Cryptococcus neoformans, a fungus under stress

Cryptococcus neoformans is a human fungal pathogen that survives exposure to stresses during growth in the human host, including oxidative and nitrosative stress, high temperature, hypoxia, and nutrient deprivation. There have been many genes implicated in resistance to individual stresses. Notably, the catalases do not have the expected role in resistance to external oxidative stress, but specific peroxidases appear to be critical for resistance to both oxidative and nitrosative stresses. Signal transduction through the HOG1 and calcineurin/calmodulin pathways has been implicated in the stress response. Microarray and proteomic analyses have indicated that the common responses to stress are induction of metabolic and oxidative stress genes, and repression of genes encoding translational machinery.     

Role of a VPS41 homologue in starvation response, intracellular survival and virulence of Cryptococcus neoformans

Previous studies have demonstrated an important role for the vacuole in the virulence of the fungus Cryptococcus and studies in yeast have implicated the vacuolar protein Vps41 in copper loading of proteins such as iron transporters. However, our studies found that a cryptococcal vps41Delta strain displayed wild-type growth on media containing iron and copper chelators and normal activity of the copper-containing virulence factor laccase as well as almost normal growth at 37 degrees C and wild-type production of the virulence factor capsule. Despite these attributes, the vps41Delta mutant strain showed a dramatic attenuation of virulence in mice and co-incubation of mutant cells with the macrophage cell line, J774.16, resulted in a dramatic loss in viability of the vps41Delta mutant strain at 10 h compared with wild-type and complemented strains. Closer examination revealed that the vps41Delta mutant displayed a dramatic loss in viability after nutrient starvation which was traced to a failure to undergo G2 arrest, but there was no defect in the formation of autophagic or proteolytic vesicles. Our results indicate that VPS41 plays a key role in regulating starvation response in this pathogenic organism and that defects in cell cycle arrest are associated with attenuated pathogenic fitness in mammalian hosts.     

Capsule structural heterogeneity and antigenic variation in Cryptococcus neoformans

Cryptococcus neoformans is a human pathogenic fungus with a capsule composed primarily of glucuronoxylomannan (GXM) that is important for virulence. Current views of GXM structure postulate a polymer composed of repeating mannose trisaccharide motifs bearing a single beta(1,2) glucuronic acid with variable xylose and O-acetyl substitutions to form six triads. GXM from different strains is notoriously variable in triad composition, but it is not known if the polymer consists of one or more motif-repeating units. We investigated the polymeric organization of GXM by using mass spectrometry to determine if its compositional motif arrangement was similar to that of bacterial capsular polysaccharides, namely, a polymer of a single repeating unit. The results were consistent with, and confirmatory for, the current view that the basic unit of GXM is a repeating mannose trisaccharide motif, but we also found evidence for the copolymerization of different GXM repeating units in one polysaccharide molecule. Analysis of GXM from isogenic phenotypic switch variants suggested structural differences caused by glucuronic acid positional effects, which implied flexibility in the synthetic pathway. Our results suggest that cryptococcal capsule synthesis is fundamentally different from that observed in prokaryotes and employs a unique eukaryotic approach, which theoretically could synthesize an infinite number of structural combinations. The biological significance of this capsule construction scheme is that it is likely to confer a powerful avoidance strategy for interactions with the immune system and phagocytic environmental predators. Consistent with this premise, the antigenic variation of a capsular epitope recognized by a nonprotective antibody was observed under different growth conditions.     

Role and mechanism of phosphatidylinositol-specific phospholipase C in survival and virulence of Cryptococcus neoformans

Phospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans . To investigate the role of phosphatidylinositol-specific phospholipase C (PI-PLC) in Plb1 secretion, we identified two putative PI-PLC-encoding genes in C. neoformans var. grubii ( PLC1 and PLC2 ), and created Δ plc1 and Δ plc2 deletion mutants. In Δ plc1 , which expressed less PI-PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37°C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Δ plc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen-activated protein kinase (MAPK) in the presence of cell wall-perturbing agents. In contrast to Δ plc2 , which was as virulent as WT, Δ plc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25°C, demonstrating that mechanism(s) independent of the 37°C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.     

