Biology subjects

Hole, C. R.

Publications and source records attributed to Hole, C. R..

7 recordsLinked to original sources

bioRxiv2026

Neutrophils Restrain CCR2-Dependent Monocyte Immunopathology During Pulmonary Cryptococcosis.

Neutrophils are essential for host defense against most invasive fungal pathogens, yet their role in cryptococcosis remains paradoxical. Neutropenia is not a recognized risk factor for cryptococcal disease, and an early study reported that neutrophil depletion improves survival in mice, supporting the prevailing view that these cells are dispensable. Here we re-examined this question using Ly6G-specific depletion strategies. Contrary to prior reports, neutrophil-depleted mice infected with Cryptococcus neoformans succumbed significantly more rapidly than isotype-treated controls. Accelerated mortality occurred without any increase in pulmonary fungal burden at 24h, 48h, or day 7, or in brain and spleen at day 7, indicating host-mediated rather than pathogen-driven death. This phenotype was independent of mouse genetic background, depletion antibody, inoculum, and fungal strain. Within 24h, neutrophil depletion provoked an early chemokine and IL-17A response, followed by pronounced pulmonary accumulation of inflammatory monocytes and macrophages by day 7. Strikingly, the increased mortality associated with neutrophil depletion was completely absent in Ccr2-/- mice, implicating CCR2-dependent inflammatory responses in disease severity. Together, these findings reveal a previously underappreciated protective function of neutrophils during cryptococcosis, whereby they constrain inflammatory monocyte-associated immunopathology and promote host survival independently of measurable changes in fungal burden.

microbiology↗

bioRxiv2025

Reactive Oxygen Species Drive the Aberrant Immune Response to a C. neoformans Chitin Synthase 3 (chs3{triangleup}) Mutant.

Cryptococcus neoformans is a globally distributed fungal pathogen that causes severe opportunistic infections, particularly in individuals with impaired cell-mediated immunity, such as AIDS patients and organ transplant recipients. Chitosan, a deacetylated derivative of chitin, plays a key role in modulating immune responses during Cryptococcus neoformans infection. We previously showed that mice inoculated with a heat-killed (HK) chitosan-deficient C. neoformans strain lacking chitin synthase 3 (chs3{Delta}) undergo rapid, neutrophil-dependent mortality within 36 hours, despite the absence of viable organisms. Here, we investigated the immune mechanisms underlying this lethal inflammatory response. We assessed the role of inflammatory cytokines, adaptive immunity, and neutrophil-derived effector functions in this lethal response. IL-6-deficient and Rag1-deficient mice exhibited only modest survival benefits, suggesting that IL-6 signaling and adaptive immune cells play limited roles. In contrast, mice lacking NADPH oxidase 2 (NOX2), the catalytic subunit of the phagocyte oxidase complex required for reactive oxygen species (ROS) production, were completely protected. Despite equivalent pulmonary neutrophil recruitment in wild-type and NOX2-deficient mice, only the former displayed elevated proinflammatory cytokine and chemokine levels and succumbed to disease. These findings indicate that neutrophils are not intrinsically pathogenic but mediate lethal immunopathology through ROS-dependent inflammatory amplification.

immunology↗

bioRxiv2025

Simple Growth Conditions Improve Targeted Gene Deletion in Cryptococcus neoformans

Cryptococcus neoformans infections are a significant cause of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. The cryptococcal cell wall is very dynamic and can be modulated depending on growth conditions. It was reported that when C. neoformans is grown in unbuffered yeast nitrogen base (YNB) for 48 hours, the pH of the media drastically drops and the cells start to shed their cell walls. With this observation, we sought to determine if YNB-grown cells could be used directly for genetic transformation. To test this, we targeted ADE2 using TRACE (transient CRISPR-Cas9 coupled with electroporation) in YNB-grown or competent cells. Deletion of the ADE2 gene results in red-pigmented colonies allowing visual confirmation of disruption. We were able to successfully delete ADE2 in YNB-grown cells and with better efficiency compared to competent cells. Recently, it was shown that gene deletion can be accomplished using short (50 bp) homology arms in place of the normal long arms ([~]1 kb). However, it was inefficient, leading to more insertions and gene disruption than gene deletions. We tested short homology with YNB-grown cells vs. competent cells and found gene deletion was significantly improved in YNB-grown cells, around 60%, compared to around 6% with the competent cells. This was also observed when we deleted LAC1 with the short arms. Altogether, using simple growth conditions, we have greatly improved the speed and efficiency of cryptococcal genetic transformations. ImportanceThe World Health Organization recently ranked C. neoformans as the #1 highest-priority fungal pathogen based on unmet research and development needs and public health importance. Understanding cryptococcal pathogenicity is key for developing treatments. We found that using simple growth conditions can greatly improve the speed and efficiency of cryptococcal genetic transformations. This finding will advance the field by expanding the ease of cryptococcal genetic manipulations.

