Tuning the innate immune response to antimicrobial resistant infections

Background
Neutrophils (innate immune white blood cells) can kill many invading pathogens using powerful artillery at their disposal. We will determine whether host neutrophils can be activated as an additional therapy to the limited antifungal drugs available. Invasive fungal infection is a major and increasing cause of mortality in the immunocompromised. The increasing prevalence and emerging antifungal drug resistance is a global emergency that threatens the treatment of many thousands of individuals with immunocompromise. This project will focus on two clinically important invasive fungal infections, candidiasis and cryptococcosis.
We will use zebrafish embryos infected with Candida and Cryptococcus as tractable in vivo models of fungal disease. We focus on genetic hypoxia signalling (via the transcription factor Hif-alpha) that has profound activating effects on neutrophil microbicidal function, and is an exciting host-derived therapeutic target. In preliminary data we have shown that upregulation of Hif-1alpha is protective against Candida infection.

Objectives
We hypothesise that Hif can aid fungal infection control by activating neutrophils.
We will use cutting-edge molecular biology and fluorescence microscopy techniques to address:
1. How Hif-alpha signalling is protective against Candida/Cryptococcal infection
2. How modulating Hif-alpha variants can fine-tune neutrophil behaviour during fungal infection

Novelty
Targeting neutrophils to augment pathogen killing is emerging as an important host-derived strategy against infections with resistant pathogens, but has not yet been applied to fungal disease.

Timeliness
The opportunity to target Hif's role(s) in emerging fungal infections will use recently-established transgenic fish lines and cutting edge methodologies in the newly opened (Autumn 2020) Wolfson Zebrafish Infection Laboratory.

Experimental Approach
Neutrophil and macrophage populations will be manipulated in zebrafish to assess the effect of each cell type on the protective Hif-alpha effect. Neutrophil migratory behaviour will be assayed using timelapse fluorescence microscopy to observe neutrophil/fungal interactions. Links between Hif-alpha signalling and neutrophil phenomena including neutrophil swarming and NET (neutrophil extracellular trap) formation will be explored to uncover novel molecular mechanisms in fungal infection. The effect on endogenous Hif-alpha levels during fungal infection will be assessed using the transgenic phd3:GFP Hif reporter fish. The stability of Hif-alpha variants during infection will be assessed in vitro in the lab of the third supervisor, a specialist in HIF-alpha imaging in living cells. The downstream targets of Hif-alpha will be determined from RNAseq data of FACS isolated neutrophils after Hif-alpha modulation. Zebrafish findings can be extrapolated to human neutrophil in vitro models if required.