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Inositol pyrophosphates (PP-IPs) comprising inositol, phosphate, and pyrophosphate (PP) are essential for multiple functions in eukaryotes. Their role in fungal pathogens has never been addressed. Cryptococcus neoformans is a model pathogenic fungus causing life-threatening meningoencephalitis. We investigate the cryptococcal kinases responsible for the production of PP-IPs (IP7/IP8) and the hierarchy of PP-IP importance in pathogenicity. Using gene deletion and inositol polyphosphate profiling, we identified Kcs1 as the major IP6 kinase (producing IP7) and Asp1 as an IP7 kinase (producing IP8). We show that Kcs1-derived IP7 is the most crucial PP-IP for cryptococcal drug susceptibility and the production of virulence determinants. In particular, Kcs1 kinase activity is essential for cryptococcal infection of mouse lungs, as reduced fungal burdens were observed in the absence of Kcs1 or when Kcs1 was catalytically inactive. Transcriptome and carbon source utilization analysis suggested that compromised growth of the KCS1 deletion strain (Δkcs1 mutant) in the low-glucose environment of the host lung is due to its inability to utilize alternative carbon sources. Despite this metabolic defect, the Δkcs1 mutant established persistent, low-level asymptomatic pulmonary infection but failed to elicit a strong immune response in vivo and in vitro and was not readily phagocytosed by primary or immortalized monocytes. Reduced recognition of the Δkcs1 cells by monocytes correlated with reduced exposure of mannoproteins on the Δkcs1 mutant cell surface. We conclude that IP7 is essential for fungal metabolic adaptation to the host environment, immune recognition, and pathogenicity.
IMPORTANCE Cryptococcus neoformans is responsible for 1 million cases of AIDS-associated meningitis and ~600,000 deaths annually. Understanding cellular pathways responsible for pathogenicity might have an impact on new drug development. We characterized the inositol polyphosphate kinases Kcs1 and Asp1, which are predicted to catalyze the production of inositol pyrophosphates containing one or two diphosphate moieties (PP-IPs). Using gene deletion analysis and inositol polyphosphate profiling, we confirmed that Kcs1 and Asp1 are major IP6 and IP7 kinases, respectively. Kcs1-derived IP7, but not Asp1-derived IP8, is crucial for pathogenicity. Global expression profiling and carbon source utilization testing suggest that IP7-deficient cryptococci cannot adapt their metabolism to allow growth in the glucose-poor environment of the host lung, and consequently, fungal burdens are significantly reduced. Persistent asymptomatic Δkcs1 mutant infection correlated with decreased mannoprotein exposure on the Δkcs1 mutant surface and reduced phagocytosis. We conclude that IP7 is crucial for the metabolic adaptation of C. neoformans to the host environment and for pathogenicity.
Cryptococcus neoformans var. grubii is a basidiomycetous opportunistic human fungal pathogen of global significance that commonly infects immunocompromised hosts, including those with AIDS. Cryptococcus neoformans is responsible for ~1 million cases of HIV-associated cryptococcal meningitis and ~600,000 deaths per year worldwide, predominantly in sub-Saharan Africa (1). Cryptococcal lung infection within an immunocompromised host can result from the inhalation of infectious propagules or the reactivation of a latent infection (2). Dissemination from the lungs to the central nervous system (CNS) occurs via the blood or lymphatic system (2). Survival of Cryptococcus neoformans within the adverse environment of the host depends on the coordinated functions of multiple stress-related signaling pathways that regulate the expression of a suite of virulence traits and remodeling of the fungal cell wall (3).
Cryptococcal virulence determinants include the ability to grow at host physiological temperature (37°C) and the production of melanin and polysaccharide capsule (for a review, see references 4to7). Melanin protects cells from damage caused by reactive oxygen species, which macrophages produce as part of their antimicrobial defense (8). It is deposited in the cell wall via the catalytic activity of the cell wall-associated enzyme laccase (9). The capsule surrounds the cell wall and is comprised of glucuronoxylomannan (GXM) (90%), galactoxylomannan (GalXM), and highly mannosylated and immunogenic mannoproteins (1%) (10, 11). It protects cryptococci within the host by having a suppressive effect on the host proinflammatory response, by inhibiting the antigen-presenting capability of monocytes, and by rendering cryptococcal cells resistant to phagocytosis by shielding cell wall glucans and mannoproteins from recognition by macrophages and dendritic cells (7, 12, 13). Many mannoproteins are tethered to the cell wall by a glycosylphosphatidylinositol (GPI) anchor (14) and are localized predominantly in the innermost layer of the capsule. However, they can become detached from the anchor and secreted through the capsular layer (15–19). Several of these mannoproteins are potential vaccine candidates against cryptococcal infection (14, 20–22).
The cryptococcal enzyme phospholipase C1 (Plc1) is a component of a stress-related signaling pathway governing the production of a suite of virulence traits, which are compromised when the Plc1-encoding gene is deleted (23). We demonstrated that recombinant Plc1 hydrolyzes the membrane lipid, phosphatidylinositol-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) (23) and that IP3 is a substrate for cryptococcal Arg1, an inositol polyphosphate kinase (IPK) (24). As with the Δplc1 mutant, multiple cryptococcal virulence traits were compromised in the Δarg1 mutant (24), suggesting that the absence of more complex inositol polyphosphates (IPs) and/or inositol pyrophosphates (PP-IPs) downstream of Arg1 contributes to the reduced virulence observed for both mutants.
PP-IPs, of which IP7 and IP8 are the most physiologically relevant, differ from IPs in that they possess a diphosphate on one or more carbons of the inositol ring. They regulate diverse cellular functions in eukaryotes (25–32). In the nonpathogenic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, different IP7 isoforms are essential for growth, vacuole biogenesis, salt tolerance, endocytic trafficking, the function of the cortical actin cytoskeleton, surface adhesion, and dimorphic switching in S. pombe (26, 33–37). PP-IPs affect cellular function at both the transcriptional and the posttranslational level via mechanisms that remain to be fully understood. These molecules modulate protein function directly through binding or indirectly by phosphorylating prephosphorylated proteins via a nonenzymatic reaction (38, 39). IP7 isoforms can also compete with membrane PI(3,4,5)P3 for binding to pleckstrin homology domain-containing proteins, affecting the association of these proteins with the plasma membrane, as observed in Dictyostelium discoideum (25). Recently, Worley et al. (40) showed that PP-IPs act in parallel with the TORC1 pathway to control the activity of the class I histone deacetylase Rpd3L and elicit global transcriptional changes in response to different types of stress (40). PP-IPs are therefore central regulators of eukaryotic cellular function, and targeting their synthesis holds promise for the development of new antifungal therapies. However, the kinases directly responsible for the synthesis of PP-IPs in fungal pathogens have not been determined, and the direct role of PP-IPs in regulating fungal pathogenicity remains unclear.
In the present study, we identified two PP-IP-producing kinases, Kcs1 and Asp1 (homolog of S. cerevisiae Vip1) in C. neoformans, characterized their enzymatic activities, and investigated the role of their products in pathogenicity and cellular function. Since IP7 was found to be the most crucial PP-IP for cryptococcal pathogenicity, we further investigated its role in cellular function using gene expression and carbon source utilization studies.
Kcs1 is an IP6 kinase in C. neoformans.We identified a putative IP6 kinase-encoding gene (CNAG_02897/KCS1) in C. neoformans var. grubii (strain H99) using Kcs1 from S. cerevisiae in a homology search. The protein encoded by KCS1 is predicted to be 1,249 amino acids long and to contain an IPK domain at the C terminus. C. neoformans Kcs1 (CnKcs1) has 53% similarity to S. cerevisiae Kcs1 (ScKcs1) at the amino acid level. While the long N-terminal sequences are highly varied among fungal Kcs1 homologs, the C-terminal IPK domains are more conserved: the CnKcs1 and ScKcs1 IPK domains share 50% similarity. Phylogenetic analysis of the fungal Kcs1 homologs using full Kcs1 sequences or IPK domains demonstrates that CnKcs1 is clustered with other Kcs1 homologs of Basidiomycetes (Fig. 1A).
