Biology subjects

Casadevall, A.

Publications and source records attributed to Casadevall, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Strain-specific thermotolerance, UV-C tolerance, and biofilm formation on clinically relevant plastic substrates in the emerging opportunistic pathogen Rhodotorula mucilaginosa

Rhodotorula mucilaginosa is an emerging opportunistic fungal pathogen increasingly associated with catheter-related bloodstream infections. Although biofilm formation is considered a major virulence trait for R. mucilaginosa, factors contributing to biofilm persistence on medical devices remain poorly understood. Here, we characterized the thermotolerance, biofilm formation, UV resistance, and cell surface hydrophobicity profiles of eight R. mucilaginosa strains representing clinical and non-clinical (laboratory, environmental, and marine mammal) isolates. All strains grew optimally at 30C and exhibited restricted growth at 35C and 37C, although one environmental isolate maintained robust growth at 37C. All strains exhibited moderate to high cell surface hydrophobicity. We then assessed biofilm formation for each strain, including adherence to two different plastic substrates, development of biofilm biomass, comparison of biofilm metabolic activity, and the effects of temperature on biofilm formation. Under static conditions, biofilm biomass of most isolates on 96-well polystyrene plates was greatest at 24C. Clinical isolates generally maintained higher biofilm metabolic activity at 37C than nonclinical isolates, while at lower temperatures, clinical and non-clinical isolates did not differ significantly in metabolic activity. All strains readily formed biofilms on polyurethane intravenous catheters under dynamic conditions, as confirmed by scanning electron microscopy and metabolic activity. While planktonic cells already displayed substantial UV-C tolerance, biofilm-associated cells remained viable following exposure to UV-C doses up to eightfold higher than those that impaired planktonic growth. These findings document differences in thermotolerance and biofilm formation by isolate origin and identify biofilm formation as a major factor promoting persistence of R. mucilaginosa on clinically relevant materials and reduced susceptibility to UV-C sterilization.

microbiology↗

bioRxiv2026

Calcium triggers Cryptococcus neoformans aggregation by forming coordination bonds with capsular glucuronoxylomannan

The cryptococcal polysaccharide capsule is a unique eukaryotic virulence factor that is a target for the immune system and the development of therapeutic antibodies. Our understanding of capsular architecture is limited to a few studies suggesting that metal dications play a role. In this work we explore a mechanism of cryptococcal aggregation that depends on calcium phosphate precipitation. We describe the chemical and biophysical properties of calcium interaction with the predominant cryptococcal polysaccharide, glucuronoxylomannan (GXM). We show that cell aggregation is a pH-dependent and occurs in a calcium dose-dependent manner. Furthermore, this cellular aggregation phenomenon as well as interpolymer capsular polysaccharide interactions are unique to calcium dications and do not occur with other mono- or dications as shown by size exclusion chromatography and circular dichroism. Diffusion ordered spectroscopy nuclear magnetic resonance and ab-initio calculations support complexation of calcium with glucuronic acid (GlcA). The ab-initio calculations also suggest that calcium ions can complex up to four GlcA monomers. Not only does calcium act as a scaffold for the cryptococcal capsule, interacting with up to four glucuronic acid residues of GXM, but calcium phosphate treatment of cells reduces the anti-phagocytic properties of the capsule, promoting ingestion by macrophages and altering antibody interactions with the capsule. This work advances our understanding of the cryptococcal capsule, its biophysical properties, by providing a model for the critical role of calcium interactions with capsular polymers of Cryptococcus neoformans including important impacts at the host-cell interface.

biochemistry↗

bioRxiv2026

Amphotericin B Resistance in Lomentospora prolificans is associated with a soluble cell wall component

