New Evidence for Differential Roles of L10 Ribosomal Proteins from Arabidopsis

The RIBOSOMAL PROTEIN L10 (RPL10) is an integral component of the eukaryotic ribosome large subunit. Besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions in the nucleus have been described for RPL10 in different organisms. Previously, we demonstrated that Arabidopsis (Arabidopsis thaliana) RPL10 genes are involved in development and translation under ultraviolet B (UV-B) stress. In this work, transgenic plants expressing Pro_RPL10:β-glucuronidase fusions show that, while AtRPL10A and AtRPL10B are expressed both in the female and male reproductive organs, AtRPL10C expression is restricted to pollen grains. Moreover, the characterization of double rpl10 mutants indicates that the three AtRPL10s differentially contribute to the total RPL10 activity in the male gametophyte. All three AtRPL10 proteins mainly accumulate in the cytosol but also in the nucleus, suggesting extraribosomal functions. After UV-B treatment, only AtRPL10B localization increases in the nuclei. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional gels followed by mass spectrometry. Overall, our data provide new evidence about the nonredundant roles of RPL10 proteins in Arabidopsis.In eukaryotes, the cytosolic ribosomes consist of large 60S and small 40S subunits. In Arabidopsis (Arabidopsis thaliana), ribosomal protein genes exist as families composed of two to seven members that could be differentially incorporated into the cytosolic ribosome under specific situations (Schmid et al., 2005; Byrne, 2009). In this way, ribosomal heterogeneity would allow selective translation of specific mRNAs under particular cell conditions (Barakat et al., 2001; Szick-Miranda and Bailey-Serres, 2001; Giavalisco et al., 2005; Carroll et al., 2008; Carroll, 2013). Arabidopsis mutants in ribosomal proteins exhibit a large range of developmental phenotypes with extreme abnormalities, including embryonic lethality, suggesting that ribosomes also have specific functions regulating the expression of developmental genes (Van Lijsebettens et al., 1994; Degenhardt and Bonham-Smith, 2008; Byrne, 2009; Horiguchi et al., 2011, 2012; Szakonyi and Byrne, 2011). Furthermore, it has been recently demonstrated that ribosomal proteins control auxin-mediated developmental programs by translational regulation of auxin response factors (Rosado et al., 2012). In addition, the characterization of single, double, and, in certain cases, triple mutants as well as complementation by paralog genes have demonstrated full, partial, and no redundancy between members of ribosomal protein families (Briggs et al., 2006; Guo and Chen, 2008; Guo et al., 2011; Horiguchi et al., 2011; Stirnberg et al., 2012).RIBOSOMAL PROTEIN L10 (RPL10) was initially identified in humans as a putative suppressor of Wilms’ tumor (Dowdy et al., 1991). Since then, RPL10 has been studied in different organisms from archaea and bacteria to eukaryotes such as mammals, insects, yeast, and plants (Marty et al., 1993; Mills et al., 1999; Hwang et al., 2000; Zhang et al., 2004; Wen et al., 2005; Singh et al., 2009). A remarkable property of this protein is its high degree of amino acid conservation, suggesting fundamental and critical conserved functions of RPL10 in different organisms (Farmer et al., 1994; Eisinger et al., 1997; Hofer et al., 2007; Nishimura et al., 2008). Likewise, the crystallographic structural similarity observed among RPL10 orthologs in eukaryotes, bacteria, and archaea (called L16) established the conservation of this universal ribosomal protein family and provided evidence of the inalterability of the ribosome during evolution (Spahn et al., 2001; Nishimura et al., 2008). Nevertheless, besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions have been described for RPL10 (Mills et al., 1999; Hwang et al., 2000; Chávez-Rios et al., 2003; Zhang et al., 2004; Singh et al., 2009). In yeast, RPL10 is essential for viability, organizes the union site of the aminoacyl-tRNA, and its incorporation into the 60S subunit is a prerequisite for subunit joining and the initiation of translation (West et al., 2005; Hofer et al., 2007). Extensive analysis of the in vivo assembly of ribosomes revealed that RPL10 is loaded to the ribosome in the cytosol with the assistance of its chaperone suppressor of QSR1 truncations (Hedges et al., 2005; West et al., 2005).Arabidopsis has three genes encoding RPL10 proteins, AtRPL10A, AtRPL10B, and, AtRPL10C. Recently, we demonstrated that Arabidopsis RPL10 genes are differentially regulated by UV-B radiation: RPL10B is down-regulated, RPL10C is up-regulated, while RPL10A is not UV-B regulated. Arabidopsis single mutants showed that RPL10 genes are not functionally equivalent. Heterozygous rpl10a mutant plants are translation deficient under UV-B conditions, knockout rpl10A mutants are not viable, and knockdown homozygous rpl10B mutants show abnormal growth. Conversely, knockout homozygous rpl10C mutants do not exhibit any visible phenotype. Overall, RPL10 genes are involved in development and translation under UV-B stress (Falcone Ferreyra et al., 2010b). Furthermore, coimmunoprecipitation studies showed an association of RPL10 with nuclear proteins, suggesting that at least one of the RPL10 isoforms could have an extraribosomal function in the nucleus (Falcone Ferreyra et al., 2010a).The aim of this work was to further investigate the contribution of each Arabidopsis RPL10 to plant development and UV-B responses. We examined the spatiotemporal expression of each AtRPL10 using transgenic plants expressing Pro_RPL10:GUS fusions. By AtRPL10-GFP fusions, we analyzed the subcellular localization of each RPL10, demonstrating that the three isoforms are mainly localized in the cytosol but also in the nucleus. In order to investigate the functional redundancy between AtRPL10 genes in more detail, we generated and characterized double rpl10 mutants. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional (2D) gels followed by mass spectrometry. Overall, our data provide new insights into the role of each RPL10 in Arabidopsis.     