Next, we assessed the role of Kcs1 in cryptococcal IP metabolism by creating a KCS1 deletion strain (Δkcs1 mutant) and comparing its IP profile with that of the wild type (WT) and Δarg1 mutant (see Text S1 and Fig. S1A and B in the supplemental material; Fig. 1B). Intracellular IPs in the WT and mutant strains were radiolabeled by adding [3H]myo-inositol to the culture medium, extracted from the cells and fractionated using anion-exchange high-performance liquid chromatography (HPLC). As in S. cerevisiae and mammalian cells, the most abundant IP species in the WT was IP6. Accumulation of IP3 in the C. neoformans Δarg1 mutant was consistent with the findings of our previous study, where raised levels of IP3 were detected using a radiometric IP3 binding assay (24). The Δarg1 mutant was also deficient in more highly phosphorylated IP species, including the predicted products of Arg1 (IP4 and IP5) and IP6 to IP8 (IP6–8). In the C. neoformans Δkcs1 mutant, the amount of IP3–6 was similar to that in the WT. However, the more highly phosphorylated PP-IP5 (IP7) and PP2-IP4 (IP8) were not detected, consistent with Kcs1 functioning as the major IP6 kinase in C. neoformans. These results also establish that Arg1 and Kcs1 are components of the same metabolic pathway.
Text S1
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Figure S1
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The mammalian IP6 kinase IP6K1 has been reported to perform physiological functions that are independent of its IP6 kinase activity (41). Furthermore, CnKcs1 and ScKcs1 contain an extended N terminus, which may have additional physiological functions. To distinguish IP6 kinase-dependent and -independent effects, we created the Δkcs1::KCS1AAA strain, in which the sequence encoding conserved amino acids in the Kcs1 catalytic domain (1032 SLL1034) was altered to encode AAA, resulting in the expression of kinase-inactive Kcs1 (Text S1; Fig. S1A. This same substitution abrogated Kcs1 catalytic activity in S. cerevisiae (34). The Δkcs1::KCS1AAA strain was created by ectopic insertion of the mutagenized KCS1 gene with its native promoter and terminator into the genome of the Δkcs1 mutant. A control strain (the Δkcs1::KCS1 strain) was similarly created by inserting the native KCS1 genomic fragment ectopically into the genome of the Δkcs1 mutant. Integration of KCS1AAA and KCS1 into a single genomic location in each strain was confirmed by Southern blotting (Fig. S1C). The levels of expression of ectopically integrated KCS1 and KCS1AAA in the respective reconstituted strains were comparable to the expression of native KCS1 in the WT (Fig. S1D). Like the Δkcs1 mutant, the Δkcs1::KCS1AAA strain did not produce IP7–8 (Fig. 1B, inset). The IP profile of the Δkcs1::KCS1 strain was similar to that of the WT (Fig. 1B, inset). The Δkcs1 and Δkcs1::KCS1AAA strains were subsequently used to identify phenotypes that were specifically associated with the absence of Kcs1 catalytic activity.
Loss of IP6 kinase activity leads to growth and cell wall integrity defects.The ability of C. neoformans to colonize host tissue is dependent on its ability to grow at mammalian physiological temperatures. Since PP-IP products generated by ScKcs1 play a role in the response to heat stress, the growth of the WT, Δkcs1, and Δkcs1::KCS1AAA strains was assessed at 30°C, 37°C, and 39°C using a drop dilution test. Like that of the ScΔkcs1 mutant, the growth of the Δkcs1 and Δkcs1::KCS1AAA strains at all three temperatures was reduced compared to that of the WT and Δkcs1::KCS1 strain (Fig. 2).
We also investigated the integrity of the cell wall in the Δkcs1 and Δkcs1::KCS1AAA mutants and their ability to produce mating filaments. Cell wall integrity was assessed by comparing the extent of growth in the presence of cell wall-perturbing agents. Figure 2 demonstrates that the growth of the Δkcs1 and Δkcs1::KCS1AAA strains was similarly reduced in the presence of SDS and caffeine, compared with that of the WT and Δkcs1::KCS1 strains, consistent with a cell wall integrity defect. The Δkcs1 and Δkcs1::KCS1AAA (mating type α) strains also produced fewer mating filaments following a unilateral cross with KN99 (mating type a), while filament production was restored to WT levels in the Δkcs1::KCS1 strain (Fig. S2). Since the cell wall integrity and mating defects persisted in the Δkcs1::KCS1AAA strain (Fig. 2; Fig. S2), it can be concluded that the PP-IPs produced by Kcs1 are essential for cell wall integrity and mating.
Figure S2
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The Δkcs1 mutant is hypersusceptible to antifungal agents.To investigate whether disruption of PP-IP synthesis alters the susceptibility of C. neoformans to antifungal drugs, the extent of growth of the WT and the Δkcs1 mutant was compared in the presence of amphotericin B, flucytosine, echinocandins, and a range of azoles (Table S1). The Δkcs1 mutant was 8 times more susceptible than the WT to posaconazole, voriconazole, itraconazole, and fluconazole and twice as susceptible to flucytosine and amphotericin B but remained resistant to the echinocandins, caspofungin, micafungin, and anidulafungin. The Δarg1 mutant demonstrated a similar drug susceptibility profile (Table S1 and Text S1).
Table S1
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The Δkcs1 mutant has defective melanin production and an increased capsule size.Next, we assessed the ability of the Kcs1-deficient strains to produce melanin. Melanin protects cryptococci from a variety of stresses, including macrophage-derived oxidative stress and UV light. The Δkcs1 and Δkcs1::KCS1AAA strains produced less melanin than the WT when grown on minimal medium (MM) without glucose and supplemented with l-3,4-dihydroxyphenylalanine (l-DOPA) (Fig. 3A). The extent of melanization in the Δkcs1::KCS1 strain was similar to that of the WT. Since the cell wall-associated enzyme laccase 1 catalyzes melanin synthesis in C. neoformans, allowing it to be deposited in the cell wall, the extracellular laccase activity associated with intact fungal cells was also measured. The results in Fig. 3B demonstrate that the reduced melanization of the Δkcs1 and Δkcs1::KCS1AAA strains (Fig. 3A) coincided with reduced extracellular laccase activity as measured by oxidation of the laccase substrate 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS). Once again, laccase activity was restored to WT levels in the Δkcs1::KCS1 strain. To determine whether reduced laccase activity in the Kcs1-deficient strains is due to the absence of LAC1 gene induction under melanin-inducing conditions, we measured LAC1 expression in the WT, Δkcs1, Δkcs1::KCS1, and Δkcs1::KCS1AAA strains following 5 h of growth in MM without glucose. Figure 3C demonstrates that the LAC1 expression profile in all strains is similar to that observed for laccase activity and melanin production. These data indicate that Kcs1-derived PP-IPs are essential for induction of LAC1.
Capsule production was also assessed following the growth of fungal strains on Sabouraud (SAB) agar. Notably, colonies formed by the Kcs1-deficient strains had a mucoid (glossy) appearance, in contrast to the dull surface of the WT (Fig. 4A). The capsules produced by the Δkcs1 and Δkcs1::KCS1AAA strains were on average larger than those produced by the WT (Fig. 4B and C). Colony appearance and capsule size were partially restored in the Δkcs1::KCS1 strain.