IntroductionLomentospora prolificans is a pathogenic filamentous fungus that causes disease primarily in people with severely compromised immune systems. It is pan-resistant to antifungal drugs, but the mechanism of its resistance to amphotericin B (AMB) is unknown. ObjectivesWe aimed to investigate the mechanism of resistance to AMB of L. prolificans. MethodsThe AMB susceptibility of L. prolificans protoplasts was measured using broth microdilution. L. prolificans, either intact, homogenized or fractionated was incubated with AMB in broth. The same activity was carried out with Aspergillus fumigatus as a control. This broth was then used to prepare microdilution plates with Saccharomyces cerevisiae to determine the activity of the conditioned AMB. ResultsAMB was 16-fold more effective in inhibiting the growth of L. prolificans protoplasts than conidia, but only two-fold more effective against A. fumigatus protoplasts than conidia. Incubation of L. prolificans hyphae with AMB in media diminished drug activity to a much greater extent than A. fumigatus, with 8-fold greater fungal mass of the latter required to achieve the effect of the former. Homogenization and fractionization of L. prolificans revealed that the factor inhibiting AMB activity was soluble with a mass >100 kda. DNase, trypsin, proteinase K, amyloglucosidase, SDS and 0.22 m had no effect on the AMB resistance factor, while treatment with urea, acetonitrile inactivated it. ConclusionWe report a different mechanism for AMB resistance based on the existence of a substance residing in the L. prolificans cell wall that can eliminate the antifungal activity of AMB.

microbiology↗

bioRxiv2026

Stochasticity and Bet Hedging Drive Cryptococcal Capsule Dynamics

Cryptococcus neoformans and related species are major human pathogens that cause cryptococcosis, a disease with high mortality and morbidity despite antifungal therapy. Pathogenic Cryptococcus spp. cells express a polysaccharide capsule, which is the most important virulence factor. In this study we analyzed the distribution of capsule sizes for several strains from Cryptococcus spp. and found that they follow stochastic dynamics, with a heavy right-hand tail distribution, favoring larger capsules. The distribution for each strain is remarkably stable despite repeated perturbation of culture conditions including media refreshment, time, and macrophage ingestion. Growth in macrophages resulted in different capsule distributions, observed in vitro, with a suggestion of different polysaccharide-like materials formed or utilized in the resident phagosome. We propose that the stability in capsule size distributions represents a capsulestat mechanism for the population. An emergent property whereby individual cells manifest capsule size variation emanating from random effects on individual capsule assembly steps. This distribution balances between cells with large capsules that are less susceptible to a variety of environmental stresses at the price of slower replication, increased size, and increased energy requirements and cells with smaller, less protective capsules that reproduce faster. Thus, Cryptococcus spp. populations establish a bet hedging strategy that can enhance the viability of the population as conditions change at the cost of optimal short-term growth.

microbiology↗

bioRxiv2026

Antigen Stimulation Reactivates HIV-1 Proviruses Despite Integration in Repressive Chromatin

Intact HIV-1 proviruses become progressively enriched in transcriptionally repressive genomic regions during long-term antiretroviral therapy (ART) and in elite controllers, raising questions about their capacity for reactivation in vivo. We used an antigen-restricted quantitative viral outgrowth assay (ag qVOA) to test whether cognate antigen stimulation can reverse latency of proviruses integrated within repressive chromatin. Using cells from two people with HIV (PWH) on ART, one on long-term treatment and one an elite controller, we show that antigen-specific stimulation induces viral outgrowth from intact proviruses integrated into a pericentromeric transition region and a zinc finger gene, respectively. These findings demonstrate that antigen recognition can overcome epigenetic constraints to reactivate proviruses with low inducibility and suggest that proviruses in so-called "deeper latency" may contribute to residual viremia and viral rebound following treatment interruption.

