage that is internalized. In addition, using our screen, we identified kinases that appear to preferentially regulate the uptake of parasites over beads, indicating that the methods required for Leishmania to be internalized by macrophages may differ significantly from generalized phagocytic mechanisms. Our findings are intended to be used as a hypothesis generation resource for the broader scientific community studying the roles of kinases in host-pathogen interactions.

microbiology↗

bioRxiv2022

ERK1/2 Activation in Macrophages Promotes Leishmania Internalization and Pathogenesis

The obligate intracellular protozoan parasite Leishmania binds several host cell receptors to trigger its uptake by phagocytic cells, ultimately resulting in visceral or cutaneous leishmaniasis. After Leishmania engages receptors on macrophages and other phagocytes, a series of signaling pathways in the host cell are activated during its internalization, which are critical for establishment and persistence of Leishmania infection. Thus, preventing Leishmania internalization by phagocytes could be a novel therapeutic strategy for leishmaniasis. However, the host cellular machinery that mediates promastigote and amastigote uptake is not well understood. Here, using small molecule inhibitors of Mitogen-activated protein kinases/Extracellular signal regulated kinases (MAPK/ERK), we demonstrate that ERK1/2 mediates Leishmania amazonensis uptake and (to a lesser extent) phagocytosis of beads by macrophages. We find that inhibition of MEK1/2 or ERK1/2 leads to inefficient amastigote uptake by macrophages. Moreover, using inhibitors and primary macrophages lacking spleen tyrosine kinase (SYK) or Abl family kinases, we show that SYK and Abl family kinases mediate Raf, MEK, and ERK1/2 activity and are necessary for efficient uptake. Finally, we demonstrate that trametinib, a MEK1/2 inhibitor used clinically to treat certain cancers, significantly reduces disease severity and parasite burden in Leishmania-infected mice, even if it is started significantly after lesions develop. Our results show that maximal Leishmania infection requires MAPK/ERK and highlight the potential for MAPK/ERK-mediated signaling pathways to be novel therapeutic targets for leishmaniasis. Lay summaryLeishmania is a single-celled parasite that causes skin ulcers or a disseminated disease in humans. Our goal is to identify new drugs to treat Leishmania infection. Leishmania must live inside human immune cells to cause disease. If Leishmania is not able to enter human immune cells, it dies. Our studies demonstrate how Leishmania infection permits a set of proteins called MAP kinases to pass signals from one protein to the next within mammalian immune cells. The resulting signals allow Leishmania to enter into these immune cells and survive within its host. Importantly, trametinib, a drug that prevents these signals from MAP kinases, decreases the development of skin ulcers when it is given to mice that are infected with Leishmania. Our findings suggest that leishmaniasis could be treated with drugs that act on kinases found in humans rather than the parasites themselves. One sentence summaryAn Abl2-SYK-Raf-MEK-ERK pathway facilitates Leishmania uptake by phagocytic cells and promotes disease severity in Leishmania-infected mice.

microbiology↗

bioRxiv2022

The Src and Abl family kinases activate the Spleen Tyrosine Kinase to maximize phagocytosis and Leishmania infection

Leishmania spp. are obligate intracellular parasites that must be internalized by phagocytic cells to evade immune responses and cause disease. The uptake of both Leishmania promastigotes (insect-stage parasites) and amastigotes (proliferative stage parasites in humans and mice) by phagocytes is thought to be mainly host cell-driven, not parasite-driven. Our previous work indicates that host Src and Abl family kinases facilitate Leishmania entry into macrophages and pathogenesis in murine cutaneous leishmaniasis. Here we demonstrate that host spleen tyrosine kinase (SYK) is required for efficient uptake of Leishmania promastigotes and amastigotes. A Src family kinase-Abl family kinase-SYK signaling cascade induces Leishmania amastigote internalization. Finally, lesion size and parasite burden during Leishmania infection is significantly decreased in mice lacking SYK in monocytes or by treatment with the SYK inhibitor entospletinib. In summary, SYK is required for maximal Leishmania uptake by macrophages and disease in mice. Our results suggest potential for treating leishmaniasis using host cell-directed agents. SUMMARY STATEMENTActivation of Spleen Tyrosine Kinase by Src and Abl family kinases is required for maximal Leishmania uptake by macrophages and disease in a mouse model of cutaneous leishmaniasis.

microbiology↗