Shining a light on Candida-induced epithelial damage with a luciferase reporter.

Host cell damage is a key parameter for research in infection biology, drug testing, and substance safety screening. In this study, we introduce a luciferase reporter system as a new and reliable assay to measure cell damage and validate it with the pathogenic yeast, Candida albicans, as a test case. We transduced human epithelial cell lines with a lentiviral vector to stably express an optimized luciferase enzyme, Nanoluc. Upon cell damage, the release of cytoplasmic luciferase into the extracellular space can be easily detected by a luminometer. We used the luciferase reporter system to investigate the damage caused by C. albicans to different newly generated epithelial reporter cell lines. We found that fungus-induced cell damage, as determined by established methods, correlated tightly with the release of the luciferase. The new luciferase reporter system is a simple, sensitive, robust, and inexpensive method for measuring host cell damage and has a sensitivity comparable to the standard assay, release of lactate dehydrogenase. It is suitable for high-throughput studies of pathogenesis mechanisms of any microbe, for antimicrobial drug screening, and many other applications.IMPORTANCEWe present a quick, easy, inexpensive, and reliable assay to measure damage to mammalian cells. To this end, we created reporter cell lines which artificially express luciferase, an enzyme that can be easily detected in the supernatant when these cells are damaged. We used infections with the well-investigated fungal pathogen of humans, Candida albicans, as a test case of our system. Using our reporter, we were able to recapitulate the known effects of strain variability, gene deletions, and antifungal treatments on host cell damage. This easily adaptable reporter system can be used to screen for damage in infection models with different microbial species, assay cell-damaging potential of substances, discover new non-toxic antibiotics, and many other damage-based applications.

A synthetic peptide mimic kills Candida albicans and synergistically prevents infection.

More than two million people worldwide are affected by life-threatening, invasive fungal infections annually. Candida species are the most common cause of nosocomial, invasive fungal infections and are associated with mortality rates above 40%. Despite the increasing incidence of drug-resistance, the development of novel antifungal formulations has been limited. Here we investigate the antifungal mode of action and therapeutic potential of positively charged, synthetic peptide mimics to combat Candida albicans infections. Our data indicates that these synthetic polymers cause endoplasmic reticulum stress and affect protein glycosylation, a mode of action distinct from currently approved antifungal drugs. The most promising polymer composition damaged the mannan layer of the cell wall, with additional membrane-disrupting activity. The synergistic combination of the polymer with caspofungin prevented infection of human epithelial cells in vitro, improved fungal clearance by human macrophages, and significantly increased host survival in a Galleria mellonella model of systemic candidiasis. Additionally, prolonged exposure of C. albicans to the synergistic combination of polymer and caspofungin did not lead to the evolution of tolerant strains in vitro. Together, this work highlights the enormous potential of these synthetic peptide mimics to be used as novel antifungal formulations as well as adjunctive antifungal therapy.

Semisynthetic Glycoconjugate Vaccine Candidates against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.

Small Extracellular Vesicles Promote Stiffness-mediated Metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiologic matrix stiffness affects the quantity and protein cargo of small extracellular vesicles (EV) produced by cancer cells, which in turn aid cancer cell dissemination. Primary patient breast tissue released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα2ß1, ITGα6ß4, ITGα6ß1, CD44) compared with EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix proteins including collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer-associated fibroblast phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment. SIGNIFICANCE: Here we show that the quantity, cargo, and function of breast cancer-derived EVs vary with mechanical properties of the extracellular microenvironment.

Semi-synthetic glycoconjugate vaccine candidate against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, posing a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semi-synthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semi-synthetic glycoconjugate vaccines contain the identical synthetic decasaccharide (M2 motif) antigen. This motif is present in serotype A strains, which constitute 95% of clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity towards M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). While these findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. It could serve as a component in a multi-valent GXM motif vaccine, enhancing both strength and breadth of immune responses.

Small extracellular vesicles promote stiffness-mediated metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiological matrix stiffness affects the quantity and protein cargo of small EVs produced by cancer cells, which in turn drive their metastasis. Primary patient breast tissue produces significantly more EVs from stiff tumor tissue than soft tumor adjacent tissue. EVs released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα 2 ß 1 , ITGα 6 ß 4 , ITGα 6 ß 1 , CD44) compared to EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix (ECM) protein collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination through enhanced chemotaxis. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer associated fibroblast (CAF) phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment.

Ecological Niche-Inspired Genome Mining Leads to the Discovery of Crop-Protecting Nonribosomal Lipopeptides Featuring a Transient Amino Acid Building Block.