immunology↗

bioRxiv2026

Fung-AI: An AI/ML-driven pipeline for antifungal peptide discovery

Emerging fungal pathogens represent a concerning threat to both global health and food security. In this study, we aimed to address our rising vulnerability to fungal pathogens through the development of the Fung-AI pipeline: an AI/ML-driven approach for antifungal discovery. A generative adversarial network (GAN) was trained to generate novel candidate antifungal peptide sequences. Next, in silico antifungal and hemolytic classifiers were built to further prioritize AI-generated peptides for experimental validation. From a pool of [~]10,000 candidates, thirteen peptides were selected for testing over two-stages of experimentation. Five peptides were found to display mild antifungal activity against the wheat pathogen, Fusarium graminearum, with minimal inhibitory concentrations (MICs) ranging from 250 {micro}g/mL to 500 {micro}g/mL. Four of the five peptides also showed activity against the human pathogen, Candida albicans (MIC: 500 {micro}g/mL). Two of our AI-generated antifungal peptides additionally demonstrated low cytotoxicity in HepG2 human liver carcinoma cells (LC50 > 704.2 {micro}g/mL) indicating that they may be useful as scaffolds for future optimization for therapeutic applications. None of our peptides were found to considerably inhibit the emerging pathogen C. auris, suggesting the need for pathogen-specific down-selection of candidate peptides. Overall, we present a proof-of-principle, generative-AI-based approach for the rapid design of de novo antifungal peptides.

synthetic biology↗

bioRxiv2026

An Inositol Receptor Orchestrates Carbon Utilization and Fungal Virulence

Nutrient sensing and utilization are critical for microbes to adapt to their environment and evade predators. The encapsuled yeast Cryptococcus neoformans has a unique ability to sense and utilize the sugar inositol as both a signaling molecule, and as a carbon source. This trait is advantageous not only in the natural environment, but to promote its pathogenesis, including invasion of the central nervous system. Unlike many other fungal species, which utilize the canonical carbon catabolite repression (CCR) mechanism to prioritize glucose metabolism, we have identified a novel inositol transporter-like receptor (transceptor) Itr4 that also regulates CCR genes to maintain inositol metabolism activity even under high glucose conditions. Itr4 binds inositol and regulates the function of other inositol transporters (ITRs) and its loss leads to a significant decrease in inositol uptake activity, resulting in a lack of mating, reduced capsule size, reduced blood brain barrier (BBB) penetration, and virulence attenuation. Mutagenesis analysis of Itr4 protein identified an essential N-terminal tail and two amino acids, Q388 and Q389, as sites of functional importance. The ITR4Q388A, Q389A allele showed a dominant phenotype with increased inositol uptake, enlarged capsule and significantly attenuated virulence. In summary, we identify a new C. neoformans inositol transceptor, Itr4, the first such example in eukaryotes, with novel regulatory roles in both inositol and glucose metabolism and fungal virulence.

microbiology↗

bioRxiv2026

Metabolic plasticity and virulence of Cryptococcus neoformans are regulated by mitochondrial homeostasis

The mitochondrion is a versatile organelle involved in diverse processes, such as cell death, metal homeostasis, plasma membrane and cell wall integrity, stress response, oxygen concentration, temperature, and metabolic adaptation, in addition to its role in generating energy. Consequently, mitochondrial fitness is essential for the pathogenicity of various organisms, including fungi. Cryptococcus neoformans is a fungal pathogen responsible for over 180,000 HIV-related deaths each year. In this study, we analyzed C. neoformans metabolic plasticity when grown with non-fermentable carbon sources and their impact on virulence and mitochondrial homeostasis. Growth on non-fermentable carbon sources increased thermotolerance, glucuronoxylomannan (GMX) content in the capsule, melanization rate, urease activity, biofilm formation, and virulence. Moreover, cells grown on non-fermentable carbon sources manifested increased mitochondrial number and activity. Conversely, mutants of the master regulator of mitochondrial biogenesis, the Hap complex, the catalytic subunit 1 of protein kinase A, or media supplementation with antioxidants, decreased the use of alternative carbon sources, capsule formation, melanin synthesis, urease activity, mitochondrial number, and resistance to both fluconazole and macrophage killing. Our results implicate mitochondrial homeostasis in virulence regulation via the PKA pathway, suggesting that targeting fungal mitochondrial homeostasis could be a therapeutic approach for cryptococcosis.

