A sensitive high-throughput assay for evaluating host-pathogen interactions in Cryptococcus neoformans infection

Background

Cryptococcus neoformans causes serious disease in immunocompromised individuals, leading to over 600,000 deaths per year worldwide. Part of this impact is due to the organism''s ability to thwart what should be the mammalian hosts'' first line of defense against cryptococcal infection: internalization by macrophages. Even when C. neoformans is engulfed by host phagocytes, it can survive and replicate within them rather than being destroyed; this ability is central in cryptococcal virulence. It is therefore critical to elucidate the interactions of this facultative intracellular pathogen with phagocytic cells of its mammalian host.

Methodology/Principal Findings

To accurately assess initial interactions between human phagocytic cells and fungi, we have developed a method using high-throughput microscopy to efficiently distinguish adherent and engulfed cryptococci and quantitate each population. This method offers significant advantages over currently available means of assaying host-fungal cell interactions, and remains statistically robust when implemented in an automated fashion appropriate for screening. It was used to demonstrate the sensitivity of human phagocytes to subtle changes in the cryptococcal capsule, a major virulence factor of this pathogen.

Conclusions/Significance

Our high-throughput method for characterizing interactions between C. neoformans and mammalian phagocytic cells offers a powerful tool for elucidating the relationship between these cell types during pathogenesis. This approach will be useful for screens of this organism and has potentially broad applications for investigating host-pathogen interactions.     

The pursuit of cryptococcal pathogenesis: heterologous hosts and the study of cryptococcal host-pathogen interactions

Analysis of the molecular mechanisms by which a pathogen interacts with the human host is most commonly performed using a mammalian model of infection. However, several virulence-related genes previously shown to be involved in mammalian infection with Cryptococcus neoformans have also been shown to play a role in the interaction of these pathogens with invertebrates, such as Acanthamoeba castellanii, Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster and Galleria mellonella. The study of host-pathogen interactions using these model hosts has allowed rapid screening of mutant libraries and can be used for the study of evolutionarily preserved aspects of microbial virulence and host response.     

Lipid metabolism in Cryptococcus neoformans

In recent years, lipids have been shown to act as signalling molecules not only in mammalian cells but also in many other eukaryotes. Whereas in mammalian cells lipids regulate cellular functions that play crucial roles in the regulation of pathobiological processes, such as cancer, cardiovascular and neurodegenerative disorders, and inflammation, in the fungus Cryptococcus neoformans lipids play key roles in the regulation of pathogenic traits required for the development of cryptococcosis, an infectious disease particularly frequent in immunocompromised individuals. In this minireview we discuss recent advances in the understanding of lipid metabolism in this important human pathogen, highlighting the potential of fungal lipid enzymatic pathways as promising new drug targets.     

Lessons from Cryptococcal Laccase: From Environmental Saprophyte to Pathogen

Cryptococcus is an opportunistic pathogen that lives in the environment as a free-living yeast and inflicts disease in humans, primarily in immunocompromised patients such as organ-transplant recipients and people with HIV/AIDS. A key factor allowing emergence of this fungal pathogen is a copper-containing laccase enzyme that facilitates nutrient foraging as a saprophyte and offers protection against environmental dangers such as free-living amoebae and mammalian macrophages during infection. The promiscuous substrate specificity of laccase facilitates the degradation of hard plant lignin polymers as well as the synthesis of lignin-like pigments from plant flavonoids and melanin pigments from dopamine. The enzyme also possesses an iron oxidase activity that prevents Fenton product formation in macrophages and another activity that allows synthesis of immune-modulatory prostaglandins that fool the host, shutting off an effective immune response. This review provides a brief overview of key points in laccase function and its role in virulence, as well as regulation and trafficking of the enzyme during the interconversion between saprophyte and pathogen, yielding insights into pathogenesis from this adaptable pathogen.     

Glycosyltransferases and their products: cryptococcal variations on fungal themes

Glycosyltransferases are specific enzymes that catalyse the transfer of monosaccharide moieties to biological substrates, including proteins, lipids and carbohydrates. These enzymes are present from prokaryotes to humans, and their glycoconjugate products are often vital for survival of the organism. Many glycosyltransferases found in fungal pathogens such as Cryptococcus neoformans do not exist in mammalian systems, making them attractive potential targets for selectively toxic agents. In this article, we present the features of this diverse class of enzymes, and review the fungal glycosyltransferases that are involved in synthesis of the cell wall, the cryptococcal capsule, glycoproteins and glycolipids. We specifically focus on enzymes that have been identified or studied in C. neoformans, and we consider future directions for research on glycosyltransferases in the context of this opportunistic pathogen.     