Requirement of a Tsp2-type tetraspanin for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans

Fungal laccases have been widely used in industry. The expression of laccase often is repressible by the primary carbon source glucose in many fungi. The underlying basis is largely unclear. We demonstrate here that a gene, TSP2-1, was required for laccase repression by glucose in the basidiomycete Cryptococcus neoformans. TSP2-1 encodes a Tsp2-type tetraspanin. The disruption of TSP2-1 resulted in constant melanin formation and the expression of the laccase gene LAC1. This derepression phenotype was restorable by 10 mM exogenous cyclic AMP (cAMP). A capsule defect in the mutant tsp2-1Δ also was restored by cAMP. The results indicate an interaction of Tsp2-1 with the cAMP-dependent protein kinase A (PKA) pathway that has been shown to modulate laccase repression and capsule biosynthesis in this fungus. Other roles of TSP2-1, e.g., in maintaining cell membrane integrity and stress resistance, also were defined. This work reveals a Tsp2-1-dependent glucose repression in C. neoformans. The function of Tsp2-type tetraspanin Tsp2-1 is described for the first time.     

新生隐球菌感染流行病学现状及耐药机制相关研究进展

新生隐球菌是一种广泛存在于环境中的酵母类真菌,主要侵犯中枢神经系统引起隐球菌性脑膜炎。HIV感染是导致隐球菌感染的主要危险因素之一,但近年来关于非HIV患者隐球菌感染的报道不断增加。体外药敏试验证实大部分新生隐球菌对棘白菌素类药物具有内在抗性。两性霉素B和氟康唑是用于隐球菌感染治疗的一线药物,而长期广泛用药引起新生隐球菌对氟康唑的耐药率逐年升高,患者临床治疗失败率居高不下。为进一步加深对新生隐球菌的认识,本文结合国内外流行病学报道及相关研究,从感染现状、生物学特征、诊治方法和耐药性等方面进行综述,以期为新生隐球菌性脑膜炎的临床诊治提供参考。     

Pbx Proteins in Cryptococcus neoformans Cell Wall Remodeling and Capsule Assembly

The cryptococcal capsule is a critical virulence factor of an important pathogen, but little is known about how it is associated with the cell or released into the environment. Two mutants lacking PBX1 and PBX2 were found to shed reduced amounts of the capsule polysaccharide glucuronoxylomannan (GXM). Nuclear magnetic resonance, composition, and physical analyses showed that the shed material was of normal mass but was slightly enriched in xylose. In contrast to previous reports, this material contained no glucose. Notably, the capsule fibers of pbxΔ mutant cells grown under capsule-inducing conditions were present at a lower than usual density and were loosely attached to the cell wall. Mutant cell walls were also defective, as indicated by phenotypes including abnormal cell morphology, reduced mating filamentation, and altered cell integrity. All observed phenotypes were shared between the two mutants and exacerbated in a double mutant. Consistent with a role in surface glycan synthesis, the Pbx proteins localized to detergent-resistant membrane domains. These results, together with the sequence motifs in the Pbx proteins, suggest that Pbx1 and Pbx2 are redundant proteins that act in remodeling the cell wall to maintain normal cell morphology and precursor availability for other glycan synthetic processes. Their absence results in aberrant cell wall growth and metabolic imbalance, which together impact cell wall and capsule synthesis, cell morphology, and capsule association. The surface changes also lead to increased engulfment by host phagocytes, consistent with the lack of virulence of pbx mutants in animal models.     

Combatting the evolution of antifungal resistance in Cryptococcus neoformans

Fungal infections are a global concern and the evolution of intrinsic resistance to current antifungals presents an alarming problem. For Cryptococcus neoformans, a human fungal pathogen of primarily immunocompromised individuals, resistance toward treatment strategies demands alternative approaches. Given the prevalence of virulence factor production during cryptococcal infection, an emerging and important field of research encompasses the development of novel antivirulence therapies proposed to improve host immune responses and promote fungal clearance. To accomplish this task, information regarding the presence and role of virulence factors, the mechanisms of action within the host, and the ability to influence fungal susceptibility to antifungals is pertinent. Research into mechanisms of antifungal resistance for C. neoformans is limited but extrapolation from successful studies in other fungal species can improve our understanding of mechanisms employed by C. neoformans and suggest targeted strategies to enhance our ability to combat the pathogen. In this Review, we highlight antifungal therapy options against Cryptococcus, explore current knowledge of underlying mechanisms promoting resistance, and present new opportunities for novel and effective strategies to overcome fungal infections and reduce, or possibly even reverse, the effects of resistance evolution.     

Pseudohyphal forms of Cryptococcus neoformans: Decreased survival in vivo

Three pseudohyphal isolates of Cryptococcus neoformans were inoculated intracranially into mice. Four weeks post-inoculation the animals showed no symptoms of disease and the number of viable cells per brain decreased to zero. Possible roles of pseudohyphal forms of C. neoformans in the immunology and pathogenesis of cryptococcosis are discussed.     