microbiology↗

bioRxiv2026

A role for aquaporin (Aqp1) in the control of Cryptococcus neoformans cell morphology

Aquaporins are small, integral membrane channels that facilitate the transport of water across cellular membranes and, in the case of aquaglyceroporins, can also conduct specific neutral solutes, such as glycerol. These proteins are conserved across biological kingdoms, yet their roles in fungal virulence remain relatively understudied. In Cryptococcus neoformans, an opportunistic fungal pathogen, we examined the organisms single aquaporin, Aqp1, and uncovered unanticipated influences on cellular morphology. Loss of Aqp1 resulted in smaller cells, whereas its presence promoted the formation of enlarged titan-like cells. This shift in size was closely linked to intracellular redox physiology. Consequently, the overexpression of the cryptococcal aquaporin increased sensitivity to oxidative stress and led to the largest titan-like cells; antioxidant supplementation suppressed this enlargement, consistent with a ROS-dependent regulatory mechanism. Additionally, Aqp1 overexpression produced vacuolar abnormalities in titan-like cells, suggesting that excessive water influx strained intracellular organization during rapid cell expansion. These findings position Aqp1 at a functional crossroads connecting membrane transport, oxidative balance, and size control, and they support a model in which an aquaporin contributes to the morphological plasticity that allows C. neoformans to adapt to environmental pressures.

microbiology↗

bioRxiv2026

The heat-ramp method to study regulated cell death in a pathogenic yeastCryptococcus neoformans

Human fungal pathogens cause a significant public health burden. While no reliable surveilence data are available, estimations suggest that 1 billion infections and over 2 million deaths are attributable to fungal infections annually worldwide. This drove the World Health Organization to generate a priority list of fungal pathogens for reearch, which includes the yeast Cryptococcus neoformans in a top critical priority. With the rise of drug-resistance and emerging fungal pathogens, new conceptual strategies for antifungal therapies are needed in addition to existing antibiotic development pipelines to meet clinical needs. Intrinsic cell death pathways encoded by pathogenic fungi are largely unstudied but could be leveraged for antifungal therapy analogous to anti-cancer therapeutics that activate apoptosis or other cell death mechanisms. Thus far, molecularly defined fungal cell death mechanisms are best characterized for only a few, predominantly model filamentous species. To extend these studies to pathogenic yeast, here we describe and demonstrate a tunable heat-ramp stimulus that when applied to small volumes of yeast cell suspensions reveals a protracted cell death process in the pathogenic yeast Cryptococcus neoformans. This low cost protocol induces robust and reproducible phenotypes to study gene-dependent mechanisms in laboratory strains and clinical isolates.

microbiology↗

bioRxiv2025

Fungal proliferation before and after the Cretaceous-Paleogene mass extinction in North America

Palynological evidence of post-catastrophe fungal proliferation after global calamities has been found for the Permian-Triassic and Cretaceous-Paleogene (K/Pg) extinction events. However, unlike the globally documented post-Permian fungal bloom, evidence of a post-Cretaceous event has previously been limited to a single site in New Zealand. Our analysis of a K/Pg boundary section from the Denver Basin in Colorado revealed a fungal proliferative spike occurring immediately after the Chicxulub impact. The discovery of a post-impact fungal bloom in North America corroborates the New Zealand finding and supports the interpretation that this was a global phenomenon. We also identified a prolonged interval of elevated fungal abundance in the Late Cretaceous, dating to approximately 30,000-10,000 years before the impact, temporally correlated to a period of climatic cooling at the site and intriguingly coincident with the Poladpur phase of the Deccan Traps. Taken together with reports of fungal expansion following prior global calamities, these findings indicate that fungi can often flourish in the aftermath of ecosystem-level collapse. Given the capacity of fungi to cause disease in both plants and animals, the occurrence of fungal proliferative events has major potential implications for the recovery of surviving species after global cataclysms. Significance StatementFungal proliferation evidenced by spikes in fungal abundance in geologic samples can signify major ecosystem disruptions. Such spikes are well documented globally for the Permian-Triassic extinction but for the Cretaceous-Paleogene extinction have been reported previously only in New Zealand. Here, we describe a North American fungal bloom that occurred immediately after the Chicxulub impact, as well as an earlier fungal spike in the Late Cretaceous that coincided with a cooling event hypothesized to have been driven by Deccan volcanism. Identification of this pre-impact fungal proliferative episode provides additional support for the uranium-lead-based model of Deccan eruptive rates, which places the high-volume Poladpur pulse tens of thousands of years before the bolide impact.