Know Your Enemy: How to Build and Vanquish a Global Fungal Scourge

The 8th International Conference on Cryptococcus and Cryptococcosis, chaired by Maurizio Del Poeta (Medical University of South Carolina), and organized together with June Kwon-Chung (National Institute of Allergy and Infectious Diseases), Stuart Levitz (University of Massachusetts Medical School), and John Perfect (Duke University), occurred in May 2011. This meeting brought together the world's leading researchers on Cryptococcus and cryptococcosis, including basic scientists, epidemiologists, and clinicians, to discuss new developments in Cryptococcus biology. With more than 60 oral presentations and 180 posters, this meeting enhanced our understanding of pathogenicity of Cryptococcus and served as a robust forum that facilitated cross-disciplinary discussions, research, and clinical collaborations. Due to space constraints, this brief overview highlights only a few of the topics discussed in this meeting, focusing on the evolution of virulence, host and pathogen interactions, fungal and host signaling, new advances of genomics studies on Cryptococcus, and the current status of the outbreak caused by C. gattii. The 8th International Conference on Cryptococcus and Cryptococcosis brought together scientists from across the globe in the beautiful historical downtown setting of Charleston to share their latest findings and highlight advances in Cryptococcus research. With more than 250 participants, this meeting was the largest gathering of the Cryptococcus international community in the 24-year history. Here, we review the advances presented and the current state of knowledge in the field.     

Functional analysis of host factors that mediate the intracellular lifestyle of Cryptococcus neoformans

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.     

The human fungal pathogen Cryptococcus can complete its sexual cycle during a pathogenic association with plants

Cryptococcus is a globally distributed human fungal pathogen that primarily afflicts immunocompromised individuals. How and why this human fungal pathogen associates with plants and how this environmental niche influences its life cycle remains a mystery. We established Cryptococcus-Arabidopsis and Cryptococcus-Eucalyptus systems and discovered that Cryptococcus proliferates and mates on plant surfaces. Mating efficiency of C. gattii was markedly enhanced on plants and myo-inositol and indole acetic acid were specific plant products that stimulated mating. On Arabidopsis, dwarfing and chlorosis were observed following infection with a fungal mixture of two opposite mating-type strains, but not with either mating-type alone. This infection process is countered by the plant jasmonate-mediated defense mechanism. These findings reveal that Cryptococcus can parasitically interact with plants to complete its sexual cycle, which may impact an understanding of the origin and evolution of both plant and animal fungal pathogens in nature.     

新生隐球菌细胞通讯研究进展

细胞通讯系统调控多细胞生物的细胞增殖与分化等多种基础生物学过程,也是调控单细胞生物群体或社会性行为的重要策略。新生隐球菌是一种重要的环境来源病原真菌,主要感染免疫缺陷人群,具有很高的致死率。作为环境致病真菌,新生隐球菌进化出丰富的环境适应性策略。新生隐球菌细胞呈现出高度的异质性和社会性,不同形态的细胞承载着不同生物学功能和病原学特征。越来越多的研究表明,通过细胞通讯系统调控其群体或社会性行为,既是新生隐球菌适应多变的外界环境和宿主环境的关键策略,也与其致病能力密切相关。本文介绍新生隐球菌中细胞通讯系统的研究进展及其在有性生殖、细胞形态转换、适应环境及宿主压力等社会性行为中的调控作用。     

Capsules from pathogenic and non-pathogenic Cryptococcus spp. manifest significant differences in structure and ability to protect against phagocytic cells

Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence.     

Inhibition of mammalian cathepsins byPlesiomonas shigelloides

To study molecular mechanisms underlying self-defense of the bacterial pathogen Plesiomonas shigelloides against host inflammatory and immune responses, we evaluated its interactions with mammalian papain-like cathepsins that are essential for host immunity. When grown under anaerobic, but not aerobic, conditions, P. shigelloides was shown to bind and inhibit papain, a model representative of the papain family of cysteine proteinases. This points to mammalian cathepsins as likely physiological targets of a novel cysteine-proteinase inhibitor expressed on bacterial cell surface. Both papain and mammalian cathepsins L and B were inhibited by periplasmic extracts of aerobically and anaerobically grown bacteria, the inhibitory activity being higher in the latter. Inhibition by both intact cells and periplasmic samples was rapid and efficient. The results suggest a possible defensive role of bacterial inhibitors of cathepsins during invasion of a mammalian host. The bacteria thus may modulate host protective responses through inhibiting cathepsins involved in antigen processing and presentation.     

Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages

Cryptococcus neoformans (Cn) is an encapsulated yeast that is a facultative intracellular pathogen and a frequent cause of human disease. The interaction of Cn with alveolar macrophages is critical for containing the infection , but Cn can also replicate intracellularly and lyse macrophages . Cn has a unique intracellular pathogenic strategy that involves cytoplasmic accumulation of polysaccharide-containing vesicles and intracellular replication leading to the formation of spacious phagosomes in which multiple cryptococcal cells are present . The Cn intracellular pathogenic strategy in macrophages and amoebas is similar, leading to the proposal that it originated as a mechanism for survival against phagocytic predators in the environment . Here, we report that under certain conditions, including phagosomal maturation, possible actin depolymerization, and homotypic phagosome fusion, Cn can exit the macrophage host through an extrusion of the phagosome, while both the released pathogen and host remain alive and able to propagate. The phenomenon of "phagosomal extrusion" indicates the existence of a previously unrecognized mechanism whereby a fungal pathogen can escape the intracellular confines of mammalian macrophages to continue propagation and, possibly, dissemination.     

