Exploring mechanisms of activation of an innate immune pathway by fungal extracellular vesicles

Massachusetts General Hospital, Boston MA

Investigators

Abstract

Project Summary Interferon (IFN) signaling plays an important role in the host defense against invading pathogens. Activation of this innate immune response can be protective or detrimental for the host, depending on the stimulating agent. Major IFN-producing pathways, like the STING pathway, have been historically studied in the context of viral and bacterial infections. However, a recent transcriptomic study revealed upregulation of type I IFN pathway components in response to Candida albicans (Ca) infection, suggesting a significant role for IFN pathways in fungal pathogenesis as well. My preliminary data revealed that deletion of essential STING pathway components improved host survival following a Ca infection. Furthermore, I demonstrated that Ca DNA packaged in extracellular vesicles (EVs) triggers the STING pathway causing a lethal and hyperinflammatory response that clears the fungal infection. Still, the questions of pathway regulation, functional consequence, and broad application to other fungal organisms remain unanswered. By completing this mentored career development award, I will gain invaluable training in EV isolation and characterization, Nanostring analysis, flow-based analytics, and bioinformatics while also fine-tuning my skills with confocal microscopy and animal work. During the mentored phase of this award, I will optimize EV and EV DNA preparation from various fungal pathogens in the Vyas laboratory, I will design and validate any knockout cell lines or visualization needed to complete these aims (including Nanostring and RNA sequencing), and I will attend bioinformatics courses and career development seminars relevant to my future. In the independent phase I will focus on the animal survival studies (including breeding) outlined in Aim 1 and dedicate time to writing manuscripts and grants for my independent research. I will also use the training I received during my K99 phase in flow-based immunophenotyping and multiplex ELISAs as well as sequencing analysis to complete the experiments outlined in this grant. This proposal will continue to uncover the mechanism of STING-dependent type I IFN induction in response to clinically relevant fungal pathogens. In Aim 1, I will identify the functional significance for fungal EV induction of a specific ISG, viperin. In Aim 2, I will elucidate the regulatory mechanisms involved in STING pathway activation by fungal EVs and fungal DNA by exploring the role of an exonuclease (Trex1) known to regulate this pathway. Finally, in Aim 3, I will assess the extent to which fungal EVs activate the STING pathway. These findings will elucidate the functional consequence of this innate immune signaling pathway activation by fungal pathogens and uncover a new role for fungal EVs in host cell reprogramming.