Biology subjects

Dragotakes, Q.

Publications and source records attributed to Dragotakes, Q..

14 recordsLinked to original sources

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↗

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↗

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↗

bioRxiv2024

The buoyancy of cryptococcal cells and its implications for transport and persistence of Cryptococcus in aqueous environments

Cryptococcus is a genus of saprophytic fungi with global distribution. Two species complexes, C. neoformans and C. gattii, pose health risks to humans and animals. Cryptococcal infections result from inhalation of aerosolized spores and/or desiccated yeasts from terrestrial reservoirs such as soil, trees, and avian guano. More recently, C. gattii has been implicated in infections in marine mammals, suggesting that inhalation of liquid droplets or aerosols from the air-water interface is also an important, yet understudied, mode of respiratory exposure. Water transport has also been suggested to play a role in the spread of C. gattii from tropical to temperate environments. However, the dynamics of fungal survival, persistence, and transport via water have not been fully studied. The size of the cryptococcal capsule was previously shown to reduce cell density and increase buoyancy. Here, we demonstrate that cell buoyancy is also impacted by the salinity of the media in which cells are suspended, with formation of a halocline interface significantly slowing the rate of settling of cryptococcal cells through water, resulting in persistence of C. neoformans within 1 cm of the air-water interface for over 60 min and C. gattii for 4-6 h. Our data also showed that during culture in yeast peptone dextrose media (YPD), polysaccharide accumulating in the supernatant formed a raft that augmented buoyancy and further slowed settling of cryptococcal cells. These findings illustrate new mechanisms by which cryptococcal cells may persist in aquatic environments, with important implications for aqueous transport and pathogen exposure. ImportanceCryptococcosis is a major fungal disease leading to morbidity and mortality worldwide. C. neoformans is a major fungal species of public health concern, causing opportunistic systemic infections in immunocompromised patients. C. gattii was traditionally a tropical pathogen, but in the 1990s emerged in the temperate climates of British Columbia and the Pacific Northwest United States. Outbreaks in these areas also led to the first host record of cryptococcosis in free-ranging cetaceans. C. gattii is particularly concerning as an emerging fungal pathogen due to its capacity to cause clinical disease in immunocompetent patients, its recent spread to a new ecological niche, and its higher resistance to antifungal therapies. Our research defines characteristics that influence transport of cryptococci through water and its persistence at the air-water interface, which improve our understanding of mechanisms for cryptococcal aqueous transport and persistence.

microbiology↗

bioRxiv2024

A food color based colorimetric assay for Cryptococcus neoformans laccase activity

Cryptococcus neoformans is a fungal pathogen that causes cryptococcosis mostly in immune compromised patients, such as those with HIV/AIDS. One survival mechanism of C. neoformans during infection is melanin production, which catalyzed by laccase, and protects fungal cells against immune attack. Hence comparative assessment of laccase activity is useful for characterizing cryptococcal strains. We serendipitously observed that culturing C. neoformans with food coloring resulted in the degradation of some dyes with phenolic structures. Consequently, we investigated the color changes for the food dyes metabolized by C. neoformans laccase and explored using this effect for the development of a colorimetric assay to measure laccase activity. We developed several versions of a food dye based colorimetric laccase assay that can be used to compare the relative laccase activities between different C. neoformans strains. We found that phenolic color degradation was glucose dependent, which may reflect changes in the reduction properties of the media. Our food color based colorimetric assay has several advantages over the commonly used 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for laccase activity, including lower cost, irreversibility, and does not require constant monitoring. This method has potential applications to bioremediation of water pollutants in addition to its use in determining laccase virulence factor expression. ImportanceCryptococcus neoformans is present in the environment and while infection is common, disease occurs mostly in immunocompromised individuals. C. neoformans infection in the lungs results in symptoms like pneumonia, and consequently cryptococcal meningitis occurs if the fungal infection spreads to the brain. The laccase enzyme catalyzes the melanization reaction that serves as a virulence factor of C. neoformans. Developing a simple and less costly assay to determine laccase activity in C. neoformans strains can be useful for a variety of procedures ranging from studying the relative virulence of cryptococci to environmental pollution studies.

