Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection.

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish-Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo. ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection.

Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish.

Neutrophils are primary phagocytes of the innate immune system that generate reactive oxygen species (ROS) and mediate host defense. Deficient phagocyte NADPH oxidase (PHOX) function leads to chronic granulomatous disease (CGD) that is characterized by invasive infections, including those by the generally non-pathogenic fungus Aspergillus nidulans The role of neutrophil ROS in this specific host-pathogen interaction remains unclear. Here, we exploit the optical transparency of zebrafish to image the effects of neutrophil ROS on invasive fungal growth and neutrophil behavior in response to Aspergillus nidulans In a wild-type host, A. nidulans germinates rapidly and elicits a robust inflammatory response with efficient fungal clearance. PHOX-deficient larvae have increased susceptibility to invasive A. nidulans infection despite robust neutrophil infiltration. Expression of subunit p22phox (officially known as CYBA), specifically in neutrophils, does not affect fungal germination but instead limits the area of fungal growth and excessive neutrophil inflammation and is sufficient to restore host survival in p22phox-deficient larvae. These findings suggest that neutrophil ROS limits invasive fungal growth and has immunomodulatory activities that contribute to the specific susceptibility of PHOX-deficient hosts to invasive A. nidulans infection.

PTP1B phosphatase dampens iPSC-derived neutrophil motility and antimicrobial function.

Neutrophils are rapidly recruited to sites of infection and are critical for pathogen clearance. Therapeutic use of primary neutrophils has been limited, as they have a short lifespan and are not amenable to genetic manipulation. Human induced pluripotent stem cells (iPSCs) can provide a robust source of neutrophils for infusion and are genetically tractable. However, current work has indicated that dampened intracellular signaling limits iPSC-derived neutrophil (iNeutrophil) cellular activation and antimicrobial response. Here, we show that protein tyrosine phosphatase 1B (PTP1B) inhibits intracellular signaling and dampens iNeutrophil effector function. Deletion of the PTP1B phosphatase increased PI3K and ERK signaling and was associated with increased F-actin polymerization, cell migration, and phagocytosis. In contrast, other effector functions like NETosis and reactive oxygen species production were reduced. PTP1B-deficient neutrophils were more responsive to Aspergillus fumigatus and displayed rapid recruitment and control of hyphal growth. Accordingly, depletion of PTP1B increased production of inflammatory factors including the neutrophil chemokine interleukin-8. Taken together, these findings suggest that PTP1B limits iNeutrophil motility and antimicrobial function.

Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection.

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish- Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo . ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection. Author Summary: Aspergillus fumigatus is a common environmental fungus that can infect immunocompromised people and cause a life-threatening disease called invasive aspergillosis. An important step during infection is the development of A. fumigatus filaments known as hyphae. A. fumigatus uses hyphae to acquire nutrients and invade host tissues, leading to tissue damage and disseminated infection. In this study we report that a regulator of gene transcription in A. fumigatus called ZfpA is important for hyphal growth during infection. We find that ZfpA activity protects the fungus from being killed by innate immune cells and decreases the efficacy of antifungal drugs during infection by regulating construction of the cell wall, an important protective layer for fungal pathogens. Our study introduces ZfpA as an important genetic regulator of stress tolerance during infection that protects A. fumigatus from the host immune response and antifungal drugs.

Switching to the cyclic pentose phosphate pathway powers the oxidative burst in activated neutrophils.

Neutrophils are cells at the frontline of innate immunity that can quickly activate effector functions to eliminate pathogens upon stimulation. However, little is known about the metabolic adaptations that power these functions. Here we show rapid metabolic alterations in neutrophils upon activation, particularly drastic reconfiguration around the pentose phosphate pathway, which is specifically and quantitatively coupled to an oxidative burst. During this oxidative burst, neutrophils switch from glycolysis-dominant metabolism to a unique metabolic mode termed 'pentose cycle', where all glucose-6-phosphate is diverted into oxidative pentose phosphate pathway and net flux through upper glycolysis is reversed to allow substantial recycling of pentose phosphates. This reconfiguration maximizes NADPH yield to fuel superoxide production via NADPH oxidase. Disruptions of pentose cycle greatly suppress oxidative burst, the release of neutrophil extracellular traps and pathogen killing by neutrophils. Together, these results demonstrate the remarkable metabolic flexibility of neutrophils, which is essential for their functions as the first responders in innate immunity.