Immunoglobulins in defense, pathogenesis, and therapy of fungal diseases

Only two decades ago antibodies to fungi were thought to have little or no role in protection against fungal diseases. However, subsequent research has provided convincing evidence that certain antibodies can modify the course of fungal infection to the benefit or detriment of the host. Hybridoma technology was the breakthrough that enabled the characterization of antibodies to fungi, illuminating some of the requirements for antibody efficacy. As discussed in this review, fungal-specific antibodies mediate protection through direct actions on fungal cells and through classical mechanisms such as phagocytosis and complement activation. Although mechanisms of antibody-mediated protection are often species-specific, numerous fungal antigens can be targeted to generate vaccines and therapeutic immunoglobulins. Furthermore, the study of antibody function against medically important fungi has provided fresh immunological insights into the complexity of humoral immunity that are likely to apply to other pathogens.     

Role for Chitin and Chitooligomers in the Capsular Architecture of Cryptococcus neoformans

Molecules composed of β-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformans varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformans cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformans in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.Cryptococcus neoformans is the etiologic agent of cryptococcosis, a disease still characterized by high morbidity and mortality despite antifungal therapy (3). Pathogenic species belonging to the Cryptococcus genus also include Cryptococcus gattii, which causes disease mostly in immunocompetent individuals (24). A unique characteristic of Cryptococcus species is the presence of a polysaccharide capsule, which is essential for virulence (7-9, 19, 25, 33).C. neoformans has a complex cell surface. The thick fungal cell wall is composed of polysaccharides (29), pigments (11), lipids (35), and proteins (36). External to the cryptococcal cell wall, capsular polysaccharides form a capsule (19). Seemingly, the assembly of the surface envelope of C. neoformans requires the interaction of cell wall components with capsular elements. Some of the cryptococcal cell wall-capsule connectors have been identified, including the structural polysaccharide α-1,3-glucan and chitooligomers (29, 30, 32).Chitin-like molecules in fungi are polymerized by chitin synthases, which use cytoplasmic pools of UDP-GlcNAc (N-acetylglucosamine) to form β-1,4-linked oligosaccharides and large polymers. In C. neoformans, the final cellular site of chitin accumulation is the cell wall. The polysaccharide is also used for chitosan synthesis through enzymatic deacetylation (1). Eight putative cryptococcal chitin synthase genes and three regulator proteins have been identified (2). The chitin synthase Chs3 and regulator Csr2 may form a complex with chitin deacetylases for conversion of chitin to chitosan (1). Key early events in the synthesis of chitin/chitosan require the activity of glucosamine 6-phosphate synthase, which promotes the glutamine-dependent amination of fructose 6-phosphate to form glucosamine 6-phosphate, a substrate used for UDP-GlcNAc synthesis (23).In a previous study, we demonstrated that β-1,4-linked GlcNAc oligomers, which are specifically recognized by the wheat germ agglutinin (WGA), form bridge-like connections between the cell wall and the capsule of C. neoformans (32). In fact, other reports indicate that molecules composed of GlcNAc or its deacetylated derivative play key roles in C. neoformans structural biology. For example, mutations in the genes responsible for the expression of chitin synthase 3 or of the biosynthetic regulator Csr2p caused the loss of the ability to retain the virulence-related pigment melanin in the cell wall (1, 2). These cells were also defective in the synthesis of chitosan, which has also been demonstrated to regulate the retention of cell wall melanin (1). Treatment of C. neoformans acapsular mutants with chitinase affected the incorporation of capsular components into the cell wall (32). Considering that melanin and capsular components are crucial for virulence, these results strongly suggest that GlcNAc-derived molecules are key components of the C. neoformans cell surface. The expression of GlcNAc-containing molecules is likely to be modulated during infection since chitinase expression by host cells is induced during lung cryptococcosis (37).In this study, we used β-1,4-linked GlcNAc oligomers and an inhibitor of UDP-GlcNAc synthesis to evaluate the role played by GlcNAc-containing molecules in the surface architecture of C. neoformans. The results point to a direct relationship between the expression of GlcNAc-containing molecules and capsular assembly, indicating that chitin and chitooligomers are required for capsule organization in C. neoformans.     

Phagocytosis of Cryptococcus neoformans by,and Nonlytic Exocytosis from,Acanthamoeba castellanii

