人类环境病原真菌——新生隐球菌的社会行为

人类病原真菌代表了病原微生物一个重要类群,主要感染免疫缺陷、受损和受抑制的患者,导致很高的死亡率。绝大多数的人类病原真菌属于环境病原微生物,它们与人类宿主并无长期共同进化史以及紧密的共生关系,这类真菌被称为环境病原真菌。新生隐球菌作为环境病原真菌的模式种,每年导致约60万人死亡。与其他环境病原真菌相似,该菌的致病能力并不由宿主因素驱动,而是衍生于其对于生境压力出色的适应性策略。近几年来的研究表明,群体或社会行为可能作为一个关键环境适应策略,广泛参与新生隐球菌的感染、耐药以及有性生殖过程。本文以新生隐球菌为例,针对生物被膜以及生物被膜样群落,总结其中的群体或社会调控方式,并对其研究前景和临床应用提出了一些思考。     

Paradoxical role of capsule in murine bronchoalveolar macrophage-mediated killing of Cryptococcus neoformans

Infections with the encapsulated fungus Cryptococcus neoformans are usually acquired via inhalation, and the presence of a capsule has been identified as a virulence factor. Therefore, we studied murine bronchoalveolar macrophage (BAM)-mediated killing and phagocytosis of encapsulated and acapsular strains of C. neoformans. After 2 h, BAM killed encapsulated strains CN52 and MP415 more readily than acapsular strains CN602 and CAP67 (54.9 and 36.2% vs 26.1 and 6.7%, respectively, p less than 0.001). Pre-incubating CN602 with purified capsular polysaccharide increased killing to 42.7% (p = 0.04). Significantly greater killing of the encapsulated strains also occurred in vivo. BAM-mediated killing of CN52 appeared to proceed by non-oxidative mechanisms, as BAM released minimal amounts of H2O2 after stimulation with CN52, and killing was not reduced by inhibitors or scavengers of the respiratory burst. The association between encapsulation and susceptibility to BAM fungicidal effects was not attributable to differences in yeast ingestion. Using the same low ratio of organisms to BAM as in the killing assay, greater than 95% of both CN52 and CN602 were phagocytosed. However, BAM phagocytosed significantly greater numbers of acapsular CN602 when incubated with a higher inoculum. Phagocytosis and killing of CN52 and CN602 required fresh serum as a source of C. Phagocytosis of CN52, but not CN602, was profoundly inhibited if BAM were plated on surfaces coated with mAb against the C3bR (CR1). mAb against the iC3b receptor (CR3) did not affect phagocytosis of either strain. These data demonstrate the innate ability of BAM to preferentially kill, by apparently non-oxidative mechanisms, an encapsulated as opposed to acapsular organism. Inasmuch as different receptors appear involved in phagocytosis of encapsulated versus acapsular C. neoformans, the disparity in killing may result from the greater ability of receptors mediating uptake of encapsulated organisms to trigger the antimicrobial armamentarium of the BAM.     

Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast-form fungus which causes pulmonary and meningeal infections preferentially in the immunocompromised host. It is thought that cell-mediated immunity is important for acquired resistance against cryptococcosis with activated macrophages as the final effector cells. However, specific polysaccharides in the capsule of C. neoformans protect the fungus from adherence to phagocytes and from subsequent phagocytosis. We have studied extracellular killing of C. neoformans by IFN-gamma-activated macrophages and their products. Murine bone marrow-derived macrophages stimulated with rIFN-gamma for 24 h were able to effectively suppress the growth of C. neoformans and the effect of IFN-gamma was augmented by LPS. Killing of C. neoformans was also achieved by cell-free supernatants from bone marrow-derived macrophages stimulated with IFN-gamma plus LPS. Our results indicate that killing of C. neoformans by activated macrophages is independent from toxic oxygen radicals and mediated by secreted protein(s) of apparent molecular mass of 15 and 30 kDa. These findings indicate that activated macrophages play a major role in host defense, although the fungus resists phagocytosis and remains in the extracellular milieu.     

真菌miRNA的发现

microRNAs (miRNAs)在植物和动物中大量存在,但是否在真菌中存在一直是个未解之谜.本研究组在担子菌新型隐球酵母Cryptococcus neoformans中发现了miRNA.两个miRNA,miR1和miR2,长度分别是22nt和18nt,前体是70nt,和动物miRNA相近.通过报告基因,证实miRl/2具有沉默功能.真菌miRNA的发现为研究其进化、功能等提供有用知识.     