A Salmonella typhimurium-translocated Glycerophospholipid:Cholesterol Acyltransferase Promotes Virulence by Binding to the RhoA Protein Switch Regions

Salmonella enterica serovar typhimurium translocates a glycerophospholipid:cholesterol acyltransferase (SseJ) into the host cytosol after its entry into mammalian cells. SseJ is recruited to the cytoplasmic face of the host cell phagosome membrane where it is activated upon binding the small GTPase, RhoA. SseJ is regulated similarly to cognate eukaryotic effectors, as only the GTP-bound form of RhoA family members stimulates enzymatic activity. Using NMR and biochemistry, this work demonstrates that SseJ competes effectively with Rhotekin, ROCK, and PKN1 in binding to a similar RhoA surface. The RhoA surface that binds SseJ includes the regulatory switch regions that control activation of mammalian effectors. These data were used to create RhoA mutants with altered SseJ binding and activation. This structure-function analysis supports a model in which SseJ activation occurs predominantly through binding to residues within switch region II. We further defined the nature of the interaction between SseJ and RhoA by constructing SseJ mutants in the RhoA binding surface. These data indicate that SseJ binding to RhoA is required for recruitment of SseJ to the endosomal network and for full Salmonella virulence for inbred susceptible mice, indicating that regulation of SseJ by small GTPases is an important virulence strategy of this bacterial pathogen. The dependence of a bacterial effector on regulation by a mammalian GTPase defines further how intimately host pathogen interactions have coevolved through similar and divergent evolutionary strategies.     

Cell wall chitosan is necessary for virulence in the opportunistic pathogen Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Its cell wall is composed of glucans, proteins, chitin, and chitosan. Multiple genetic approaches have defined a chitosan-deficient syndrome that includes slow growth and decreased cell integrity. Here we demonstrate chitosan is necessary for virulence and persistence in the mammalian host.     

Reconciling West Nile virus with the autophagic pathway

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for recurrent outbreaks of meningitis and encephalitis. Several studies analyzing the interactions of this pathogen with the autophagic pathway have reported opposite results with evidence for and against the upregulation of autophagy in infected cells. In this regard, we have recently reported that minimal genetic changes (single amino acid substitutions) in nonstructural proteins of WNV can modify the ability of the virus to induce autophagic features such as LC3 modification and aggregation in infected cells. We think that these results could help explain some of the previously reported discrepancies. These findings could also aid in deciphering the interactions of this pathogen with the autophagic pathway at the molecular level aimed to develop feasible antiviral strategies to combat this pathogen, and other related flaviviruses.     

Unisexual Reproduction of Cryptococcus gattii

Cryptococcus gattii is a basidiomycetous human fungal pathogen that typically causes infection in tropical and subtropical regions and is responsible for an ongoing outbreak in immunocompetent individuals on Vancouver Island and in the Pacific Northwest of the US. Pathogenesis of this species may be linked to its sexual cycle that generates infectious propagules called basidiospores. A marked predominance of only one mating type (α) in clinical and environmental isolates suggests that a-α opposite-sex reproduction may be infrequent or geographically restricted, raising the possibility of an alternative unisexual cycle involving cells of only α mating type, as discovered previously in the related pathogenic species Cryptococcus neoformans. Here we report observation of hallmark features of unisexual reproduction in a clinical isolate of C. gattii (isolate 97/433) and describe genetic and environmental factors conducive to this sexual cycle. Our results are consistent with population genetic evidence of recombination in the largely unisexual populations of C. gattii and provide a useful genetic model for understanding how novel modes of sexual reproduction may contribute to evolution and virulence in this species.     

Mechanisms of unisexual mating in Cryptococcus neoformans

Sex serves a pivotal role in genetic exchange and it contributes to the fitness and genetic diversity for eukaryotic populations. Although the importance of the canonical bisexual mating has been widely accepted, the significance of the evolution and maintenance of unisexual mating observed in some eukaryotes is unclear. The recent discovery of same-sex mating in the human fungal pathogen Cryptococcus neoformans and the revelation of its impact on the Cryptococcus global population structure provide a platform to elucidate the molecular mechanisms and significance of unisexual mating. Here, we review the evidence of unisexual mating in Cryptococcus and provide some perspective on the biological significance of this life style on the survival of this important fungal pathogen in the environment and in animal hosts. We also summarize our current understanding of the molecular mechanisms governing this unconventional mode of reproduction.     

NO way to live; the various roles of nitric oxide in plant-pathogen interactions

Nitric oxide has attracted considerable interest from plant pathologists due its established role in regulating mammalian anti-microbial defences, particularly via programmed cell death (PCD). Although NO plays a major role in plant PCD elicited in response to certain types of pathogenic challenge, the race-specific hypersensitive response (HR), it is now evident that NO also acts in the regulation of non-specific, papilla-based resistance to penetration by plant cells that survive attack and, possibly, in systemic acquired resistance. Equally, the potential roles of NO signalling/scavenging within the pathogen are being recognized. This review will consider key defensive roles played by NO in living cells during plant-pathogen interactions, as well as in those undergoing PCD.     

Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.