Phenotypic defects in the Δkcs1 mutant are caused by the absence of IP7, not IP8.Kcs1 is essential for the production of the PP-IPs IP7 and IP8, as neither species was detected in the Δkcs1 and Δkcs1::KCS1AAA strains (Fig. 1B). To determine the relative contribution of IP8 to the Kcs1-dependent traits, we identified a homolog of the IP6–7 kinase of S. cerevisiae (ScVip1) in C. neoformans, designated Asp1. Asp1 protein (encoded by CNAG_02161) is predicted to be 1,117 amino acids long and is 33% identical and 24% similar to ScVip1. Like ScVip1 and Asp1 in Schizosaccharomyces pombe, Asp1 of C. neoformans contains an N-terminal ATP grasp domain responsible for the kinase activity and a C-terminal histidine acid phosphatase domain of unknown function. C. neoformans ASP1 was deleted as described for KCS1, creating the Δasp1 mutant. IP7 accumulated and PP2-IP4 (IP8) was not detected in the Δasp1 mutant (Fig. 5), consistent with C. neoformans Asp1 functioning as an IP7 kinase, as reported for S. cerevisiae Vip1 (42, 43). A reduction in IP5 and IP6 was also noted in the Δasp1 strain (Fig. 5). The growth of the Δasp1 mutant was assessed under the same conditions as those for the Δksc1 mutant, including 37°C, in the presence of cell wall-perturbing agents, and on mating agar (Fig. S3). The Δasp1 strain was also assessed for the production of melanin and capsule. Under all growth conditions, the Δasp1 strain exhibited a phenotype similar to that of the WT, indicating that Kcs1-derived PP-IP5 (IP7), rather than Asp1-derived PP2-IP4 (IP8), is the most crucial PP-IP for cryptococcal cellular function.
Figure S3
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The Δkcs1 mutant is less pathogenic than the WT in animal models.The pathogenicity of the Δkcs1 mutant was compared to that of the WT in an invertebrate model, the greater wax moth (Galleria mellonella), and a mouse inhalation model. Larvae of Galleria mellonella were infected by inoculation of fungi into the lower prolegs and incubated at 30°C. The average survival of Δkcs1 mutant-infected larvae was 14 days, compared with 5 days for WT-infected larvae (Fig. 6A). Mice were inoculated via the nares. The median survival time of mice infected with the WT was 14 days. However, no mice succumbed to infection with the Δkcs1 mutant for up to 50 days postinoculation (Fig. 6B). Virulence was almost restored by the introduction of KCS1 into the Δkcs1 mutant, with the median survival time of Δkcs1::KCS1 strain-infected mice being 18 days. Despite the avirulent phenotype, Δkcs1 cells were detected in mouse lungs (3 × 103 to 90 × 103 CFU per total lung tissue) over the 50-day postinfection period (Fig. 6C). The Δkcs1 mutant infection burden was ~8-fold lower than that of the WT at 3 days postinfection. However, the difference between the WT and the Δkcs1 mutant pulmonary burdens increased to ~140-fold at the onset of the inflammatory response 7 days postinfection (see Fig. 7) and to ~286-fold 14 days postinfection. These increases were due primarily to the more rapid increase in WT pulmonary burden than in Δkcs1 mutant burden over this time. Despite the persistence of Δkcs1 mutant in lungs up to 50 days postinfection, no colonization of the brain was observed during this time (Fig. 6D). This was in contrast to what occurred with mice infected with the WT, which developed a CNS infection by day 14 (the median survival time of WT-infected mice).
Periodic acid-Schiff (PAS)-stained sections of WT- and Δkcs1 mutant-infected lungs at day 7 and day 14 are shown in Fig. S4 in the supplemental material. A dense WT infection was observed on both days within the alveolar spaces and bronchi, with evidence of extensive infiltration of monocytes and polymorphs. In contrast, but consistent with organ burden analysis, Δkcs1 mutant infection was sparse and lung tissue was healthier than in WT-infected lung, as indicated by large areas of well-aerated alveoli and minimal evidence of inflammation. Despite the death of Δkcs1 mutant cells over the infection time course, budding cells were observed in the infected lung tissue.
Figure S4
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The Δasp1 mutant was also tested for virulence in a mouse inhalation model, and the progression of disease in Δasp1 mutant-infected animals was found to be similar to that of WT-infected animals. This was expected, as no phenotypic differences were observed between the WT and the Δasp1 strain (Fig. S3 and S5).
Figure S5
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Δkcs1 mutant infection elicits a weak host immune response.The persistent, asymptomatic Δkcs1 mutant infection observed in the mouse model (Fig. 6C) prompted us to compare the immune responses elicited by the WT and the Δkcs1 mutant. The cytokine profile in infected lung tissue at 3, 7, and 14 days postinoculation was determined by analyzing the supernatants of homogenized lung extracts prepared from WT-, Δkcs1 mutant-, and mock-infected animals using a cytokine bead array (CBA) and flow cytometry (Fig. 7).
WT infection elicited a strong proinflammatory response in the lung (gamma interferon [IFN-γ] and tumor necrosis factor [TNF]), which peaked at day 7 postinfection; the day 7 response for IFN-γ and TNF-α was 36- and 3-fold higher than the day 3 response, respectively, and ~688- and 27-fold higher than the response observed in mock-infected animals (Fig. 7). TNF is required for neutrophil recruitment into pulmonary tissue during a cryptococcal infection (44), and IFN-γ maintains monocyte-derived macrophages in an activated state conducive to fungal killing and clearance. The strong proinflammatory response in WT-infected mice was accompanied by a strong monocyte chemoattractant protein-1 (MCP-1) response, which also peaked at day 7. MCP-1 is a crucial mediator of monocyte and T cell recruitment to the sites of infection. At day 7, the MCP-1 response was 22-fold higher than the day 3 response and 40-fold higher than the response observed in mock-infected animals. Despite the decrease in MCP-1 levels at day 14, significant numbers of monocytes were present at this time point in WT-infected lung (Fig. S4). In contrast to WT, no increase in the level of any of these three cytokines was observed in Δkcs1 mutant-infected lung at 7 days, despite evidence of persistent Δkcs1 mutant infection. Interleukin 6 (IL-6) production also peaked in WT-infected lung at day 7, with the response being ~4-fold higher than at day 3 and ~84-fold higher than the response observed in mock-infected animals. In contrast to the proinflammatory cytokines and MCP-1, IL-6 production peaked at day 7 in Δkcs1 mutant-infected lungs and was ~36-fold higher than the day 3 response and ~17-fold higher than the response observed in mock-infected animals.
IL-2 levels increased in Δkcs1 mutant-infected lungs over the time course but particularly from day 3 to day 7. However, the day 3 level was lower than the mock-infected control and WT values, while the day 7 level was similar to that of the mock-infected control but lower than the WT value. Interestingly, similar IL-2 responses were obtained for WT- and Δkcs1 mutant-infected lungs at day 14 despite the significantly lower Δkcs1 mutant fungal burden. In contrast to that of the other cytokines, IL-4 and IL17-A production was higher at day 14 than at day 7 in WT-infected lungs. However, the IL-4 response observed for Δkcs1 mutant-infected lungs did not change significantly between days 7 and 14 but was still higher than the response observed in mock-infected lungs (Fig. 7).
For both the WT and Δkcs1 strains, IL-17A levels at day 3 were below the level of detection and are not shown. At day 7, the IL-17A level in Δkcs1 mutant-infected lung was lower than that in WT-infected lung, while at day 14, a similar IL-17A response occurred in WT- and Δkcs1 mutant-infected lungs, despite the significantly lower Δkcs1 mutant burden.