microbiology↗

bioRxiv2024

Immunological correlates of protection mediated by a whole organism Cryptococcus neoformans vaccine deficient in chitosan

The global burden of infections due to the pathogenic fungus Cryptococcus is substantial in persons with low CD4+ T cell counts. Previously, we deleted three chitin deacetylase genes from C. neoformans to create a chitosan-deficient, avirulent strain, designated cda1{Delta}2{Delta}3{Delta} which, when used as a vaccine, protected mice from challenge with virulent C. neoformans strain KN99. Here, we explored the immunological basis for protection. Vaccine-mediated protection was maintained in mice lacking B cells or CD8+ T cells. In contrast, protection was lost in mice lacking /{beta} T cells or CD4+ T cells. Moreover, CD4+ T cells from vaccinated mice conferred protection upon adoptive transfer to naive mice. Importantly, while monoclonal antibody-mediated depletion of CD4+ T cells just prior to vaccination resulted in complete loss of protection, significant protection was retained in mice depleted of CD4+ T cells after vaccination, but prior to challenge. Vaccine-mediated protection was lost in mice genetically deficient in IFN{gamma}, TNF, or IL-23p19. A robust influx of leukocytes and IFN{gamma}- and TNF-expressing CD4+ T cells was seen in the lungs of vaccinated and challenged mice. Finally, a higher level of IFN{gamma} production by lung cells stimulated ex vivo correlated with lower fungal burden in the lungs. Thus, while B cells and CD8+ T cells are dispensable, IFN{gamma} and CD4+ T cells have overlapping roles in generating protective immunity prior to cda1{Delta}2{Delta}3{Delta} vaccination. However, once vaccinated, protection becomes less dependent on CD4+ T cells, suggesting a strategy for vaccinating HIV+ persons prior to loss of CD4+ T cells. ImportanceThe fungus Cryptococcus neoformans is responsible for >100,000 deaths annually, mostly in persons with impaired CD4+ T cell function such as AIDS. There are no approved human vaccines. We previously created a genetically engineered avirulent strain of C. neoformans, designated cda1{Delta}2{Delta}3{Delta}. When used as a vaccine, cda1{Delta}2{Delta}3{Delta} protects mice against a subsequent challenge with a virulent C. neoformans strain. Here, we defined components of the immune system responsible for vaccine-mediated protection. We found that while B cells and CD8+ T cells were dispensible, protection was lost in mice genetically deficient in CD4+ T cells, and the cytokines IFN{gamma}, TNF, or IL-23. A robust influx of cytokine-producing CD4+ T cells was seen in the lungs of vaccinated mice following infection. Importantly, protection was retained in mice depleted of CD4+ T cells following vaccination, suggesting a strategy to protect persons who are at risk for future CD4+ T cell dysfunction.

immunology↗

bioRxiv2019

Cryptococcus neoformans Chitin Synthase 3 (Chs3) Plays a Critical Role in Dampening Host Inflammatory Responses

Cryptococcus neoformans infections are significant causes of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. One of the main interfaces between the fungus and the host is the fungal cell wall. The cryptococcal cell wall is unusual among human pathogenic fungi in that the chitin is predominantly deacetylated to chitosan. Chitosan deficient strains of C. neoformans were found to be avirulent and rapidly cleared from the murine lung. Moreover, infection with a chitosan deficient C. neoformans lacking three chitin deacetylases (cda1{Delta}2{Delta}3{Delta}) was found to confer protective immunity to a subsequent challenge with a virulent wild type counterpart. In addition to the chitin deacetylases, it was previously shown that chitin synthase 3 (Chs3) is also essential for chitin deacetylase mediated formation of chitosan. Mice inoculated with chs3{Delta} at a dose previously shown to induce protection with cda1{Delta}2{Delta}3{Delta} die within 36 hours after installation of the organism. Mortality was not dependent on viable fungi as mice inoculated with heat-killed preparation of chs3{Delta} died at the same rate as mice inoculated with live chs3{Delta}, suggesting the rapid onset of death was host mediated likely caused by an over exuberant immune response. Histology, cytokine profiling, and flow cytometry indicates a massive neutrophil influx in the mice inoculated with chs3{Delta}. Mice depleted of neutrophils survived chs3{Delta} inoculation indicating that death was neutrophil mediated. Altogether, these studies lead us to conclude that Chs3, along with chitosan, plays critical roles in dampening cryptococcal induced host inflammatory responses.\n\nIMPORTANCECryptococcus neoformans is the most common disseminated fungal pathogen in AIDS patients, resulting in [~]200,000 deaths each year. There is a pressing need for new treatments for this infection, as current antifungal therapy is hampered by toxicity and/or the inability of the hosts immune system to aid in resolution of the disease. An ideal target for new therapies is the fungal cell wall. The cryptococcal cell wall is different than many other pathogenic fungi in that it contains chitosan. Strains that have decreased chitosan are less pathogenic and strains that are deficient in chitosan are avirulent and can induce protective responses. In this study we investigated the host responses to chs3{Delta}, a chitosan-deficient strain, and found mice inoculated with chs3{Delta} all died within 36 hours and death was associated with an aberrant hyperinflammatory immune response driven by neutrophils, indicating that chitosan is critical in modulating the immune response to Cryptococcus.