immunology↗

bioRxiv2023

The Size of Fields in Biomedical Sciences

Scientific research output has increased exponentially over the past few decades, but not equally across all fields of study, and we lack clear methods for estimating the size of any given field of research. Understanding how fields grow, change, and are organized is essential to understanding how human resources are allocated to the investigation of scientific problems. In this study we estimated the size of certain biomedical fields from the number of unique author names appearing in field relevant publications in the PubMed database. Focusing on microbiology, where the size of fields is often associated with those who work on a particular microbe, we find large differences in the size of its subfields. We found that plotting the number of unique investigators as a function of time can show changes consistent with growing or shrinking fields. We envision using unique author count to measure the strength of a workforce in any given field, analyze the overlap of workforce between fields, and compare how workforce correlates to available research funds and public health burden of a field.

systems biology↗

bioRxiv2023

Scientific civility and academic performance

In modern science, interdisciplinary and collaborative research is encouraged among scientists to solve complex problems. However, when the time comes to measure an individuals academic productivity, collaborative efforts are hard to conceptualize and quantify. In this study, we hypothesized that a social behavior coined "scientific civility", which encompasses civility, collaboration, cooperation, or a combination of these, enhances an individuals productivity influencing their academic performance. To facilitate recognition of this unique attribute within the scientific environment, we developed a new indicator: the C score. We examined publicly available data from 1000 academic scientists at the individual-level, focusing on their scholarly output and collaborative networks as a function of geographic distribution and time. Our findings strongly suggest that the C score gauges academic performance from an integral perspective based on a synergistic interaction between productivity and collaborative networks, prevailing over institutionally limited economic resources and minimizing inequalities related to the length of individuals academic career, field of investigation, and gender. Author SummaryThe increased connectivity between fields and specialties of science is undeniable. We propose a new indicator, the C score, to assess the value of collaborative efforts and research output to a scientists academic performance. This indicator reflects collaborative and interdisciplinary efforts and provides a measure of "scientific civility" and teamwork. The C score may be used as a decision-making tool to track career advancement within the academic environment. Along with other indicators such as the h index, the C score supports a more integrative and holistic assessment of an individuals academic performance.

scientific communication and education↗

bioRxiv2022

Human and murine Cryptococcus neoformans infection selects for common genomic changes in an environmental isolate.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from the cerebral spinal fluid (CSF) of an immunocompromised patient with cryptococcosis based on the molecular analyses available in 2000. Here we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MAT strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including 8 nonsynonymous changes involving 7 genes. To ascertain whether changes in these genes are selected during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frame-shift mutation in one of the seven genes altered in the human sample, a gene predicted to encode a SWI-SNF chromatin-remodeling complex protein. Both cockatoo and patient strains as well as mouse passaged isolates obtained from brain tissue had a premature stop codon in a homolog of ZFC3, a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and appears to have reverted to a full-length protein in the mouse passaged isolates obtained from lung tissue. The patient strain and mouse passaged isolates show variability in the expression of virulence factors, with differences in capsule size, melanization, and rates on non-lytic expulsion from macrophages observed. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

genomics↗

bioRxiv2022

Melanization is an important antifungal defense mechanism in Galleria mellonella hosts

