Adoptive Transfer of Cryptococcus neoformans-Specific CD4 T-Cells to Study Anti-fungal Lymphocyte Responses In Vivo.

CD4 T-cells are important for long-term control and clearance of several fungal infections in humans, particularly those caused by Cryptococcus species. Understanding the mechanisms underlying protective T-cell immunity against fungal infection is critical for developing mechanistic insights into the pathogenesis of the disease. Here, we describe a protocol that enables analysis of fungal-specific CD4 T-cell responses in vivo, using adoptive transfer of fungal-specific T-cell receptor (TCR) transgenic CD4 T-cells.

Similar evolutionary trajectories in an environmental isolate after human and murine infection.

A pet cockatoo was the suspected source of recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure.

The Cyclin Cln1 Controls Polyploid Titan Cell Formation following a Stress-Induced G Arrest in Cryptococcus.

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the and cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells.

Amoeba Predation of Cryptococcus neoformans Results in Pleiotropic Changes to Traits Associated with Virulence.

Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as ; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of (1 clinical and 2 environmental) to predation by for prolonged times and then analyzed surviving colonies phenotypically and genetically.

Cryptococcus neoformansInfected Macrophages Release Proinflammatory Extracellular Vesicles: Insight into Their Components by Multi-omics.

causes deadly mycosis in immunocompromised individuals. Macrophages are key cells fighting against microbes. Extracellular vesicles (EVs) are cell-to-cell communication mediators.

The Diverse Roles of Monocytes in Cryptococcosis.

Monocytes are considered to play a central role in the pathogenesis of infection. Monocytes and monocyte-derived macrophages and dendritic cells are key components for the control of infection, but paradoxically they can also contribute to detrimental host responses and may even support fungal proliferation and dissemination. Simultaneously, the polysaccharide capsule can impair the functions of monocytes.

Macrophages use a bet-hedging strategy for antimicrobial activity in phagolysosomal acidification.

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.

Melanin deposition in two species depends on cell-wall composition and flexibility.

and are two species complexes in the large fungal genus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In , the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall.

The enigmatic role of fungal annexins: the case of Cryptococcus neoformans.

Annexins are multifunctional proteins that bind to phospholipid membranes in a calcium-dependent manner. Annexins play a myriad of critical and well-characterized roles in mammals, ranging from membrane repair to vesicular secretion. The role of annexins in the kingdoms of bacteria, protozoa and fungi have been largely overlooked.

Dragotcytosis: Elucidation of the Mechanism for Macrophage-to-Macrophage Transfer.

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. In this study, we imaged mouse macrophages with CellTracker Green 5-chloromethylfluorescein diacetate-labeled cytosol to ascertain whether cytosol was shared between donor and acceptor macrophages.

The 'Amoeboid Predator-Fungal Animal Virulence' Hypothesis.

The observation that some aspects of amoeba-fungal interactions resemble animal phagocytic cell-fungal interactions, together with the finding that amoeba passage can enhance the virulence of some pathogenic fungi, has stimulated interest in the amoeba as a model system for the study of fungal virulence. Amoeba provide a relatively easy and cheap model system where multiple variables can be controlled for the study of fungi-protozoal (amoeba) interactions. Consequently, there have been significant efforts to study fungal⁻amoeba interactions in the laboratory, which have already provided new insights into the origin of fungal virulence as well as suggested new avenues for experimentation.

The Capsule of Cryptococcus neoformans Modulates Phagosomal pH through Its Acid-Base Properties.

Phagosomal acidification is a critical cellular mechanism for the inhibition and killing of ingested microbes by phagocytic cells. The acidic environment activates microbicidal proteins and creates an unfavorable environment for the growth of many microbes. Consequently, numerous pathogenic microbes have developed strategies for countering phagosomal acidification through various mechanisms that include interference with phagosome maturation.

Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH.

Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized.

The Outcome of the Macrophage Interaction Depends on Phagolysosomal Membrane Integrity.

is a fungal pathogen with worldwide distribution. resides within mature phagolysosomes where it often evades killing and replicates. induces phagolysosomal membrane permeabilization (PMP), but the mechanism for this phenomenon and its consequences for macrophage viability are unknown.

Conservation of Intracellular Pathogenic Strategy among Distantly Related Cryptococcal Species.

The genus includes several species pathogenic for humans. Until recently, the two major pathogenic species were recognized to be and We compared the interaction of murine macrophages with three species complex strains (WM179, R265, and WM161, representing molecular types VGI, VGIIa, and VGIII, respectively) and one species complex strain (H99, molecular type VNI) to ascertain similarities and differences in the yeast intracellular pathogenic strategy. The parameters analyzed included nonlytic exocytosis frequency, phagolysosomal pH, intracellular capsular growth, phagolysosomal membrane permeabilization, and macrophage transcriptional response, assessed using time-lapse microscopy, fluorescence microscopy, flow cytometry, and gene expression microarray analysis.

Divalent Metal Cations Potentiate the Predatory Capacity of Amoeba for Cryptococcus neoformans.

Among the best-studied interactions between soil phagocytic predators and a human-pathogenic fungus is that of and The experimental conditions used in amoeba-fungus confrontation assays can have major effects on whether the fungus or the protozoan is ascendant in the interaction. In the presence of Mg and Ca in phosphate-buffered saline (PBS), was consistently killed when incubated with survived better in the presence of Mg and Ca, even when incubated with In the absence of Mg and Ca, survived when incubated with , and the percentage of dead amoebae was higher than when incubated without yeast cells. These results show that the presence of Mg and Ca can make a decisive contribution toward tilting the outcome of the interaction in favor of the amoeba.

Lipids Affect the Cryptococcus neoformans-Macrophage Interaction and Promote Nonlytic Exocytosis.

Many microbes exploit host cellular lipid droplets during the host-microbe interaction, but this phenomenon has not been extensively studied for fungal pathogens. In this study, we analyzed the role of lipid droplets during the interaction of with macrophages in the presence and the absence of exogenous lipids, in particular, oleate. The addition of oleic acid increased the frequency of lipid droplets in both and macrophages responded to oleic acid supplementation by faster growth inside and outside macrophages.

Polyploid titan cells produce haploid and aneuploid progeny to promote stress adaptation.

Unlabelled: Cryptococcus neoformans is a major life-threatening fungal pathogen. In response to the stress of the host environment, C. neoformans produces large polyploid titan cells.

Functional characterization of the small heat shock protein Hsp12p from Candida albicans.

Hsp12p is considered to be a small heat shock protein and conserved among fungal species. To investigate the expression of this heat shock protein in the fungal pathogen Candida albicans we developed an anti-CaHsp12p antibody. We show that this protein is induced during stationary phase growth and under stress conditions including heat shock, osmotic, oxidative and heavy metal stress.

Candida albicans HSP12 is co-regulated by physiological CO2 and pH.

Global transcriptional analysis of Candida albicans exposed to elevated ambient CO(2) revealed a statistically significant differential regulation of 14 genes. Subsequent RNA hybridisation analysis of one gene, HSP12, confirmed CO(2)-regulation via a cAMP-dependent mechanism. Additionally, Northern analyses and gel mobility shift assays demonstrate the co-regulation of HSP12 by environmental pH via a Rim101-dependent mechanism.

Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1.

Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C.