microbiology↗

bioRxiv2025

Peroxisomes regulate virulence and cell density sensing in Cryptococcus neoformans

Cryptococcus neoformans, a ubiquitous environmental fungus that causes cryptococcosis, survives in diverse environments including human hosts due to metabolic flexibility. Consequently, identifying how C. neoformans connects diverse metabolic pathways and virulence factor expression is important for understanding fungal pathogenesis. Peroxisomes play an essential role in metabolic homeostasis and regulation of carbon and lipid metabolism. In this article, we report a link between nickel exposure, a known hypoxia-mimetic and mitochondrial respiration inhibitor in yeast, and peroxisomal {beta}-oxidation. Loss of the last two genes involved in the peroxisomal {beta}-oxidation pathway, MFE2 (CNAG_05721) and POT1 (CNAG_00490), resulted in cell density-dependent virulence factor defects and growth inhibition attributed to a metabolic state involving large peroxisomes. We found that increasing cell density rescued virulence factor phenotypes and growth. Our results implicate mitochondrial retrograde signaling (RTG), a previously uncharacterized pathway in C. neoformans, in cell density sensing, peroxisomal {beta}-oxidation pathway expression, and virulence, thus highlighting a critical role for metabolism in cryptococcal virulence.

microbiology↗

bioRxiv2025

Unexpected variability in laboratory and clinical assays used to quantify Cryptococcus neoformans polysaccharide.

The measurement of cryptococcal capsular polysaccharide (PS) amount and concentration is essential for laboratory studies of its structure and function. Clinically, the detection of cryptococcal capsular polysaccharide as cryptococcal antigen is a foundational assay for the diagnosis of cryptococcosis. In this study we compared the results of four laboratory assays for measuring cryptococcal polysaccharide (gravimetric determination, phenol-sulfuric acid reactivity, Size Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS), and ELISA) and two clinical assays (lateral flow assay (LFA) (IMMY) and the cryptococcal latex agglutination assay (CLAA) (Remel)) using four cryptococcal strains that each express single repeating polysaccharide motifs. The results show that the sensitivity of laboratory assays for PS was relatively consistent but manifested significant variation with strain. Phenol-sulfuric acid determination was highly variable unless the PS was pre-digested with HCl, suggesting that differences in the structural complexity of the polysaccharide could affect timing and intensity of the colorimetric reaction. Serological assays were the most sensitive, but these showed the most strain-to-strain variation on the threshold of PS detection consistent with antigenic differences. Differences in the reactivity of LFA and CLAA for cryptococcal PS from different strains imply that comparable cryptococcal antigen titers could reflect different concentrations of PS in bodily fluids. In summary, we find that variation in PS structure amongst strains can affect the sensitivity of the various tests used to measure cryptococcal PS. (Word count: 224)

