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

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.

Grant Number: 1K99AI185158-01A1
NIH Institute/Center: NIH
Principal Investigator: Hannah Brown