The phagocytosis of the Δkcs1 mutant by monocytes and subsequent monocyte activation are compromised.Monocytes and macrophages are the first line of defense against cryptococcal infection. Since we observed an altered surface phenotype in the Kcs1-deficient mutants (Fig. 4A), which may affect cryptococcal recognition by monocytes, we compared the efficiencies of phagocytosis of the WT and Δkcs1 mutant in vitro, using the THP-1 monocytic cell line. Fungal cells were labeled with fluorescein isothiocyanate (FITC) and opsonized with human serum (containing complement and antifungal antibodies), and their uptake was measured by flow cytometry. Figure 8A demonstrates that after 4 h of coincubation, ~40% of THP-1 cells were associated with WT C. neoformans. However, only ~10% of THP-1 cells were associated with Δkcs1 cells. Figure S6A shows a representative scatterplot used for quantification of adhesion/uptake. The extent of THP-1 cell association with the WT and Δkcs1 mutant was also measured when IgG antibodies purified from human blood were used as the opsonizing agent. The results in Fig. 8A show that only ~20% of THP-1 cells were associated with WT C. neoformans, indicating that the combination of complement and antibody is a more potent opsonin of WT cryptococci than antibody alone. As with the results observed for serum opsonization, the number of THP-1 cells associated with IgG-opsonized Δkcs1 cells was reduced. Notably, the difference in THP-1 cell association between the WT and mutant was even greater when IgG was used as an opsonin than when serum was used (9-fold versus 4-fold). The extent of THP-1 cell association with the WT and Δkcs1 strains was also measured when anticapsular antibodies were used as the opsonizing agent (Fig. 8A). However, in this case, the extents of THP-1 association with the WT and Δkcs1 mutant were similar.
Figure S6
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To rule out the possibility that Δkcs1 cells are taken up less by THP-1 monocytes due to their lower rate of proliferation, we compared the phagocytosis of heat-killed WT and Δkcs1 mutant. As with live cryptococci, the phagocytosis of dead Δkcs1 cells was reduced (Fig. 8B). The extents of adhesion/uptake of heat-killed cryptococcal cells were also compared in primary human CD14-positive monocytes in a peripheral blood mononuclear cell (PBMC) preparation. Monocytes were distinguished from T and B cells by the binding of fluorescent anti-CD14. As with THP-1 cells (Fig. 8A and B), monocyte uptake of heat-killed, serum-opsonized Δkcs1 cells by CD14-positive monocytes was reduced (Fig. 8C). Microscopy of the cocultures also revealed reduced association of the Δkcs1 mutant with THP-1 cells and PBMCs (Fig. S6B), consistent with the flow cytometry analysis of fungal adhesion/uptake.
To determine whether reduced phagocytosis of Δkcs1 cells by monocytes coincided with reduced monocyte activation, we compared levels of THP-1 activation in the presence of live WT or Δkcs1 cells after 4 h of coincubation, using an RT2 profiler PCR array. The results in Fig. 8D indicate that the expression of proinflammatory cytokine- and interleukin-encoding genes is significantly higher in THP-1 cells coincubated with WT than with Δkcs1 cells, consistent with the stronger cytokine induction profile observed in the WT-infected mouse lung (Fig. 7). For example, the induction of the gene encoding the monocyte chemoattractant protein (Mcp1/Ccl2) was >9-fold higher in THP-1 cells coincubated with the WT than in cells coincubated with the Δkcs1 mutant. Thus, the reduced rate of Δkcs1 cell phagocytosis correlated with reduced monocyte activation.
Mannoprotein exposure at the Δkcs1 cell surface is reduced.The similar rates of phagocytosis of anti-GXM-opsonized WT and Δkcs1 cells suggest that the antibody binds similarly to the surfaces of both strains despite their microscopic (larger capsule size) and macroscopic (mucoid colony morphology) differences. The difference in phagocytosis of WT and Δkcs1 cells observed when serum or human IgG were used for opsonization suggests that the respective opsonins bind to surface epitopes other than the sugar moiety binding targets of anti-GXM antibodies. Furthermore, the data suggest that the targets of serum and human IgG opsonins are present in greater amounts on WT than on Δkcs1 cells. The capsule contains mannoproteins, in addition to polysaccharides, and human serum contains antibodies against fungal mannoproteins that can initiate classical pathway activation upon binding to macrophages (45). To investigate mannoprotein exposure at the surfaces of WT and Δkcs1 cells, the extents of concanavalin A (ConA) binding were compared. ConA is a lectin that reacts with surface mannose sugars found on many microbes and is available as an FITC conjugate so that binding can be assessed by flow cytometry. The results in Fig. 8E show that ConA-FITC binding to the Δkcs1 cell surface is decreased in comparison with binding to WT cells, suggesting that mannosylated proteins are not displayed normally on the surface of the mutant cells.
To determine whether the reduced presence of mannoproteins on the Δkcs1 cell surface correlates with reduced expression of genes encoding mannoproteins, we performed transcriptome sequencing (RNA-seq) of the WT and Δkcs1 strains. We focused on genes encoding proteins with a signal peptide which targets them to the endoplasmic reticulum (ER)/Golgi complex and then to the cell periphery, excluding transmembranal proteins and proteins likely to be mitochondrial, peroxisomal, or ER resident. The expression of 22 extracellular proteins was down-regulated in the mutant, with 17 predicted to be glycosylated and 4 containing a GPI anchor (Table 1). Taken together, the data indicate that Kcs1-derived IP7 affects the fungal surface composite at the transcriptional level, influencing the extent of opsonization and phagocytosis and the ensuing immune response.
Transcriptome analysis identifies metabolic changes in the Δkcs1 mutant.The gene expression profiles of the WT and Δkcs1 strains obtained by RNA-seq were further analyzed to identify changes in gene expression that could be responsible for the altered phenotype and virulence profile of the Δkcs1 mutant. Initial functional classification of differentially expressed genes was made using gene ontology (GO) term assignment generated by the Joint Genome Institute (JGI) and then refined further based on published literature and by performing a homology search. We found that the absence of IP7 resulted in an 11-fold reduction in the expression of the LAC1 gene involved in melanization, consistent with the results in Fig. 3. However, the most significant changes were observed in metabolic function. Tables 2 and S3 demonstrate that genes encoding proteins involved in glycolysis are more highly expressed in the Δkcs1 mutant than in the WT. Genes involved in mRNA translation and ribosomal biosynthesis are also more highly expressed in the Δkcs1 mutant. However, expression of genes encoding numerous sugar transporters and enzymes that comprise metabolic pathways involved in the utilization of alternative carbon sources (citric acid and glyoxylate cycles, gluconeogenesis, and fatty acid β-oxidation) is reduced in the Δkcs1 mutant. These metabolic differences suggest that WT, but not Δkcs1, cells actively express genes involved in the utilization of carbon sources other than glucose, including lactate and fatty acids, which require mitochondrial function.
Table S3
Copyright © 2015 Lev et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
The environment of the host lung is low in glucose (46), and enzymes involved in the use of alternative carbon sources are essential for cryptococcal pathogenicity (47–49). We therefore compared the extent of growth of both strains on glucose and three nonfermentable carbon sources, lactate, glycerol, and oleic acid, each present as the sole carbon source. We also used minimal medium (MM), which is more physiological than yeast extract-peptone-dextrose (YPD) (Fig. 2) . In MM supplemented with glucose Δkcs1 mutant grew similar to the WT strain (Fig. 9A). Glycerol is incorporated into the glycolytic/gluconeogenesis pathway following conversion to dihydroacetone phosphate by mitochondrial flavin adenine dinucleotide (FAD)-dependent glycerol-3-phosphate dehydrogenase. Lactate is converted to pyruvate, which is channeled into the citric acid cycle and gluconeogenesis. Oleic acid undergoes β-oxidation in mitochondria and/or peroxisomes to generate acetyl coenzyme A (acetyl-CoA), which can be used for energy generation via the citric acid cycle. The results in Fig. 9A demonstrate that in contrast to that on glucose, the growth of Δkcs1 cells on the alternative carbon sources was severely compromised.
