浆细胞样树突状细胞在宿主抵御隐球菌肺部感染中的作用及机制研究进展

肺隐球菌病是由隐球菌感染引起的常见真菌病,由于症状的非特异性,临床上诊断较为困难。作为条件致病性真菌感染,肺隐球菌病的结局主要与宿主免疫力有关。目前肺隐球菌病免疫学发病机制研究主要局限在T细胞和巨噬细胞。近年研究表明,作为树突状细胞亚群之一的浆细胞样树突细胞,由于其激活后可以产生大量的I型干扰素并活化相关的T细胞,所以在机体抵抗病毒和细菌免疫中发挥着重要的作用。但是浆细胞样树突细胞在真菌病,尤其是在隐球菌病的发生发展中发挥的作用尚不明确。本文将介绍肺隐球菌病的临床表现、诊治及T细胞和巨噬细胞在肺隐球菌病中的免疫机制,并通过介绍肺隐球菌病和浆细胞样树突细胞及二者之间已有报道的联系,初步阐述浆细胞样树突细胞在肺隐球菌病免疫学发病机制中的相关作用。     

Calcium: a central player in Cryptococcus biology

Adaptation to the host environment is crucial for fungal pathogenesis. Calcium (Ca²⁺) signals are essential for fungal cells to respond rapidly to stress stimuli. In eukaryotic cells, Ca²⁺ is the main intracellular secondary messenger and regulates a myriad of processes, including the cellular fitness of the fungal pathogen Cryptococcus neoformans. In this minireview, we highlight the main cryptococcal processes regulated by Ca²⁺. Moreover, we underline all the characterized proteins responsible for intracellular calcium homeostasis in this yeast, such as Ca²⁺ transporters and binding proteins. These elements, in general, are essential for C. neoformans’ growth and adaptation to the host environment, as well as to virulence mechanisms. We also revisit the specific traits of the calcineurin signaling pathway in C. neoformans, which is the major pathway regulated by calcium and is crucial for yeast pathogenesis, adaptation, and growth at 37 °C. Notably, several Ca²⁺-related functions are highly conserved throughout fungal cells. Moreover, C. neoformans exhibits exclusive, significant features that are required for disease progression, thus attracting attention as feasible targets for antifungal drug development. Collectively, all the available data related to Ca²⁺ processes clarify the complex role that Ca²⁺ plays within cryptococcal cells, participating in host adaptation, transmigration, antifungal resistance, cell growth, and more.     

Polysaccharide diversity in VNI isolates of Cryptococcus neoformans from Roraima,Northern Brazil

Species of the Cryptococcus genus comprise environmental, encapsulated fungal pathogens that cause lethal meningitis in immunosuppressed individuals. In humans, fungal uptake of hypocapsular Cryptococcus by macrophages was associated with high fungal burden in the cerebrospinal fluid and long-term patient survival. On the basis of the key role of the cryptococcal capsule in disease, we analyzed the diversity of capsular structures in 23 isolates from pigeon excreta collected in the cities of Boa Vista, Bonfim and Pacaraima, in the state of Roraima (Northern Brazil). All isolates were identified as Cryptococcus neoformans (VNI genotype) by MALDI-TOF mass spectrometry. Through a combination of fluorescence microscopy, flow cytometry, ELISA and spectrophotometric methods, each isolate was characterized at the phenotypical level, which included measurements of growth rates at 30 and 37 °C, pigmentation, cell body size, capsular dimensions, serological reactivity, urease production and ability to produce extracellular glucuronoxylomannan (GXM), the main capsular component of C. neoformans. With the exception of melanization, a formidable diversity was observed considering all parameters tested in our study. Of note, hyper and hypo producers of GXM were identified, in addition to isolates with hyper and hypo profiles of reactivity with a polysaccharide-binding monoclonal antibody. Capsular dimensions were also highly variable in the collection of isolates. Extracellular GXM production correlated positively with capsular dimensions, urease activity and cell size. Unexpectedly, GXM concentrations did not correlate with serological reactivity with the cryptococcal capsule. These results reveal a high diversity in the ability of environmental C. neoformans to produce capsular components, which might impact the outcome of human cryptococcosis.     

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.     

Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features

In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.     