Relation of the pigeon to cryptococcosis: Natural carrier state,heat resistance and survival of Cryptococcus neoformans

Summary The feral pigeon in New York City was found to serve as a mechanical carrier of pathogenic strains ofCryptococcus neoformans. Of 94 feral pigeons freshly trapped in the city, 7 were found to carryC. neoformans on their beaks and feet, while their rectal swabs were negative. Following crop instillation ofC. neoformans in 3 feral pigeons, the fungus survived passage through the gastrointestinal tract and appeared in the fresh feces within one hour after inoculation and was still present 24 hours later. The internal body (rectal) temperatures of 57 feral pigeons recorded soon after capture in two seasons of the year, ranged from 41.5° C to 43.3° C and averaged 42.5° ± 0.39° C. The birds were able to maintain their high temperatures in the face of sustained cold, indicating the presence of a strong thermoregulating mechanism. A study of the growth and survival of 60 human strains ofC. neoformans on Sabouraud dextrose yeast extract agar slants revealed that 100 % of the cultures were able to grow at 39° C, 92 % at 40° C, 30 % at 41° C, 17 % at 42° C and 8 % at 43° C. Despite exposure to these temperatures for 7 days, 100 % of the human strains survived at 40° C, 97 % at 41° C, 95 % at 42° C, 91 % at 43° C, 88 % at 44° C, 47 % at 45° C and none at 47° C. Seventy strains ofC. neoformans of pigeon excreta origin showed a similar pattern of heat resistance, except that 11 % of these strains survived 47° C. The inability ofC. neoformans to grow at 44° C, a property shared by all 130 strains, provides a new species characteristic.C. neoformans was found to multiply rapidly in moist pigeon excreta extract, reaching counts up to 60 million per ml within 3 weeks, and to be still viable in moist as well as dessicated pigeon excreta extract after more than two years of storage at room temperature.Presented in part at the 26th VA-Armed Forces Pulmonary Disease Research Conference, Cleveland, Ohio, Jan. 23–26, 1967, and the 67th Annual Meeting American Society for Microbiology, New York, April 30–May 4, 1967. Supported by Grants GB-4722 from the National Science Foundation and U-1367 from The Health Research Council of the City of New York.Acknowledgment is gratefully given toThomas J. Dalton, Division of Environmental Sanitation, Department of Health, New York City andMabel Halloran for their valuable assistance and to the ASPCA of Manhattan and Brooklyn for their cooperation in this study.     

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival. However, relatively little is known concerning this functional dichotomy, particularly the regulation of p38-dependent survival pathways. Here, we demonstrate that the Rit GTPase promotes cell survival by directing an unexpected p38 MAPK-dependent AKT survival pathway. Following stress exposure, Rit small hairpin RNA interference (shRNAi)-treated cells display increased apoptosis and selective disruption of p38 MAPK signaling, while expression of constitutively activated Rit promotes p38-AKT-dependent cell survival. Rit, but not Ras or Rap GTPases, can associate with, and is critical for, stress-mediated activation of the scaffolded p38-MK2-HSP27-AKT prosurvival signaling complex. Together, our studies establish Rit as a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular survival mechanism in response to stress.     

Blood vessel occlusion by Cryptococcus neoformans is a mechanism for haemorrhagic dissemination of infection

Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis.     

Amphotericin B mediates killing in Cryptococcus neoformans through the induction of a strong oxidative burst

We studied the effects of Amphotericin B (AmB) on Cryptococcus neoformans using different viability methods (CFUs enumeration, XTT assay and propidium iodide permeability). After 1h of incubation, there were no viable colonies when the cells were exposed to AmB concentrations ≥ 1 mg/L. In the same conditions, the cells did not become permeable to propidium iodide, a phenomenon that was not observed until 3h of incubation. When viability was measured in parallel using XTT assay, a result consistent with the CFUs was obtained, although we also observed a paradoxical effect in which at high AmB concentrations, a higher XTT reduction was measured than at intermediate AmB concentrations. This paradoxical effect was not observed after 3h of incubation with AmB, and lack of XTT reduction was observed at AmB concentrations higher than 1mg/L. When stained with dihydrofluorescein, AmB induced a strong intracellular oxidative burst. Consistent with oxidative damage, AmB induced protein carbonylation. Our results indicate that in C. neoformans, Amphotericin B causes intracellular damage mediated through the production of free radicals before damage on the cell membrane, measured by propidium iodide uptake.