Using quantitative PCR (qPCR) and minimal medium containing glucose or lactate, we compared the levels of expression of a gene encoding a key enzyme in gluconeogenesis, phosphoenolpyruvate carboxykinase (PCK1; CNAG_04217) (Fig. 9B). Pck1 catalyzes an irreversible step of gluconeogenesis, and its expression was 20-fold lower in Δkcs1 cells grown overnight in YPD than in WT cells under the same conditions (Table S3). In minimal medium supplemented with glucose, PCK1 expression was 4.5-fold lower in Δkcs1 than in WT cells (Fig. 9B), consistent with the global expression data. In the absence of glucose (minimal medium with lactate), PCK1 was induced in both strains, but its expression was 2-fold lower in the Δkcs1 mutant, indicating that gluconeogenesis is not fully activated in the absence of IP7. This result confirms that the mutant is compromised in its ability to metabolize alternative carbon sources and that the defect is caused, at least in part, by inadequate transcriptional regulation of metabolic enzymes.
Delineation of the IP biosynthesis pathway in C. neoformans.Using gene deletion analysis and HPLC-based radiometric IP profiling, we have confirmed that Arg1 is an IP3 kinase. Furthermore, we have demonstrated that the role of Arg1 is to provide IP precursors for the PP-IP-producing kinase Kcs1, which, like Arg1, is essential for cryptococcal virulence. We determined that Kcs1 is the major IP6 kinase in C. neoformans, since it is essential for the synthesis of PP-IP5 (IP7) and PP2-IP4 (IP8). In particular, neither the Δkcs1 mutant nor the kinase-inactive mutant produced detectable amounts of IP7 (or IP8). We also showed that Asp1 functions primarily as an IP7 kinase, converting Kcs1-generated IP7 to IP8. In S. cerevisiae, IP7 is also produced by an Asp1 homolog, Vip1 (42). However, our IP profiling revealed that Asp1 does not contribute significantly to IP7 production in C. neoformans. Based on the IP profile obtained for the Δarg1, Δkcs1, and Δasp1 mutants, we propose a model of the IP biosynthesis pathway in C. neoformans depicted in Fig. 10. The presence of a putative IP5 kinase (Ipk1) in the middle of the pathway can be inferred from its homology to Ipk1 in S. cerevisiae; its role in C. neoformans is currently being investigated in our laboratory.
The IP biosynthetic pathway as a drug target.Two observations support investigation of the fungal PP-IP biosynthetic pathway as a potential antifungal drug target: (i) the absence of redundancy in the fungal pathway compared with the mammalian pathway and (ii) low sequence similarity between fungal and mammalian enzymes. For example, while Arg1 in C. neoformans is the only enzyme able to convert IP3 to IP4, three IP3 kinase isoforms and the inositol polyphosphate multikinase (IPMK) catalyze this reaction in humans. Arg1 differs structurally and functionally from mammalian IP3 kinases, implying potential selectivity of inhibition (50, 51). Arg1 and IPMK are only 19% identical, while Kcs1 is even less similar (13% identity) to its mammalian equivalent, inositol hexakisphosphate kinase 1 isoform 1. The hypersusceptibility of Δarg1 and Δkcs1 cells to antifungal agents (Table S1) also suggests that inhibitors of fungal Kcs1 or Arg1 may act synergistically with these marketed antifungal drugs to provide a better treatment outcome.
Kcs1-derived IP7, but not Asp1-derived IP8, is required for optimal growth and the production of virulence traits.We observed delayed growth of the CnΔkcs1 mutant under standard growth conditions and a cell wall integrity defect but no high-temperature growth defect. In a cross with the KN99 MATa strain, the Δkcs1 mutant, but not the Δasp1 mutant, produced stunted mating filaments. Defective Δkcs1 cell walls may affect retention of the sexual agglutinins and adhesins, which are required for efficient cell contact prior to filamentation (52), as well as capsule attachment. A mucoid phenotype, typical of the Δkcs1 mutant, is likely to be associated with increased capsule size. Such a correlation was also observed in C. neoformans following phenotypic switching (53) and in the allergen 1 (ALL1) deletion mutant (54). During glucose deprivation, the Δkcs1 strain, but not the Δasp1 strain, fails to produce extracellular laccase and, consequently, melanin. We established that Kcs1-derived IP7 affects melanization at the transcriptional level, as the gene encoding the major melanin-producing enzyme in C. neoformans, laccase 1 (LAC1), was not induced in Δkcs1 cells. Transcriptional profiling data supported this finding.
IP7-dependent metabolic adaptation affects growth and pathogenicity.The inability of the Δkcs1 mutant to achieve high infection burdens in mouse lung is most likely multifactorial, due to its weakened cell walls, inability to produce melanin, and reduced growth rate. High-throughput analysis of gene expression in the WT and Δkcs1 mutant suggested that the Δkcs1 mutant has a reduced ability to utilize carbon sources other than glucose and that this metabolic dysfunction may be a potential reason for its reduced ability to colonize glucose-deficient mouse lungs (46, 55). It has been demonstrated that WT cryptococci harvested during pulmonary infection exhibit a gene expression profile consistent with a starvation response (47, 56, 57). Metabolic dysfunction due to the absence of IP7, as suggested by our gene expression data, was supported by our observation of the severely reduced growth of the Δkcs1 mutant on alternative carbon sources, such as oleic acid, glycerol, and lactate (Fig. 9A). Utilization of these carbon sources requires functional mitochondria and, in the case of oleic acid, peroxisomes (58). A similar metabolic imbalance was reported for the Δkcs1 mutant of S. cerevisiae (59), whose cells did not grow on ethanol or glycerol as a sole carbon source.
Characterization of cryptococcal mutants defective in key metabolic enzymes established unequivocally the importance of β-oxidation and gluconeogenesis for the survival of C. neoformans in the nutrient-poor environment of the host and for pathogenicity. For example, a mutant in the peroxisomal β-oxidation pathway (Δmfe1 mutant) failed to grow on fatty acids as a carbon source and displayed hypovirulence in mice and reduced dissemination to the brain (49). Consistent with this report and with our observation that Δkcs1 mutant growth was reduced on oleic acid as the sole carbon source, MFE1 expression in Δkcs1 cells was 21-fold lower than in WT cells (Table S3). Similarly, a gluconeogenesis-defective cryptococcal mutant deficient in phosphoenolpyruvate carboxykinase Pck1(Δpck1 mutant) failed to grow on lactate as a sole carbon source and was also markedly less virulent in mice (48). Figure 9B and Table S3 demonstrate that the expression of PCK1 was lower in Δkcs1 cells than in the WT; the difference was 20-fold when the WT and Δkcs1 cells were grown in YPD and 2-fold when the strains were grown in minimal medium with lactate as a sole carbon source. Taken together, our findings suggest that the reduced pathogenicity of the Δkcs1 mutant may be due to a failure to adjust its metabolic response to a nutrient-poor environment, and this failure is caused, at least in part, by compromised regulation of gene expression. Such metabolic adaptation is essential for successful host infection.
Persistence of Δkcs1 mutant infection correlates with reduced surface mannoprotein.Despite the reduced pathogenicity and metabolic defects observed in the Δkcs1 mutant, a low level of apparently asymptomatic infection persisted in vivo. Our results suggest that this persistence of Δkcs1 mutant infection in lung is due primarily to the reduction in the display of surface mannoproteins in the Δkcs1 strain, as evidenced by decreased ConA binding and reduced expression of genes encoding immunogenic mannoproteins, leading to reduced recognition by phagocytes. It is likely that the majority of the opsonic anticryptococcal antibodies in serum target mannoproteins (45, 60). Thus, the reduced mannoprotein content on the surfaces of Δkcs1 cells is expected to result in reduced opsonization by antibodies against mannoprotein and less efficient recognition of the fungal cells by phagocytic Fc receptors (45). Reduced phagocytosis of Δkcs1 cells indeed coincided with evidence of less phagocyte activation. The levels of phagocytosis of heat-killed WT and Δkcs1 cells were also compared to rule out the possibility that the reduction in phagocytosis of Δkcs1 cells by THP-1 monocytes was due to the lower proliferation rate of Δkcs1 cells. Again, the phagocytosis of killed Δkcs1 cells was reduced relative to that of WT cells (Fig. 8B). This reduction was even greater than that observed for live cells (~16-fold versus ~4-fold), possibly because a different concentration of serum was used for opsonization of the killed cells, as indicated in the legend of Fig. 8.