A Single Protein S-acyl Transferase Acts through Diverse Substrates to Determine Cryptococcal Morphology,Stress Tolerance,and Pathogenic Outcome

Cryptococcus neoformans is an opportunistic yeast that kills over 625,000 people yearly through lethal meningitis. Host phagocytes serve as the first line of defense against this pathogen, but fungal engulfment and subsequent intracellular proliferation also correlate with poor patient outcome. Defining the interactions of this facultative intracellular pathogen with host phagocytes is key to understanding the latter’s opposing roles in infection and how they contribute to fungal latency, dissemination, and virulence. We used high-content imaging and a human monocytic cell line to screen 1,201 fungal mutants for strains with altered host interactions and identified multiple genes that influence fungal adherence and phagocytosis. One of these genes was PFA4, which encodes a protein S-acyl transferase (PAT), one of a family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of PFA4 caused dramatic defects in cryptococcal morphology, stress tolerance, and virulence. Bioorthogonal palmitoylome-profiling identified Pfa4-specific protein substrates involved in cell wall synthesis, signal transduction, and membrane trafficking responsible for these phenotypic alterations. We demonstrate that a single PAT is responsible for the modification of a subset of proteins that are critical in cryptococcal pathogenesis. Since several of these palmitoylated substrates are conserved in other pathogenic fungi, protein palmitoylation represents a potential avenue for new antifungal therapeutics.     

Equine Pulmonary Cryptococcosis: A Comparative Literature Review and Evaluation of Fluconazole Monotherapy

Cryptococcus is the most common fungal respiratory pathogen in Australian horses, manifesting primarily as pulmonary granulomas. Disease severity at presentation is dependent on the athletic use of the horse. The diagnosis and estimation of disease severity are centred around clinical findings, cytological evaluation of respiratory tract secretions, diagnostic imaging, and antigen titre testing. Both the lateral flow assay and the latex cryptococcal antigen titre are used, and important similarities and differences between species are discussed. Cryptococcus gattii occurs with greater frequency than Cryptococcus neoformans in equine pulmonic cryptococcosis and can be successfully treated with enteral fluconazole monotherapy, with disease severity determining treatment length.     

Functional activation of T cells by dendritic cells and macrophages exposed to the intracellular parasite Neospora caninum

Neospora caninum is an intracellular protozoan pathogen that causes abortion in cattle. We studied how the interaction between murine conventional dendritic cells or macrophages and N. caninum influences the generation of cell-mediated immunity against the parasite. We first explored the invasion and survival ability of N. caninum in dendritic cells and macrophages. We observed that protozoa rapidly invaded and proliferated into these two cell populations. We then investigated how Neospora-exposed macrophages or dendritic cells distinguish between viable and non-viable (heat-killed tachyzoites and antigenic extract) parasites. Viable tachyzoites and antigenic extract, but not killed parasites, altered the phenotype of immature dendritic cells. Dendritic cells infected with viable parasites down-regulated the expression of MHC-II, CD40, CD80 and CD86 whereas dendritic cells exposed to N. caninum antigenic extract up-regulated the expression of MHC-II and CD40 and down-regulated CD80 and CD86 expression. Moreover, only viable tachyzoites and antigenic extract induced IL-12 synthesis by dendritic cells. MHC-II expression was up-regulated and CD86 expression was down-regulated at the surface of macrophages, regardless of the parasitic form was encountered. However, IL-12 secretion by macrophages was only observed under conditions using viable and heat-killed parasite. We then analysed how macrophages and dendritic cells were involved in inducing T-cell responses. T lymphocyte IFN-γ-secretion in correlation with IL-12 production occurred after interactions between T cells and dendritic cells exposed to viable tachyzoites or antigenic extract. By contrast, for macrophages IFN-γ production was IL-12-independent and only occurred after interactions between T cells and macrophages exposed to antigenic extract. Thus, N. caninum-induced activation of murine dendritic cells, but not that of macrophages, was associated with T cell IFN-γ production after IL-12 secretion.     

Brain Inositol Is a Novel Stimulator for Promoting Cryptococcus Penetration of the Blood-Brain Barrier

Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, we found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection.     