microbiology↗

bioRxiv2025

The Dynamics of Cryptococcus neoformans infection in Galleria mellonella

Galleria mellonella has emerged as an important host for the study of fungal virulence, insect immune responses, and the evaluation of antifungal agents. In this study we investigated the dynamics of fungal infections in G. mellonella using Cryptococcus neoformans, a human pathogenic fungus. Since the analysis of infection dynamics requires a fine temporal resolution of larval death, we employed a photographic timelapse technique that allowed us to simultaneously measure death by proxy of larval melanization and absence of movement. Larval mortality occurred in two phases, early and late, which differed in their timing of melanization. Early phase deaths occurred with rapid whole-body onset of melanization, followed by sudden cessation of movement several hours later. Contrastingly, late phase deaths occurred with a gradual cessation of movement, followed by melanization, typically radiating from one location on the larva. The differences in mortality kinetics suggests differences in fungal pathogenesis with one population succumbing early while the rest linger for later death. Subsequent analysis of mortality data using the inversion method revealed predictable deterministic dynamics without evidence for chaotic signatures, indicating that this C. neoformans-G. mellonella infection model behaves differently than bacterial-insect models. ImportanceThe ability to predict the course of an infection is critical to anticipating disease progression and effectively treating patients. Similarly, the ability to make predictions about pathogenesis in laboratory infection models could further our understanding of pathogenesis and lead to new treatments. As fungal diseases are expected to rise, understanding the dynamics of fungal infections will be important to anticipate and mitigate future threats. Here, we developed a timelapse method to visualize infections of Galleria mellonella larvae with the fungal pathogen Cryptococcus neoformans. This method provided insight into infection progression that are not apparent from standard survival measurement protocols, including the relationship between melanization and death. Further, it enabled us to explore the dynamics of disease progression in this system, which revealed deterministic dynamics without evidence of chaos, implying predictability in the outcome of cryptococcal infection in this moth.

microbiology↗

bioRxiv2025

Cross-Species Communication via Fungal Extracellular Vesicles

Extracellular vesicles (EVs) play crucial roles in fungal communication and host immune modulation, representing potential therapeutic targets for fungal infections. This study investigated the role of fungal EVs in both intra- and interspecies communication, focusing on their effects on virulence and immune responses. Co-incubation experiments were performed using EVs derived from Candida albicans and Candida auris to assess interactions with C. albicans planktonic cells and biofilms, as well as Cryptococcus neoformans and Cryptococcus gattii EVs interacting with C. neoformans cultures. EVs were observed associating with recipient cell surfaces, suggesting subsequent internalization. Functional assays revealed that EV exposure led to increased expression of Cap59, Lac1, Ure1, and Erg11 genes, correlating with reduced antifungal susceptibility in both planktonic and biofilm forms. Additionally, EVs facilitated cross-species communication, enhancing biofilm adhesion and dispersion, which underscores their role in phenotypic modulation. Macrophages stimulated with fungal EVs exhibited receptor-specific gene expression changes, notably the upregulation of galectin-3, along with a pro-inflammatory phenotype marked by increased iNOS expression and elevated cytokine levels (IL-1{beta}, IL-6, and IL-8). Collectively, these findings underscore a critical role for fungal EVs in interspecies communication, biofilm regulation, and immune modulation, offering valuable insights into fungal pathogenicity mechanisms. ImportanceCurrently, no vaccines exist to prevent fungal infections, underscoring the need for new therapies. As fungal diseases increase globally, understanding fungal biology is essential to identifying treatment targets. Fungi use EVs to communicate and evade immune responses. EVs mediate cell-cell communication, transporting proteins, polysaccharides, lipids, and nucleic acids - serving as "messages" exchanged within a fungal network. Understanding how these vesicles facilitate communication not only within a single species but also across different fungal species can shed light on their contribution to infection persistence and cross-species adaptability. Moreover, EVs may have a broader role in inter-kingdom communication, influencing how fungi interact with host immune cells. The impact of fungal EVs on human innate immune responses remains a largely underexplored area, with significant gaps in our knowledge. This study aims to examine how fungal EVs affect immune responses and whether their signaling varies across species, potentially revealing new therapeutic targets.