Investigating the ecological context of microbial predator-prey interactions enables the identification of microorganisms, which produce multiple secondary metabolites to evade predation or to kill the predator. In addition, genome mining combined with molecular biology methods can be used to identify further biosynthetic gene clusters that yield new antimicrobials to fight the antimicrobial crisis. In contrast, classical screening-based approaches have limitations since they do not aim to unlock the entire biosynthetic potential of a given organism. Here, we describe the genomics-based identification of keanumycins A-C. These nonribosomal peptides enable bacteria of the genus Pseudomonas to evade amoebal predation. While being amoebicidal at a nanomolar level, these compounds also exhibit a strong antimycotic activity in particular against the devastating plant pathogen Botrytis cinerea and they drastically inhibit the infection of Hydrangea macrophylla leaves using only supernatants of Pseudomonas cultures. The structures of the keanumycins were fully elucidated through a combination of nuclear magnetic resonance, tandem mass spectrometry, and degradation experiments revealing an unprecedented terminal imine motif in keanumycin C extending the family of nonribosomal amino acids by a highly reactive building block. In addition, chemical synthesis unveiled the absolute configuration of the unusual dihydroxylated fatty acid of keanumycin A, which has not yet been reported for this lipodepsipeptide class. Finally, a detailed genome-wide microarray analysis of Candida albicans exposed to keanumycin A shed light on the mode-of-action of this potential natural product lead, which will aid the development of new pharmaceutical and agrochemical antifungals.

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 reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and 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.

Inositol Metabolism Regulates Capsule Structure and Virulence in the Human Pathogen Cryptococcus neoformans.

The environmental yeast Cryptococcus neoformans is the most common cause of deadly fungal meningitis in primarily immunocompromised populations. A number of factors contribute to cryptococcal pathogenesis. Among them, inositol utilization has been shown to promote C. neoformans development in nature and invasion of central nervous system during dissemination. The mechanisms of the inositol regulation of fungal virulence remain incompletely understood. In this study, we analyzed inositol-induced capsule growth and the contribution of a unique inositol catabolic pathway in fungal development and virulence. We found that genes involved in the inositol catabolic pathway are highly induced by inositol, and they are also highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. This pathway in C. neoformans contains three genes encoding myo-inositol oxygenases that convert myo-inositol into d-glucuronic acid, a substrate of the pentose phosphate cycle and a component of the polysaccharide capsule. Our mutagenesis analysis demonstrates that inositol catabolism is required for C. neoformans virulence and deletion mutants of myo-inositol oxygenases result in altered capsule growth as well as the polysaccharide structure, including O-acetylation. Our study indicates that the ability to utilize the abundant inositol in the brain may contribute to fungal pathogenesis in this neurotropic fungal pathogen. IMPORTANCE The human pathogen Cryptococcus neoformans is the leading cause of fungal meningitis in primarily immunocompromised populations. Understanding how this environmental organism adapts to the human host to cause deadly infection will guide our development of novel disease control strategies. Our recent studies revealed that inositol utilization by the fungus promotes C. neoformans development in nature and invasion of the central nervous system during infection. The mechanisms of the inositol regulation in fungal virulence remain incompletely understood. In this study, we found that C. neoformans has three genes encoding myo-inositol oxygenase, a key enzyme in the inositol catabolic pathway. Expression of these genes is highly induced by inositol, and they are highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. Our mutagenesis analysis indeed demonstrates that inositol catabolism is required for C. neoformans virulence by altering the growth and structure of polysaccharide capsule, a major virulence factor. Considering the abundance of free inositol and inositol-related metabolites in the brain, our study reveals an important mechanism of host inositol-mediated fungal pathogenesis for this neurotropic fungal pathogen.

Uncharted territories in the discovery of antifungal and antivirulence natural products from bacteria.

Many fungi can cause deadly diseases in humans, and nearly every human will suffer from some kind of fungal infection in their lives. Only few antifungals are available, and some of these fail to treat intrinsically resistant species and the ever-increasing number of fungal strains that have acquired resistance. In nature, bacteria and fungi display versatile interactions that range from friendly co-existence to predation. The first antifungal drugs, nystatin and amphotericin B, were discovered in bacteria as mediators of such interactions, and bacteria continue to be an important source of antifungals. To learn more about the ecological bacterial-fungal interactions that drive the evolution of natural products and exploit them, we need to identify environments where such interactions are pronounced, and diverse. Here, we systematically analyze historic and recent developments in this field to identify potentially under-investigated niches and resources. We also discuss alternative strategies to treat fungal infections by utilizing the antagonistic potential of bacteria to target fungal stress pathways and virulence factors, and thereby suppress the evolution of antifungal resistance.

Variation in Cell Surface Hydrophobicity among Cryptococcus neoformans Strains Influences Interactions with Amoebas.