A key component of insect immunity is melanin encapsulation of microbes. Melanization is also a part of an immune process known as nodulation, which occurs when insect hemocytes surround microbes and produce melanin. Insect nodules are analogous to mammalian immune granulomas. Melanin is believed to kill microbes through the production of toxic intermediates and oxidative damage. However, it is unclear to what extent immune melanin is directly fungicidal during infections of insect hosts. We reported previously that C. neoformans cells are encapsulated with host-derived melanin within hemocyte nodules. Here we report an association between melanin-based immune responses by Galleria mellonella wax moth larvae and fungal cell death of C. neoformans during infection. To monitor melanization in situ, we applied a tissue-clearing technique to G. mellonella larvae, revealing that nodulation occurs throughout the organism. Further, we developed a protocol for time-lapse microscopy of extracted hemolymph following exposure to fungal cells, which allowed us to visualize and quantify the kinetics of the melanin-based immune response. Using this technique, we found evidence that cryptococcal melanins and laccase enhance immune melanization in hemolymph. We used these techniques to also study the fungal pathogen Candida albicans infections of G. mellonella. We find that the yeast form of C. albicans was the primary targets of host melanization, while filamentous structures were melanin-evasive. Approximately 23% of melanin-encapsulated C. albicans yeast survive and break through the encapsulation. Overall, our results provide direct evidence that the melanization reaction functions as a direct antifungal mechanism in insect hosts.

microbiology↗

bioRxiv2021

Bet-hedging antimicrobial strategies in macrophage phagosome acidification drive the dynamics of Cryptococcus neoformans intracellular escape mechanisms

The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress abrogated Dragotcytosis frequency, that ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH and oxidative stresses and correlated with the macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.

microbiology↗

bioRxiv2021

Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction

Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host-pathogen interactions. We chose to study further Yor1, a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1{Delta} strains revealed defects in non-lytic exocytosis and capsule size, when compared to wild-type strains. We detected no difference in growth rates, cell body size and vesicle secretion. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal-macrophage interaction.

microbiology↗

bioRxiv2018

Chance is an important element in phagolysosomal acidification that favors the macrophage

Microbial ingestion by a macrophage results in the formation of an acidic phagolysosome but the host cell has no information on the pH susceptibility of the ingested organism. This poses a problem for the macrophage and raises the fundamental question of how the phagocytic cell optimizes the acidification process to prevail. We analyzed the dynamical distribution of phagolysosomal pH in murine and human macrophages that had ingested live or dead Cryptococcus neoformans cells, or inert beads. Phagolysosomal acidification produced a range of pH values that approximated normal distributions, but these differed from normality depending on ingested particle type. Analysis of the increments of pH reduction revealed no forbidden ordinal patterns, implying that phagosomal acidification process was a stochastic dynamical system. Using simulation modeling, we determined that by stochastically acidifying a phagolysosome to a pH within the observed distribution, macrophages sacrificed a small amount of overall fitness to gain the benefit of reduced variation in fitness. Hence, chance in the final phagosomal pH introduces unpredictability to the outcome of the macrophage-microbe, which implies a bet-hedging strategy that benefits the macrophage. While bet hedging is common in biological systems at the organism level, our results show its use at the organelle and cellular level.

microbiology↗

bioRxiv2018

Dragotcytosis: Elucidation of the Mechanism for Cryptococcus neoformans Macrophage-to-Macrophage Transfer

Cryptococcus neoformans is a pathogenic yeast capable of a unique and intriguing form of cell-to-cell transfer between macrophage cells. The mechanism for cell-to-cell transfer is not understood. Here we imaged macrophages with CellTracker Green CMFDA-labeled cytosol to ascertain whether cytosol was shared between donor and acceptor macrophages. Analysis of several transfer events detected no transfer of cytosol from donor to acceptor macrophages. However, blocking Fc and complement receptors resulted in a major diminution of cell-to-cell transfer events. The timing cell-to-cell transfer (11.17 min) closely approximated the sum of phagocytosis (4.18 min) and exocytosis (6.71 min) times. We propose that macrophage cell-to-cell transfer represents a non-lytic exocytosis event followed by phagocytosis into a macrophage that is in close proximity and name this process Dragotcytosis (Dragot is a Greek surname meaning Sentinel) as it represents sharing of a microbe between two sentinel cells of the innate immune system.

microbiology↗