Persistent Δkcs1 strain lung infection may also be due to impaired sampling of mannoproteins by resident macrophages and dendritic cells for subsequent presentation to T cells, once again as a result of diminished mannoprotein production in Δkcs1 cells. Notably, the secreted polysaccharide deacetylase homolog Fpd1/D21 (CNAG_06291), which stimulates T-cell activation in mice and therefore contributes to the adaptive immune response to WT C. neoformans (61, 62), was down-regulated in Δkcs1 cells (Table 1). Transcription of many genes encoding proteases, including aspartic proteases (CNAG_06213, CNAG_04380, and CNAG_00581), carboxypeptidases (CNAG_01040 and CNAG_05973), serine-type endopeptidase (CNAG_04625), and elastinolytic metalloproteinase (CNAG_04735), was also reduced. Fungal proteases are known to elicit inflammatory responses via activation of protease-activated receptors (PARs), leading to allergic inflammation (63, 64). Thus, reduced secretion of proteases by the Δkcs1 mutant potentially contributes to the weak proinflammatory cytokine response and impaired infiltration of inflammatory neutrophils and monocytes observed in Δkcs1 mutant-infected lung (Fig. 7 and S4).
The persistence of Δkcs1 strain infection also correlated with a reduced recognition of Δkcs1 cells by the host immune system, as indicated by the lung cytokine profile (Fig. 7). First, in contrast to what occurred in WT-infected lung, no peak at day 7 was observed for the TNF, IFN-γ, and MCP-1 responses in Δkcs1 mutant-infected lung. This is consistent with a greater reduction in the proinflammatory response to Δkcs1 cells at day 7 when fungal burden is taken into account. Interestingly, however, similar IL-2 and IL-17A responses were obtained in WT- and Δkcs1-infected lungs at day 14 despite the significantly lower Δkcs1 mutant burden. Taken together, the phagocytosis, lung cytokine, and histological data suggest that the persistence of a low-grade, asymptomatic Δkcs1 mutant infection is attributable to reduced recruitment of monocytes to sites of Δkcs1 mutant infection and reduced uptake of the Δkcs1 mutant by resident lung macrophages and infiltrating monocytes.
In summary, we have demonstrated for the first time that the IP biosynthetic pathway is essential for cryptococcal pathogenicity in a mammalian infection model. We have shown that Kcs1 is the major IP6 kinase in C. neoformans producing IP7, while Asp1 is an IP7 kinase essential for the production of IP8. We have established that, of the two PP-IPs, IP7 exerts the greatest influence on cryptococcal pathogenicity. Our findings indicate that IP7 affects cryptococcal cellular functions, at least in part, via gene expression and that successful host infection requires IP7 to activate starvation-related metabolic pathways.
Strains and media.Wild-type C. neoformans var. grubii strain H99 (serotype A, MATa) was used in this study. The ARG1 (CNAG_06500) deletion mutant (Δarg1 mutant) was described previously (24). The KN99 (MATα) strain was a kind gift from Joseph Heitman's laboratory (Duke University, Durham, NC, USA) (65). C. neoformans strains were maintained on SAB agar plates (4% dextrose, 1% peptone, 2% agar, pH 5.6), and incubated in YNB (6.7 g/liter yeast nitrogen base, 0.5% glucose), YPD (1% yeast extract, 2% peptone, and 2% dextrose), or MM (minimal medium; 15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, 3 µM thiamine) broth in preparation for the experiments. Mating was induced on 5% V8 juice (pH 5), 2% agar plates. MM without glucose was used for laccase induction in broth culture and for the preparation of broth and plates with different carbon sources (1% oleic acid, sodium lactate, or glycerol). Minimal medium plates supplemented with 1 mM l-DOPA (l-3,4-dihydroxyphenylalanine) were used to assess melanization.
Quantification of extracellular laccase activity.The protocol was adapted from reference 66. C. neoformans strains were grown overnight in YPD at 30°C. Cells were pelleted, washed with water, resuspended in MM without glucose at an optical density at 600 nm (OD600) of 1, and incubated for 4 h at 30°C. To measure cell-associated laccase activity, 200 µl of the culture was removed and the cells were pelleted by centrifugation. The pellet was resuspended in 1 ml of a 3 mM concentration of a chromogenic laccase substrate, ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)], and incubated for 30 min at 30°C until a light blue color was produced, indicative of ABTS oxidation. The cells were pelleted, and the OD of the supernatant was measured at 436 nm. Laccase-specific activity was expressed as micromoles of ABTS substrate oxidized/minute/milliliter of culture at an OD600 of 1. The concentration of oxidized ABTS was calculated using the molar extinction coefficient (ε = 29.3 mM−1 cm−1).
[3H]inositol labeling.[3H]inositol labeling of cryptococcal cells was performed according to the protocol of reference 67, with several modifications. Briefly, fungal strains from overnight YPD culture were diluted in 5 ml fresh YPD to an OD600 of 0.01 (WT versus the Δkcs1 and Δarg1 mutants) or to an OD600 of 0.05 (WT versus the Δkcs1::KCS1, Δkcs1::KCS1AAA, and Δasp1 strains), supplemented with 10 µCi/ml [3H]myo-inositol (PerkinElmer), and incubated until the OD of the cultures reached at least 6 (16 to 76 h). The cells were pelleted, washed twice with 1 ml of fresh YPD, and snap-frozen in liquid nitrogen. To extract inositol polyphosphates, the cells were resuspended in extraction buffer (1 M HClO4, 3 mM EDTA, 0.1 mg/ml IP6) and homogenized in the presence of glass beads using a bead beater (four 30-s cycles with 1-min rests in between cycles). After the debris was pelleted, supernatants were neutralized using neutralization buffer (1 M K2CO3, 3 mM EDTA) and incubated on ice for 2 h. HPLC inositol phosphate analysis was performed as previously described (67).
Murine inhalation model of cryptococcosis.All procedures were approved by the Sydney West Local Health District Animal Ethics Committee, Department of Animal Care. Survival and organ burden/cytokine analyses were conducted using 7-week-old female BALB/c mice obtained from the Animal Resource Centre, Floreat Park, Western Australia. For both sets of analyses, mice were anesthetized using isoflurane (in oxygen) delivered via an isoflurane vaporizer attached to a Stinger small-animal anesthetic machine (Advanced Anaesthesia Specialists). Groups of 8 to 10 mice were then inoculated intranasally with the C. neoformans WT or IPK mutant strain (5 × 105 yeast cells in 20 µl phosphate-buffered saline [PBS]) and observed daily for signs of ill health. The number of viable yeast cells inoculated into the nares was later confirmed by quantitative culture.
For the survival study, mice which had lost 20% of their preinfection weight or which showed debilitating clinical signs prior to losing this weight, including hunching, respiratory distress, excessive fur ruffling, or sluggish/unsteady movement, were euthanized by CO2 inhalation followed by cervical dislocation. Δkcs1 mutant-infected mice were sacrificed 50 days postinoculation. Differences in survival were determined with SPSS version 21 statistical software, using the Kaplan-Meier method (log rank test), where a P value of <0.05 was considered statistically significant.