Cryptococcus neoformans and Cryptococcus gattii are pathogenic fungi that cause significant morbidity and mortality. Cell surface hydrophobicity (CSH) is a biophysical parameter that influences the adhesion of fungal cells or spores to biotic and abiotic surfaces. C. neoformans is encased by polysaccharide capsule that is highly hydrophilic and is a critical determinant of virulence. In this study, we report large differences in the CSH of some C. neoformans and C. gattii strains. The capsular polysaccharides of C. neoformans strains differ in repeating motifs and therefore vary in the number of hydroxyl groups, which, along with higher-order structure of the capsule, may contribute to the variation in hydrophobicity that we observed. We found that cell wall composition, in the context of chitin-chitosan content, does not influence CSH. For C. neoformans, CSH correlated with phagocytosis by natural soil predator Acanthamoeba castellanii Furthermore, capsular binding of the protective antibody (18B7), but not the nonprotective antibody (13F1), altered the CSH of C. neoformans strains. Variability in CSH could be an important characteristic in comparing the biological properties of cryptococcal strains.IMPORTANCE The interaction of a microbial cell with its environment is influenced by the biophysical properties of a cell. The affinity of the cell surface for water, defined by the cell surface hydrophobicity (CSH), is a biophysical parameter that varies among different strains of Cryptococcus neoformans The CSH influences the phagocytosis of the yeast by its natural predator in the soil, the amoeba. Studying variation in biophysical properties like CSH gives us insight into the dynamic host-predator interaction and host-pathogen interaction in a damage-response framework.

Study of Microbial Extracellular Vesicles:Separation by Density Gradients, Protection Assays and Labelling for Live Tracking.

Extracellular vesicles (EVs) are produced by all domains of life including Bacteria, Archaea and Eukarya. EVs are critical for cellular physiology and contain varied cargo: virulence factors, cell wall remodeling enzymes, extracellular matrix components and even nucleic acids and metabolites. While various protocols for isolating EVs have been established for mammalian cells, the field is actively developing tools to study EVs in other organisms. In this protocol we describe our methods to perform density gradient purification of EVs in bacterial cells, allowing for separation of EV subpopulations, followed by protection assays for EV cargo characterization. Furthermore, we devised a protocol which incorporates a fluorescent conjugate of fatty acids into EVs, the first to allow live-cell EV tracking to observe release of EVs, including during infection of mammalian cells by pathogenic bacteria. These protocols are powerful tools for EV researchers as they enable the observation of EV release and the study of the mechanisms of their formation and release.

The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans.

Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins, such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-associated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus Cryptococcus neoformans We observed that melanin is assembled into the cryptococcal cell wall in spherical structures ∼200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres ∼30 nm in diameter, termed fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for C. neoformans' melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.

The capsule of Cryptococcus neoformans.

The capsule of Cryptococcus neoformans is its dominant virulence factor and plays a key role in the biology of this fungus. In this essay, we focus on the capsule as a cellular structure and note the limitations inherent in the current methodologies available for its study. Given that no single method can provide the structure of the capsule, our notions of what is the cryptococcal capsule must be arrived at by synthesizing information gathered from very different methodological approaches including microscopy, polysaccharide chemistry and physical chemistry of macromolecules. The emerging picture is one of a carefully regulated dynamic structure that is constantly rearranged as a response to environmental stimulation and cellular replication. In the environment, the capsule protects the fungus against desiccation and phagocytic predators. In animal hosts the capsule functions in both offensive and defensive modes, such that it interferes with immune responses while providing the fungal cell with a defensive shield that is both antiphagocytic and capable of absorbing microbicidal oxidative bursts from phagocytic cells. Finally, we delineate a set of unsolved problems in the cryptococcal capsule field that could provide fertile ground for future investigations.

Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles.

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

The Buoyancy of Cryptococcus neoformans Is Affected by Capsule Size.

Cryptococcus neoformans is an environmental pathogenic fungus with a worldwide geographical distribution that is responsible for hundreds of thousands of human cryptococcosis cases each year. During infection, the yeast undergoes a morphological transformation involving capsular enlargement that increases microbial volume. To understand the factors that play a role in environmental dispersal of C. neoformans and C. gattii, we evaluated the cell density of Cryptococcus using Percoll isopycnic gradients. We found differences in the cell densities of strains belonging to C. neoformans and C. gattii species complexes. The buoyancy of C. neoformans strains varied depending on growth medium. In minimal medium, the cryptococcal capsule made a major contribution to the cell density such that cells with larger capsules had lower density than those with smaller capsules. Removing the capsule, by chemical or mechanical methods, increased the C. neoformans cell density and reduced buoyancy. Melanization of the C. neoformans cell wall, which also contributes to virulence, produced a small but consistent increase in cell density. Encapsulated C. neoformans sedimented much more slowly in seawater as its density approached the density of water. Our results suggest a new function for the capsule whereby it can function as a flotation device to facilitate transport and dispersion in aqueous fluids.IMPORTANCE The buoyancy of a microbial cell is an important physical characteristic that may affect its transportability in fluids and interactions with tissues during infection. The polysaccharide capsule surrounding C. neoformans is required for infection and dissemination in the host. Our results indicate that the capsule has a significant effect on reducing cryptococcal cell density, altering its sedimentation in seawater. Modulation of microbial cell density via encapsulation may facilitate dispersal for other important encapsulated pathogens.

Microbial melanins for radioprotection and bioremediation.

Microbial melanins provide a biocompatible and scalable approach for bioremediation and radioprotection technologies due to their physicochemical properties.