Cryptococcus neoformans, an encapsulated, pathogenic yeast, is endowed with a variety of virulence factors, including a polysaccharide capsule. During mammalian infection, the outcome of the interaction between C. neoformans and macrophages is central to determining the fate of the host. Previous studies have shown similarities between the interaction of C. neoformans with macrophages and with amoebae, resulting in the proposal that fungal virulence for mammals originated from selection by amoeboid predators. In this study, we investigated the interaction of C. neoformans with the soil amoeba Acanthamoeba castellanii. Comparison of phagocytic efficiency of the wild type, nonencapsulated mutants, and complemented strains showed that the capsule was antiphagocytic for amoebae. Capsular enlargement was associated with a significant reduction in phagocytosis, suggesting that this phenomenon protects against ingestion by phagocytic predators. C. neoformans var. neoformans cells were observed to exit amoebae several hours after ingestion, in a process similar to the recently described nonlytic exocytosis from macrophages. Cryptococcal exocytosis from amoebae was dependent on the strain and on actin and required fungal viability. Additionally, the presence of a capsule was inversely correlated with the likelihood of extrusion in certain strains. In summary, nonlytic exocytosis from amoebae provide another parallel to observations in fungus-macrophage interactions. These results provide additional support for the notion that some mechanisms of virulence observed during mammalian infection originated, and were selected for, by environmental interactions.The encapsulated yeast Cryptococcus neoformans is an environmental organism that is capable of causing human disease. This fungus is a facultative intracellular pathogen with a unique pathogenic strategy, despite no obvious need for replication in an animal host as part of its life cycle (10). C. neoformans is known to interact with protozoa, some of which have been shown to be effective predators for this fungus (6, 26), and amoebae appear to be important for the control of C. neoformans in the environment (28). Previously, we reported that the interaction of C. neoformans with Acanthamoeba castellanii directly paralleled the interaction with human macrophages (33). Similarities between C. neoformans interactions with amoebae and macrophages included intracellular replication in a phagosome and the release of polysaccharide-containing vesicles into the cytoplasm (33). Furthermore, passage of avirulent C. neoformans and Histoplasma capsulatum through slime mold and amoebae was shown to increase virulence in mice (31, 32). On the basis of these observations, it was proposed that the capacity for mammalian virulence emerged from interactions with phagocytic predators, such as amoebae and slime mold, in the environment (7, 17, 30). Consequently, single-cell protists have emerged as important systems for the study of C. neoformans virulence, and subsequent studies have investigated the interaction of this fungus with slime mold and paramecia (9, 31). Additional evidence for this concept comes from studies of insect fungal pathogens, which suggest that the capacity for insect pathogenicity may follow preadaptation from interactions with amoebae in the environment (4). Understanding the mechanisms by which virulence emerges in environmental microbes is important considering that global warming has been hypothesized to bring about new fungal diseases in the coming century (13).Recent work in our laboratory and in that of Robin May simultaneously uncovered a novel strategy of avoiding macrophage killing whereby yeast cells were expulsed without lysis of the host cell (2, 19). The process is remarkable in that extrusion of the C. neoformans-filled phagosome is accompanied by the survival of both the host cells and the yeast cells. Phagosome extrusion or fungal exocytosis appears to be a C. neoformans-dictated event that is dependent on both the presence of the polysaccharide capsule and on the depolymerization of actin. A corollary of the hypothesis that C. neoformans virulence emerged from interactions with environmental predators is that phenomena observed with mammalian cells are likely to have a counterpart in free-living phagocytic cells. Consequently, the observation of an apparently unique event such as phagosomal extrusion from mammalian macrophages suggested a need to search for similar events in C. neoformans interactions with environmental phagocytic predators.In this study, we investigated parallels between the intracellular pathogenic strategy of C. neoformans in both macrophages and A. castellanii, focusing on characterizing the impact of the capsule on protozoan phagocytosis and on ascertaining whether fungal cells could also exit amoebae, including the role of the capsule in that possible mechanism. Using time-lapse microscopy, we observed the exocytosis of C. neoformans from A. castellanii. While there are significant differences in the nonlytic exocytosis process when comparing amoebae and macrophages, the observation of this phenomenon in amoebae provides additional support for the idea that the virulence of C. neoformans was selected for, and is maintained, by interactions in the environment with other soil organisms.(This research was conducted by Cara Chrisman in partial fulfillment of the requirements for a Ph.D. from the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY [awarded in 2010].)     