microbiology↗

bioRxiv2025

Three annotated chromosome-level de novo genome assemblies of Lomentospora prolificans provide evidence for a chromosomal translocation event

Lomentospora prolificans is a fungal pathogen responsible for serious, often fatal, illness in patients with compromised immune systems. Treatment is rarely successful because L. prolificans is inherently resistant to all major classes of antifungal drugs. In this study we publish three chromosome-level de novo genome assemblies, including the first complete-level assembly of L. prolificans, along with genome annotations. The L. prolificans genome is packaged in 11 nuclear chromosomes and one mitochondrial chromosome, has 36.7-37.1 Mb, and encodes for a putative 7357-7640 genes. The length and composition of contigs in one stain varied from those of the other two strains, supporting the hypothesis that a chromosomal translocation took place. These assemblies were confirmed with pulsed-field gel electrophoresis. The availability of more complete genomes will hopefully help the search for new antifungal drugs and provides insights into the evolutionary history of this pathogenic fungus.

genomics↗

bioRxiv2024

A method for authenticating the fidelity of Cryptococcus neoformans knockout collections

Gene knockout strain collections are important tools for discovery in microbiology. Cryptococcus neoformans is the only human pathogenic fungus with an available genome-wide deletion collection, and this resource is widely used by the research community. We uncovered mix-ups in the assembly of the commercially available C. neoformans deletion collection of [~]6,000 unique strains acquired by our lab. While pursuing the characterization of a gene-of-interest, the corresponding deletion strain from the C. neoformans KO collection displayed several interesting phenotypes associated with virulence. However, RNAseq analysis identified transcripts for the putative knockout gene, and the absence of transcripts for a different knockout strain found in the same plate position in an earlier partial knockout collection, raising the possibility that plates from one collection were substituted for the other. This was supported by determining the size of the nourseothricin (NAT)-resistance cassette used to generate the two separate knockout libraries and was confirmed by RNAseq and genome sequencing. Here we report that our KN99 collection is comprised of mixed plates from two independent KO libraries and present a simple authentication method that other investigators can use to distinguish the identities of these KO collections. ImportanceGene knockout strain collections are important tools for discovery in microbiology. Cryptococcus neoformans is the only human pathogenic fungus with an available genome-wide deletion collection, and this resource is widely used by the research community. Here we report that our KN99 collection is comprised of mixed plates from two independent KO libraries and present a simple authentication method that other investigators can use to distinguish the identities of these KO collections. Above all, this article serves as a reminder to 2015 library collection strains before undertaking large phenotyping experiments.

microbiology↗

bioRxiv2024

Commonalities and differences in the Cryptococcus neoformans response to ingestion by protozoal and mammalian phagocytic cells suggests host cell metabolism as a target for intracellular survival

The outcome of the interaction between Cryptococcus neoformans and infected hosts can be determined by whether the fungal cell survives ingestion by phagocytic cells. This applies to both unicellular and multicellular hosts such as amoeba and animals, respectively. Ingestion by phagocytic cells results in the formation of the cryptococcal phagosome but this structure has proved difficult to isolate. In this study, we report the successful isolation of cryptococcal phagosomes from murine and human phagocytes, followed by their characterization using proteomic and transcriptional analysis. Comparison of cryptococcal proteins from Acanthamoeba castellanii, Mus musculus, and Homo sapiens phagocytes revealed the existence of a shared set suggesting a conserved fungal response to ingestion by phagocytic cells. Given that the cryptococcal intracellular pathogenic strategy is ancient, dating to at least to the cretaceous epoch, these results are consistent with the notion that the fungal response to ingestion reflects the result of selection pressures by environmental ameboid predators over eons of evolutionary time. We propose the existence of a conserved cryptococcal toolkit for intracellular survival that includes metabolic enzymes. We propose that host cell metabolic disruption provides a common strategy for the cryptococcal survival after ingestion by phylogenetically distant phagocytic hosts.

microbiology↗