Cch1 mediates calcium entry in Cryptococcus neoformans and is essential in low-calcium environments

The ability of Cryptococcus neoformans to grow at the mammalian body temperature (37 degrees C to 39 degrees C) is a well-established virulence factor. Growth of C. neoformans at this physiological temperature requires calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase. When cytosolic calcium concentrations are low ( approximately 50 to 100 nM), calcineurin is inactive and becomes active only when cytosolic calcium concentrations rise ( approximately 1 to 10 microM) through the activation of calcium channels. In this study we analyzed the function of Cch1 in C. neoformans and found that Cch1 is a Ca(2+)-permeable channel that mediates calcium entry in C. neoformans. Analysis of the Cch1 protein sequence revealed differences in the voltage sensor (S4 regions), suggesting that Cch1 may have diminished voltage sensitivity or possibly an alternative gating mechanism. The inability of the cch1 mutant to grow under conditions of limited extracellular calcium concentrations ([Ca(2+)](extracellular), approximately 100 nM) suggested that Cch1 was required for calcium uptake in low-calcium environments. These results are consistent with the role of ScCch1 in mediating high-affinity calcium uptake in Saccharomyces cerevisiae. Although the growth defect of the cch1 mutant under conditions of limited [Ca(2+)](extracellular) ( approximately 100 nM) became more severe with increasing temperature (25 degrees C to 38.5 degrees ), this temperature sensitivity was not observed when the cch1 mutant was grown on rich medium ([Ca(2+)](extracellular), approximately 0.140 mM). Accordingly, the cch1 mutant strain displayed only attenuated virulence when tested in the mouse inhalation model of cryptococcosis, further suggesting that C. neoformans may have a limited requirement for Cch1 and that this requirement appears to include ion stress tolerance.     

Growth of Cryptococcus neoformans in a thiamine-free medium

The growth of Cryptococcus neoformans in a minimal liquid synthetic medium with or without thiamine (10 g/ml) was investigated. In these media the presence or absence of thiamine had no effect on the development of C. neoformans. To check these results, we performed a series of experiments on a solid form of the minimal synthetic medium. In this study a series of six serial transfers were carried out to starve the cells of nutrients that may have been carried over from their growth on rich media. In each of the transfers on the solid synthetic medium, C. neoformans showed a similar and scarce growth. This finding indicates that C. neoformans could be autotrophic in respect to thiamine.     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

Cryptococcus neoformans arthritis in a renal transplant recipient

We present a Cryptococcus neoformans knee joint infection in a diabetic renal transplant patient treated with steroids, cyclosporin, and mycofenolate mofetil. We discuss reported cases of cryptococcal arthritis.     

Optimizing efficacy of Amphotericin B through nanomodification

The polyene antibiotic Amphotericin B (AMB) is one of the first therapeutic agents to be marketed commercially as nanosized formulations in which the drug is associated with lipids as liposomes or complexes. In this way, its renal toxicity is reduced and its therapeutic index improved. This review summarizes the particular properties of AMB which justify this type of formulation and the early work leading up to their development. The clinical results obtained in the treatment of fungal infections are reviewed and their activity against leishmaniasis is also evoked. Some newer formulations of AMB, based on both lipids and polymers are described. In particular, their potential by the oral and pulmonary routes are discussed. Finally, the development of targeted systems to deliver the drug to specific cells and tissues is considered.     

Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival

Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.     

A Ras1-Cdc24 signal transduction pathway mediates thermotolerance in the fungal pathogen Cryptococcus neoformans

Pathogenic microorganisms must precisely regulate morphogenesis to survive and proliferate within an infected host. This regulation is often controlled by conserved signal transduction pathways that direct morphological changes in varied species. One such pathway, whose components include Ras proteins and the PAK kinase Ste20, allows the human fungal pathogen Cryptococcus neoformans to grow at high temperature. Previously, we found that Ras1 signalling is required for differentiation, thermotolerance and pathogenesis in C. neoformans. We show here that the guanine nucleotide exchange factor Cdc24 is a Ras1 effector in C. neoformans to mediate the ability of this fungus to grow at high temperature and to cause disease. In addition, we provide evidence that the Ras1-Cdc24 signalling cascade functions specifically through one of the three Cdc42/Rac1 homologues in C. neoformans. In conclusion, our studies illustrate how components of conserved signalling cascades can be specialized for different downstream functions, such as pathogenesis.     

Profiling a killer, the development of Cryptococcus neoformans

The ability of fungi to transition between unicellular and multicellular growth has a profound impact on our health and the economy. Many important fungal pathogens of humans, animals, and plants are dimorphic, and the ability to switch between morphological states has been associated with their virulence. Cryptococcus neoformans is a human fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised and, in some cases, immunocompetent hosts. Cryptococcus neoformans grows vegetatively as a budding yeast and switches to hyphal growth during the sexual cycle, which is important in the study of cryptococcal pathogenicity because spores resulting from sexual development are infectious propagules and can colonize the lungs of a host. In addition, sexual reproduction contributes to the genotypic variability of Cryptococcus species, which may lead to increased fitness and virulence. Despite significant advances in our understanding of the mechanisms behind the development of C. neoformans, our knowledge is still incomplete. Recent studies have led to the emergence of many intriguing questions and hypotheses. In this review, we describe and discuss the most interesting aspects of C. neoformans development and address their impact on pathogenicity.     