Modulation of I-A and I-E expression in macrophages by T-suppressor cells induced inCryptococcus neoformans infected rats

When the I-A and I-E expressions were assessed in peritoneal macrophages fromCryptococcus neoformans infected animals, a significant decrease in the former was observed when compared with normal macrophages (p<0.001) whereas a significant increase in the I-E expression was observed when compared with controls (p<0.005). On the other hand, when studying the in vitro action of Ts cells on the macrophages, it was observed that the I-A expression was significantly reduced in macrophages upon contact with Ts cells. Similar results were obtained when Ts cells were replaced by a soluble factor. In contrast, the I-E expression was significantly increased by in vitro action of the Ts cell or its soluble factor. Indomethacin partially restored I-A and I-E expression in the macrophages to control levels.     

Repurposing benzimidazoles to fight Cryptococcus

The human diseases caused by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are associated with high rates of mortality and toxic or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Benzimidazoles are potentially attractive antifungal compounds that were introduced in clinical practice nearly 60 years ago to treat helminthic infections. In addition to being safe, their cost of treatment is extraordinarily low. Several studies suggested benzimidazoles as promising anticryptococcal agents combining low-cost and high antifungal efficacy. So far, anti-cryptococcal activities were demonstrated for 16 different benzimidazoles. In particular, albendazole, mebendazole, flubendazole, and fenbendazole have potent in vitro antifungal activity against C. neoformans and C. gattii. Mice lethally infected with C. neoformans and treated with fenbendazole had 100 % survival when the drug was administered intranasally. In this review, we discuss the potential of benzimidazoles as potential anti-cryptococcal agents, including a general literature overview, most recent findings, mechanism of antifungal action, costs, toxicity, and antifungal potential in vivo.     

Role of the PTEN protein in the multidrug resistance of prostate cancer cells

At present, there is no doubt that the signal transduction pathway P13K/Akt/PTEN/mTOR, controlled by phosphatidylinositol-3-kinase, is involved in tumor cell resistance to a number of drugs. Another well-known mechanism determining drug resistance in tumors is associated with the activity of drug transporters of the ABC superfamily (first of all, P-glycoprotein (Pgp), MRP1, BCRP, and LRP). Several mechanisms of cell defense can simultaneously operate in one cell. The interplay of different mechanisms involved in drug resistance is poorly understood. The PC3 and DU145 human prostate cell lines were used to show that the PTEN functional status determined the cell resistance to some drugs and that correlated with the levels of MRP1 and BCRP. Pgp was not involved in drug resistance of these cells. Introduction of PTEN into PTEN-deficient PC3 cells, as well as rapamycin treatment, inhibited Akt and mTOR and sensitized cells to doxorubicin and vinblastine. Exogenous PTEN altered the MRP1 and BCRP expression. The results indicate that at least two mechanisms of drug resistance operate in prostate cancer cells: the PI3K/Akt/PTEN/mTOR pathway and an elevated MRP1 expression. The mechanisms are interconnected: PTEN and mTOR signaling is involved in MRP1 and BCRP expression regulation.     

Titan cells confer protection from phagocytosis in Cryptococcus neoformans infections

The human fungal pathogen Cryptococcus neoformans produces an enlarged "titan" cell morphology when exposed to the host pulmonary environment. Titan cells exhibit traits that promote survival in the host. Previous studies showed that titan cells are not phagocytosed and that increased titan cell production in the lungs results in reduced phagocytosis of cryptococcal cells by host immune cells. Here, the effect of titan cell production on host-pathogen interactions during early stages of pulmonary cryptococcosis was explored. The relationship between titan cell production and phagocytosis was found to be nonlinear; moderate increases in titan cell production resulted in profound decreases in phagocytosis, with significant differences occurring within the first 24 h of the infection. Not only were titan cells themselves protected from phagocytosis, but titan cell formation also conferred protection from phagocytosis to normal-size cryptococcal cells. Large particles introduced into the lungs were not phagocytosed, suggesting the large size of titan cells protects against phagocytosis. The presence of large particles was unable to protect smaller particles from phagocytosis, revealing that titan cell size alone is not sufficient to provide the observed cross-protection of normal-size cryptococcal cells. These data suggest that titan cells play a critical role in establishment of the pulmonary infection by promoting the survival of the entire population of cryptococcal cells.