For calculating lung and brain infection burdens and determining lung cytokine profiles, 3 from a group of 9 mice infected with a particular strain (the WT or Δkcs1 mutant) were sacrificed 3, 7, and 14 days postinoculation. For lung cytokine analysis, 3 mock-infected mice sacrificed on days 3, 7, and 14, respectively, served as controls. For determining infection burdens, lungs and brains were harvested from infected mice, weighed, and homogenized in a total of 1 ml sterile PBS. One hundred microliters of each homogenate was removed and serially diluted 10-fold in sterile PBS, and 100 µl of each dilution was plated onto SAB agar. The plates were incubated for 2 to 3 days at 30°C, and the number of CFU was determined. The remainder of each lung homogenate (900 µl) and 900 µl of lung homogenate obtained from each of the control mice were centrifuged at 10,000 × g to pellet tissue debris, and the supernatants were collected and frozen for cytokine analysis.
Cytokine profiling in infected lung supernatant.Cytokine concentrations in lung homogenates collected postinfection were determined using mouse Th1/Th2/Th17 cytokine bead arrays (CBA; Becton, Dickinson Biosciences). MCP-1 was measured in a separate assay using the mouse MCP-1 flex set (bead B7) (BD Biosciences). CBA kits were used according to the manufacturer's instructions, data were collected using an LSRII flow cytometer, and results were analyzed using FCAP software version 3.0 (BD Biosciences).
Virulence in Galleria mellonella.C. neoformans WT and Δkcs1 mutant cells were grown overnight in YPD broth, pelleted by centrifugation, and resuspended in water at a concentration of 108 cells/ml. G. mellonella larvae (10 per strain) were inoculated with 10 µl of cell suspension (106 yeast cells) by injection into the hemocoel via the lower prolegs. The viability of each inoculum was assessed by performing serial 10-fold dilutions, plating the lowest dilutions on SAB agar plates, and counting the CFU after a 3-day incubation at 30°C. Inoculated larvae were monitored daily for 10 days after daily removal of their cocoon. Larvae were considered dead when they no longer responded to touch (68).
Phagocytosis experiments.THP-1 cells were maintained in RPMI 1640-10% fetal bovine serum (FBS) medium. IFN-γ (10 ng/ml) was added 24 h prior to the phagocytosis experiment to activate the monocytes. Cryptococcal cells were grown overnight in YNB broth, pelleted by centrifugation, and resuspended in staining solution (0.5 mg/ml FITC in PBS) for 30 min at 30°C. To heat kill fungal cells in some of the experiments, YNB cultures were centrifuged and the cell pellets were incubated at 56°C for 30 min prior to FITC staining. Following FITC staining, fungal cell pellets were washed three times with PBS and opsonized for 30 min at 30°C with human serum (10 to 100%, as indicated in the figures) or human IgG (Sigma-Aldrich I4506) or anti-GXM antibody 18B7 (a kind gift from Arturo Casadevall) in PBS (69). Opsonized fungal cells were pelleted and mixed with THP-1 monocytes in a ratio of 3:1 or 5:1 in THP-1 growth medium. Fungal and mammalian cells were coincubated for 3 to 5 h at 37°C in 5% CO2. To quantify the adhesion/uptake of the fungal cells by THP-1, the cultures were mixed by pipetting, diluted with PBS, and analyzed by flow cytometry and microscopy. Reverse transcription-PCR (RT-PCR) was used to assess the THP-1 activation response to WT and Δkcs1 cells after 4 h of coincubation, using an RT2 profiler PCR array designed for analysis of the antifungal immune response (SABiosciences). PCR was performed using a QuantStudio6 Flex real-time PCR system (Life Technologies). The expression data were normalized to the expression of the housekeeping GAPDH, ACTB, B2M, HPRT1, and RPLPO genes and then calculated as the fold change from expression in activated THP-1 cells that had not been coincubated with cryptococci.
Peripheral blood monocytes (PBMCs) were isolated from fresh human blood using a Ficoll-Paque gradient. Up to 20 ml of blood was layered on top of 15 ml of Ficoll-Paque and centrifuged at 400 × g for 30 min. The interphase containing monocytes and T and B lymphocytes was collected in a fresh tube and washed twice with PBS before resuspension of the final pellet in RPMI 1640-10% FBS medium. PBMCs were visualized with crystal violet stain and counted using a hemocytometer. PBMCs (4 × 106) were mixed with 4 × 106 heat-killed, FITC-labeled, 10%-serum-opsonized cryptococci in 100 µl of RPMI 1640-10% FBS medium and coincubated for 2 h at 37°C in 5% CO2. The cell pellets were washed once with PBS. Nonspecific Fc receptor-mediated antibody binding was blocked before cells were stained with antigen-presenting cell (APC)-H7-conjugated anti-CD14 antibody (monocyte marker) (BD Biosciences; catalog number 560180). Adhesion/uptake of cryptococci by the CD14-positive cell population was quantified by flow cytometry and visualized by microscopy (70, 71; see Text S1 in the supplemental material).
Mannoprotein staining with ConA-FITC.Cryptococcal cells were grown overnight in YNB, washed with PBS, and resuspended in 100 µl PBS supplemented with 0.5% gelatin at an OD600 of 0.5. Each suspension was incubated for 30 min at room temperature. Ten microliters of ConA-FITC (Sigma; C7642) from a 2.5-mg/ml stock solution in water was added to each culture sample, followed by incubation for 30 min at room temperature. The extent of ConA-FITC binding was assessed using flow cytometry.
High-throughput gene expression analysis.Fungal strains were grown in YPD broth for 22 h. RNA was isolated using TRIzol and further purified using a Qiagen RNA isolation kit. Library preparation and sequencing were carried out by the Ramaciotti Centre for Genomics (UNSW, Sydney, Australia) using a TruSeq sample preparation kit and the Illumina HiSeq2000 sequencing platform to generate 100-bp paired-end reads. The resulting data were analyzed using the Galaxy platform (https://usegalaxy.org/). The analysis included data from three biological replicates for each strain; a ≥2-fold difference in gene expression between WT and Δkcs1 cells was considered to be significant. The presence of a signal peptide and N- and O-linked glycosylation were predicted using CBS prediction tools (http://www.cbs.dtu.dk/services/). The presence of a GPI anchor was predicted using the Big-PI fungal predictor (http://mendel.imp.ac.at/sat/gpi/fungi_server.html). Initial functional classification of cryptococcal genes was performed using GO term assignment from the Joint Genome Institute (JGI) database (http://genome.jgi.doe.gov/cgi-bin/ToGo?species=Cryne_ H99_1) and further improved based on the literature and by performing a homology search. A hypergeometric test was used to assess whether Kcs1-dependent genes were overrepresented in specific functional groups.
Table S2
Copyright © 2015 Lev et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
This work was supported by funding from a National Health and Medical Research Council of Australia project grant (APP1058779) and by Bioplatforms Australia through the Commonwealth Government National Collaborative Research Infrastructure Strategy (RNA-seq). A.S. is supported by the Medical Research Council (MRC core support to the MRC/UCL Laboratory for the Molecular Cell Biology University Unit [grant MC_U122680443]), and T.C.S. is a Sydney Medical School Foundation Fellow.
We thank Ana Traven for helpful suggestions and Robert Lev for generous help with the RNA-seq data analysis. We also thank Virginia James and the Histology Department at WMIMR for performing mouse lung histology, Christabel F. Wilson for help with the animal infection studies, the WMI genomics facility for assistance with qPCR, the WMI flow cytometry and microscopy facilities for assistance with phagocytosis studies, and the sequencing team at the Ramaciotti Centre for Genomics (UNSW, Sydney, Australia).
- Received 14 April 2015
- Accepted 4 May 2015
- Published 2 June 2015
- Copyright © 2015 Lev et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.