新生隐球菌嗜中枢性感染机制的研究进展

新生隐球菌是临床上最重要的侵袭性病原真菌之一,可感染免疫抑制和免疫正常人群引发具有致命威胁的隐球菌性脑膜脑炎.近年来,隐球菌嗜中枢神经系统感染的机制研究取得了长足的进展,隐球菌参与侵袭中枢神经系统的相关毒力因子及多条宿主细胞应答信号通路相继被发现.     

Particle size of airborn Cryptococcus neoformans in a tower

Nearly 10(6) cells of Cryptococcus neoformans were cultured per g of pigeon droppings in a vacant tower. The air in the tower contained an average of 45 viable cells of C. neoformans per 100 liters: 60% of the cells were less than 4.7 micron in diameter. It is estimated that a human exposed to this atmosphere for 1 h would have 41 cells of c. neoformans deposited in the lungs. Sweeping resulted in the aerosolization of large numbers of cells of C. neoformans from 4.7 to 11 micron in diameter, the number of cells less than 4.7 micron remained relatively constant. One minute after sweeping, 4.4% of viable airborne cells of C. neoformans were less than 1.1 micron in diameter. We believe that this is the first report of isolating such small cells of C. neoformans from a natural site.     

The sphingolipid pathway regulates Pkc1 through the formation of diacylglycerol in Cryptococcus neoformans

The sphingolipid biosynthetic pathway generates bioactive molecules crucial to the regulation of mammalian and fungal physiological and pathobiological processes. In previous studies (Luberto, C., Toffaletti, D. L., Wills, E. A., Tucker, S. C., Casadevall, A., Perfect, J. R., Hannun, Y. A., and Del Poeta, M. (2001) Genes Dev. 15, 201-212), we demonstrated that an enzyme of the fungal sphingolipid pathway, Ipc1 (inositol-phosphorylceramide synthase-1), regulates melanin, a pigment required for the pathogenic fungus Cryptococcus neoformans to cause disease. In this study, we investigated the mechanism by which Ipc1 regulates melanin production. Because Ipc1 also catalyzes the production of diacylglycerol (DAG), a physiological activator of the classical and novel isoforms of mammalian protein kinase C (PKC), and because it has been suggested that PKC is required for melanogenesis in mammalian cells, we investigated whether Ipc1 regulates melanin in C. neoformans through the production of DAG and the subsequent activation of Pkc1, the fungal homolog of mammalian PKC. The results show that modulation of Ipc1 regulates the levels of DAG in C. neoformans cells. Next, we demonstrated that C. neoformans Pkc1 is a DAG-activated serine/threonine kinase and that the C1 domain of Pkc1 is necessary for this activation. Finally, through both pharmacological and genetic approaches, we found that inhibition of Pkc1 abolishes melanin formation in C. neoformans. This study identifies a novel signaling pathway in which C. neoformans Ipc1 plays a key role in the activation of Pkc1 through the formation of DAG. Importantly, this pathway is essential for melanin production with implications for the pathogenicity of C. neoformans.     

巨噬细胞对新生隐球菌B3501标准株的吞噬作用

目的检测巨噬细胞对新生隐球菌活力的影响。方法新生隐球菌标准株B3501与小鼠巨噬细胞系J774细胞共孵育后,检测其出芽率,并通过电镜观察B3501在J774细胞内的超微结构。结果被吞噬的B3501超微结构完好,J774细胞对B3501菌株的吞噬指数在5.67%±1.29%~8.76%±3.09%,而B3501菌在J774细胞内的出芽率较高,可达46.85%±6.63%,出芽率随共孵育时间延长而下降,但4hrs组和8hrs组无明显差别(P>0.05)。超微结构观察显示细胞内的新生隐球菌细胞壁完整。结论虽然巨噬细胞存在着胞内和胞外的抗隐球菌活性,但新生隐球菌仍可在其细胞内外存活并繁殖。     

Identification of Cryptococcus neoformans in a routine clinical laboratory

Summary Eight taxonomic tests were compared for their ability to distinguishCryptococcus neoformans from the non-pathogenic species ofCryptococcus. Eight isolates ofCryptococcus were obtained from the American Type Culture Collection and 43 isolates were obtained directly from human and natural sources. The tests which appeared to be most valuable to the routine diagnostic laboratory were growth at 37° C, characteristic growth on Guizotia seed agar and virulence for mice.     

Culture of Cryptococcus neoformans in the nonencapsulated state

Forty-one strains of Cryptococcus neoformans were examined after 3 days growth on a fresh and aged medium at p_H 5 & p_H 7 for comparison of capsule formation. Over one-half of the strains did not form visible capsules on aged medium at p_H 5. Serotypes and source of isolation did not correlate with ability or inability to form capsules. Growth of C. neoformans in the nonencapsulated state makes it possible to culture many strains of C. neoformans in the form that more closely simulates the true infectious particles.