immunology↗

bioRxiv2019

Chitosan biosynthesis and virulence in the human fungal pathogen Cryptococcus gattii

Cryptococcus gattii R265 is a hyper-virulent fungal strain responsible for the major outbreak of cryptococcosis in Vancouver Island of British Columbia in 1999. It differs significantly from C. neoformans in its natural environment, its preferred site in the mammalian host, and in the nature and mode of pathogenesis. Our previous studies in C. neoformans have shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall of C. gattii R265 contains 2-3-fold higher amount of chitosan compared to that of C. neoformans. The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDA) have however, been modified. To deduce their roles, single, double and a triple CDA deletion strains were constructed in a R265 background and were subjected to mammalian infection studies. Unlike C. neoformans where Cda1 has a discernible role in fungal pathogenesis, in R265 Cda3 is critical for virulence. Deletion of either CDA3 alone (cda3{Delta}) or in combination with either CDA1 (cda1{Delta}3{Delta}) or CDA2 (cda2{Delta}3{Delta}) or both (cda1{Delta}2{Delta}3{Delta}) rendered the yeast cells avirulent and were cleared from the infected host. Moreover, the cda1{Delta}2{Delta}3{Delta} strain of R265 induced a protective response to a subsequent infection with R265. These studies shed more light into the regulation of chitosan biosynthesis of C. gattii and its subsequent effect on fungal virulence.\n\nImportanceThe fungal cell wall is an essential organelle whose components provide the first line of defense against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient strains are avirulent, implicating chitosan as a critical virulence factor. C. gattii R265 is an important fungal pathogen of concern due to its ability to cause infections in individuals with no apparent immune dysfunction and an increasing geographical distribution. Characterization of the fungal cell wall and understanding the contribution of individual molecules of the cell wall matrix to fungal pathogenesis offers new therapeutic avenues for intervention. In this report, we show that the C. gattii R265 strain has evolved alternate regulation of chitosan biosynthesis under both laboratory growth conditions and during mammalian infection compared to that of C. neoformans.

microbiology↗

bioRxiv2019

Cryptococcus neoformans evades pulmonary immunity by modulating xylose transport

Cryptococcus neoformans is a fungal pathogen that kills almost 200,000 people each year and is distinguished by abundant and unique surface glycan structures that are rich in xylose. A mutant strain of C. neoformans that cannot transport xylose precursors into the secretory compartment is severely attenuated in virulence in mice, yet surprisingly is not cleared. We found that this strain failed to induce the non-protective T helper cell type 2 (Th2) responses characteristic of wild-type infection, instead promoting sustained Interleukin (IL)-12p40 induction and increased IL-17A (IL-17) production. It also stimulated dendritic cells to release high levels of pro-inflammatory cytokines, a behavior we linked to xylose expression. We further discovered that inducible bronchus associated lymphoid tissue (iBALT) forms in response to infection with either wild-type cryptococci or the mutant strain with reduced surface xylose; although iBALT formation is slowed in the latter case, the tissue is better organized. Finally, our temporal studies suggest that lymphoid structures in the lung restrict the spread of mutant fungi for at least 18 weeks after infection, in contrast to ineffective control of the pathogen after infection with wild-type cells. These studies demonstrate the role of xylose in modulation of host response to a fungal pathogen and show that cryptococcal infection triggers iBALT formation.

microbiology↗