Guide to the Larval Zebrafish-Aspergillus Infection Model.

The larval zebrafish is an increasingly popular host model for the study of Aspergillosis. The visual accessibility, genetic resources, small size, and ease of handling make zebrafish larvae compatible with higher-throughput investigation of fungal virulence and host resistance mechanisms. This article provides the protocols needed to prepare Aspergillus fumigatus spore inocula and use microinjection to infect the hindbrain ventricle of zebrafish larvae. Furthermore, we include protocols for analyzing host survival, immobilizing larvae for live imaging, and suggestions for image analysis. © 2021 Wiley Periodicals LLC. Support Protocol 1: Preparing Aspergillus spores Support Protocol 2: Dechorionating zebrafish embryos Support Protocol 3: Generating transparent larvae with 1-phenyl 2-thiourea (PTU) Basic Protocol 1: Hindbrain microinjection of zebrafish larvae with Aspergillus spores Basic Protocol 2: Survival analysis Basic Protocol 3: Multi-day imaging of infected larvae Alternate Protocol: Embedding larvae in low-melting-point agarose.

Immune Cell Paracrine Signaling Drives the Neutrophil Response to A. fumigatus in an Infection-on-a-Chip Model.

INTRODUCTION: Neutrophils act as first responders during an infection, following signals from the pathogen as well as other host cells to migrate from blood vessels to the site of infection. This tightly regulated process is critical for pathogen clearance and, in many cases, eliminates the pathogen without the need for an additional immune response. It is, therefore, critical to understand what signals drive neutrophil migration to infection in a physiologically relevant environment. METHODS: In this study, we used an infection-on-a-chip model to recapitulate many important aspects of the infectious microenvironment including an endothelial blood vessel, an extracellular matrix, and the environmental fungal pathogen Aspergillus fumigatus. We then used this model to visualize the innate immune response to fungal infection. RESULTS: We found that A. fumigatus germination dynamics are influenced by the presence of an endothelial lumen. Furthermore, we demonstrated that neutrophils are recruited to and swarm around A. fumigatus hyphae and that the presence of monocytes significantly increases the neutrophil response to A. fumigatus. Using secreted protein analysis and blocking antibodies, we found that this increased migration is likely due to signaling by MIP-1 family proteins. Finally, we demonstrated that signal relay between neutrophils, mediated by LTB4 signaling, is also important for sustained neutrophil migration and swarming in response to A. fumigatus infection in our system. CONCLUSIONS: Taken together, these results suggest that paracrine signaling from both monocytes and neutrophils plays an important role in driving the neutrophil response to A. fumigatus.

Candida auris Cell Wall Mannosylation Contributes to Neutrophil Evasion through Pathways Divergent from Candida albicans and Candida glabrata.

Candida auris, a recently emergent fungal pathogen, has caused invasive infections in health care settings worldwide. Mortality rates approach 60% and hospital spread poses a public health threat. Compared to other Candida spp., C. auris avoids triggering the antifungal activity of neutrophils, innate immune cells that are critical for responding to many invasive fungal infections, including candidiasis. However, the mechanism underpinning this immune evasion has been largely unknown. Here, we show that C. auris cell wall mannosylation contributes to the evasion of neutrophils ex vivo and in a zebrafish infection model. Genetic disruption of mannosylation pathways (PMR1 and VAN1) diminishes the outer cell wall mannan, unmasks immunostimulatory components, and promotes neutrophil engagement, phagocytosis, and killing. Upon examination of these pathways in other Candida spp. (Candida albicans and Candida glabrata), we did not find an impact on neutrophil interactions. These studies show how C. auris mannosylation contributes to neutrophil evasion though pathways distinct from other common Candida spp. The findings shed light on innate immune evasion for this emerging pathogen. IMPORTANCE The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high. Previous study has demonstrated that neutrophils, leukocytes critical for the antifungal host defense, do not efficiently recognize and kill C. auris. Here, we show how the outer cell wall of C. auris promotes immune evasion. Disruption of this mannan polysaccharide layer renders C. auris susceptible to neutrophil killing ex vivo and in a zebrafish model of invasive candidiasis. The role of these mannosylation pathways for neutrophil evasion appears divergent from other common Candida species.