Empowering Women: Moving from Awareness to Action at the Immunology of Fungal Infections Gordon Research Conference

This is the final version. Available on open access from MDPI via the DOI in this recordDespite the high prevalence of women in graduate degree programs and equal or more women earning PhDs, MDs, and MD/PhDs, and despite efforts at individual and institutional levels to promote women in STEM fields, there remains a disparity in pay and academic advancement of women. Likewise, there is a paucity of women in top scientific and academic leadership positions. The causes of this gender disparity are complex and multi-factorial and to date no "magic bullet" approach has been successful in changing the landscape for women in academic and scientific fields. In this report we detail our experiences with a novel mechanism for promoting discussion and raising awareness of the challenges of gender disparity in the sciences. The Gordon Research Conferences (GRC) launched the Power Hour at its meetings in 2016: a dedicated, scheduled session held during the scientific meeting to facilitate discussion of challenges specific to women in science. Here we share our experience with hosting the second Power Hour at the 2019 GRC Immunology of Fungal Infections (IFI) meeting held in Galveston, TX. We will discuss the overall structure, key discussion points, and feedback from participants with the aim of supporting future efforts to empower women and underrepresented minority groups in science.National Institutes of Health (NIH)Wellcome TrustRoyal SocietyMedical Research Council (MRC)Department of Veterans Affair

Characterisation of the spatio-temporal localisation of a pan-Mucorales specific antigen during germination and immunohistochemistry

This is the author accepted manuscript. The final version is available on open access from Oxford University Press via the DOI in this recordBACKGROUND: Mucormycosis is an aggressive, invasive fungal infection caused by moulds in the order Mucorales. Early diagnosis is key to improving patient prognosis, yet relies on insensitive culture or non-specific histopathology. A pan-Mucorales specific monoclonal antibody (mAb), TG11, was recently developed. Here, we investigate the spatio-temporal localisation of the antigen and specificity of the mAb for immunohistochemistry. METHODS: We use immunofluorescence (IF) microscopy to assess antigen localisation in eleven Mucorales species of clinical importance and live imaging of Rhizopus arrhizus germination. Immunogold transmission electron microscopy (immunoTEM) reveals the sub-cellular location of mAb TG11 binding. Finally, we perform immunohistochemistry of R. arrhizus in an ex vivo murine lung infection model alongside lung infection by Aspergillus fumigatus. RESULTS: IF revealed TG11 antigen production at the emerging hyphal tip and along the length of growing hyphae in all Mucorales except Sakasenea. Timelapse imaging revealed early antigen exposure during spore germination and along the growing hypha. ImmunoTEM confirmed mAb TG11 binding to the hyphal cell wall only. The TG11 mAb specifically stained Mucorales but not Aspergillus hyphae in infected murine lung tissue. CONCLUSIONS: TG11 detects early hyphal growth and has valuable potential for diagnosing mucormycosis by enhancing discriminatory detection of Mucorales in tissue.Noah’s Pink Balloon Leukaemia FundBiotechnology and Biological Sciences Research Council (BBSRC)National Science Foundation (NSF)Wellcome TrustRoyal SocietyLister InstituteMedical Research Council (MRC

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype

Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordCryptococcus neoformans causes lethal meningitis and accounts for approximately 10%-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this fungus invades the mammalian brain. To investigate the dynamics of C. neoformans tissue invasion, we mapped fungal localization and host cell interactions in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an in situ imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. We confirm high fungal burden in mouse upper airway after nasal inoculation. Yeast in turbinates were frequently titan cells, with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of the upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of C. neoformans, by finding viable fungi in the bloodstream of mice a few days after intranasal infection. As early as 24 h post systemic infection, the majority of C. neoformans cells traversed the blood-brain barrier, and were engulfed or in close proximity to microglia. Our work presents a new method for investigating microbial invasion, establishes that C. neoformans can breach multiple tissue barriers within the first days of infection, and demonstrates microglia as the first cells responding to C. neoformans invasion of the brain.IMPORTANCECryptococcal meningitis causes 10%-15% of AIDS-associated deaths globally. Still, brain-specific immunity to cryptococci is a conundrum. By employing innovative imaging, this study reveals what occurs during the first days of infection in brain and in airways. We found that titan cells predominate in upper airways and that cryptococci breach the upper airway mucosa, which implies that, at least in mice, the upper airways are a site for fungal dissemination. This would signify that mucosal immunity of the upper airway needs to be better understood. Importantly, we also show that microglia, the brain-resident macrophages, are the first responders to infection, and microglia clusters are formed surrounding cryptococci. This study opens the field to detailed molecular investigations on airway immune response, how fungus traverses the blood-brain barrier, how microglia respond to infection, and ultimately how microglia monitor the blood-brain barrier to preserve brain function

Pleiotropic Effects of Deubiquitinating Enzyme Ubp5 on Growth and Pathogenesis of Cryptococcus neoformans

Ubiquitination is a reversible protein modification that influences various cellular processes in eukaryotic cells. Deubiquitinating enzymes remove ubiquitin, maintain ubiquitin homeostasis and regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immunocompromised population. In order to understand the possible influence deubiquitinases have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of seven putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 is likely the major deubiquitinating enzyme for stress responses in C. neoformans, which further delineates the evolutionary divergence of Cryptococcus from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans. Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host

Fungal G-protein-coupled receptors::mediators of pathogenesis and targets for disease control

G-protein signalling pathways are involved in sensing the environment, enabling fungi to coordinate cell function, metabolism and development with their surroundings, thereby promoting their survival, propagation and virulence. G-protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in fungi. Despite the apparent importance of GPCR signalling to fungal biology and virulence, relatively few GPCR–G-protein interactions, and even fewer receptor-binding ligands, have been identified. Approximately 40% of current pharmaceuticals target human GPCRs, due to their cell surface location and central role in cell signalling. Fungal GPCRs do not belong to any of the mammalian receptor classes, making them druggable targets for antifungal development. This Review Article evaluates developments in our understanding of fungal GPCR-mediated signalling, while substantiating the rationale for considering these receptors as potential antifungal targets. The need for insights into the structure–function relationship of receptor–ligand interactions is highlighted, which could facilitate the development of receptor-interfering compounds that could be used in disease control

An ionothermally prepared S=1/2 vanadium oxyfluoride kagome lattice

Frustrated magnetic lattices offer the possibility of many exotic ground states that are of great fundamental importance. Of particular significance is the hunt for frustrated spin-1/2 networks as candidates for quantum spin liquids, which would have exciting and unusual magnetic properties at low temperatures. The few reported candidate materials have all been based on d9 ions. Here, we report the ionothermal synthesis of [NH4]2[C7H14N][V7O6F18], an inorganic-organic hybrid solid that contains a S = 1/2 kagome network of d1 V4+ ions. The compound exhibits a high degree of magnetic frustration, with significant antiferromagnetic interactions but no long-range magnetic order or spin-freezing above 2 K, and appears to be an excellent candidate for realizing a quantum spin liquid ground state in a spin-1/2 kagome network.PreprintPostprintPeer reviewe