Fungal Cell Gigantism during Mammalian Infection

The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 µm in diameter and capsules resistant to stripping with γ-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20–50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.     

Antibody action after phagocytosis promotes Cryptococcus neoformans and Cryptococcus gattii macrophage exocytosis with biofilm-like microcolony formation

Antibody-mediated phagocytosis was discovered over a century ago but little is known about antibody effects in phagolysosomes. We explored the consequences of antibody-mediated phagocytosis for two closely related human pathogenic fungal species, Cryptococcus neoformans and Cryptococcus gattii , of which C. neoformans encompasses two varieties: neoformans and grubii. The interaction between C. neoformans varieties grubii and neoformans and host cells has been extensively studied, but that of C. gattii and macrophages remains largely unexplored. Like C. neoformans , antibody-mediated phagocytosis of C. gattii cells was followed by intracellular replication, host cell cytoplasmic polysaccharide accumulation and phagosomal extrusion. Both C. gattii and C. neoformans cells exited macrophages in biofilm-like microcolonies where the yeast cells were aggregated in a polysaccharide matrix that contained bound antibody. In contrast, complement-opsonized C. neoformans variety grubii cells were released from macrophages dispersed as individual cells. Hence, both antibody- and complement-mediated phagocytosis resulted in intracellular replication but the mode of opsonization affected the outcome of exocytosis. The biofilm-like microcolony exit strategy of C. neoformans and C. gattii following antibody opsonization reduced fungal cell dispersion. This finding suggests that antibody agglutination effects persist in the phagosome to entangle nascent daughter cells and this phenomenon may contribute to antibody-mediated protection.     

Passive administration of monoclonal antibodies to Anthrolysin O prolong survival in mice lethally infected with <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

Background  

Bacillus anthracis has two major virulence factors: a tripartite toxin that produces lethal and edema toxins and a polyglutamic acid capsule. A recent report suggested that a toxin belonging to the cholesterol dependant cytolysin (CDC) family, anthrolysin O (ALO) was a new virulence factor for B. anthracis but subsequent studies have questioned its relevance in pathogenesis. In this study, we examined the immunogenicity of recombinant anthrolysin O (rALO) in mice.