text:
Checking for Matches…
3 years 8 weeks ago
|Single by Tenacious D
|from the album Tenacious D
|July 16, 2002
|The Boat in Silver Lake, Los Angeles
|Hard rock, heavy metal, comedy rock
|Jack Black, Kyle Gass
|The Dust Brothers
|Tenacious D singles chronology
"Tribute" is the first single of Tenacious D's self-titled debut album. It was released July 16, 2002. The song is a tribute to what Gass and Black refer to as "The Greatest Song in the World" (often confused as the song's title),[1][2] which Tenacious D themselves came up with, but have since forgotten.[3] It was released as a downloadable track for Rock Band in addition to appearing as a playable track for Guitar Hero Live.
- 1 History
- 2 Synopsis
- 3 Music video
- 4 Reception
- 5 Track listings
- 6 Chart performance
- 7 Personnel
- 8 See also
- 9 References
Tribute was the first song Black and Gass played live as Tenacious D.[4] The song, like many other songs that were recorded on Tenacious D, was originally played on the TV series.[5] During earlier performances of this song Kyle Gass played the opening to "Stairway to Heaven".[6] The two songs are both in A minor and have very similar chord progressions, and critics have said the songs sound alike.[1][7][8] A slightly different version of the song first appeared on the band's TV show on HBO.
The song chronicles the band members' encounter with a demon who demands the duo play "the best song in the world" or have their souls eaten. Having nothing to lose from trying, they play "the first thing that came to our heads", and it "just so happened to be the best song in the world."
Given the "Stairway to Heaven" interlude in the original TV series version, along with the similarity of the chord progression in both songs, Tribute at first implies that the best song in the world is indeed that song. However, the lyrics make clear that Tribute sounds nothing like the song they came up with to please the demon; as Black describes: "And the peculiar thing is this my friends: The song we sang on that fateful night it didn't actually sound anything like this song."
In an interview the band claimed that the inspiration from the song came after Jack Black played Metallica's "One" for Kyle Gass, describing it as "the best song in the world",[9][10] leading to a failed attempt to themselves write an even better song, and a discussion of the meaninglessness of labeling any song that way. Tribute was written as a paean to the impossibility of reaching musical perfection, making the claim that the greatest song in the world had in fact been theirs, but that they had forgotten it.
In Tenacious D in The Pick of Destiny, it is implied that the climax of the movie, a rock-off challenge between Satan and Tenacious D told through the song Beelzeboss (The Final Showdown), is the incident chronicled in Tribute, and that the portion of Beelzeboss performed by Tenacious D is the song receiving tribute. After defeating Satan, Jack and Kyle recall the incident once more, describing it as, "the greatest song in the world". Unfortunately, they are unable to remember how it went. Also in the song Beelzeboss, they mention that they know the demon's weakness, referred to as their "rocket sauce", which is what they use in their HBO television series to defeat the demon. However, unlike the demon in the television episode, Satan says they are "Fuckin' lame" and tries to take Kyle Gass to hell after all, while the demon asks if they are angels. However, considering that they do not remember the song, they may have forgotten this as well. This was confirmed in a commentary with Jack Black for the movie.
Music video
The music video for "Tribute", directed and edited by Liam Lynch, features Jack Black and Kyle Gass, in a cheap shopping mall karaoke booth, rewired to record their tribute to their encounter with a demon and the song they sang for him, which saved their lives.
The video cuts between footage of the pair walking down the "long, lonesome road" and the encounter with the demon played by Dave Grohl. The demon also performs the electric guitar solo in the music video. After the solo, Black and Gass jump out of the booth and start shouting the lyrics at mall shoppers and dancing flamboyantly. Passersby include cameos from Lynch and Ben Stiller. While most pay no attention, an old lady, played by Linda Porter assaults Black with her purse after he grabs her while singing. A policeman, played by JR Reed, who played "Lee" in the band's television series, takes Black and Gass away. At the end the same old lady who was frightened by the duo picks up the now-recorded CD, eyes glowing red, and cackles demonically.[11]
Although the video had huge success on UK television, the song was never actually released there as a single. "Tribute" was nominated for two Music Video Production Association Awards: "Alternative Video of the Year" and "Directorial Debut of the Year".[12] In addition, it was a nominee for best video in the 2002 Kerrang! Awards.[13]
Some critics described the plot of the song as being similar to the song "The Devil Went Down to Georgia" by the Charlie Daniels Band.[1][7][14]
Track listings
- CD single
- "Tribute" – 4:08
- "Jesus Ranch (demo)" – 2:14
- CD single
- "Tribute" – 4:08
- "Cosmic Shame (live)" – 4:28 Recorded at the Cox Arena in San Diego on November 20, 2001.
- "Friendship" – 2:00
- "Tribute (Video)" – 4:08
- "Wonderboy (Video)" – 4:07
- Australia and New Zealand CD single
- "Tribute" (Radio Edit) – 4:08
- "Cave Intro" – 0:46
- "Jesus Ranch (demo)" – 2:14
- "Kyle Quit the Band (demo)" – 2:10
- "Explosivo (Mocean Worker's Megamix)" – 5:02
- Promotional CD
- "Tribute" – 4:08 (Radio Edit)
- Jack Black (vocals/acoustic guitar)
- Kyle Gass (backing vocals/acoustic guitar)
- Page McConnell (keys)
- Dave Grohl (drums)
- Warren Fitzgerald (electric guitar solo)
- Alfredo Ortiz (additional drums)
- The Dust Brothers (production)
- Ken Andrews (mixer)
See also
- Devil's Trill Sonata
- Best Song Ever (song), which also revolves around an unnamed best song of all time
- ^ a b c Strauss, Neil (2001-08-20). "Foraging for Music in the Digital Jungle". The New York Times. Retrieved 2008-03-23.
- ^ Baltin, Steve (2004-07-07). "Phair, Grohl Sing for Dems". Rolling Stone. Retrieved 2008-03-23.
- ^ Williams, Mary (2001-08-26). "Tenacious D 'rocks' the Wiltern". dailybruin.ucla.edu. Retrieved 2008-03-23.
- ^ Kaiser, Lindsey (2006-11-21). "'Pick'ing out their Hollywood destiny". dailytrojan.com. Retrieved 2008-03-23.
- ^ Scott Thurlow, James Caverly (2003-05-22). "HBOwesome". dailytargum.com. Retrieved 2008-03-23.
- ^ Scott, Aaron. "Tenacious D". Slant Magazine. Retrieved 2008-07-04.
- ^ a b Price, Simon (2006-12-24). "Iron Maiden, Metro Radio Arena, Newcastle". The Independent. Retrieved 2008-03-23.
- ^ Anderson, Eric (2001-10-04). "The 'D' drops". fairfieldmirror.com. Retrieved 2008-03-23.
- ^ Emily Watson and Lauren Thompson (2006-11-22). "The D contemplates rock-off for soul and the perfect groupie". The Daily Texan. Archived from the original on September 30, 2007. Retrieved 2007-01-01.
- ^ JimmyO (2006-11-22). "INT: Tenacious D!". JoBlo.com. Retrieved 2007-01-01.
- ^ Video on YouTube
- ^ "News Archive". Retrieved January 24, 2007.
- ^ "The Kerrang! Awards 2002". contactmusic.com. Retrieved 2008-03-23.
- ^ Waldman, Dave (2001-09-27). "Tenacious D makes move into music". gwhatchet.com. Retrieved 2008-03-23.
- ^ "Nederlandse Top 40, week 8 2003" (in Dutch). Top40.nl. Retrieved 2009-07-26.
- ^ a b "Tenacious D". australian-charts.com. Retrieved 2008-02-14.
- ^ "Discography Tenacious D". Charts.org.nz. Retrieved 2008-02-14.
- ^ "Top 50 Singles Chart #1333". Recording Industry Association of New Zealand. Retrieved 2008-01-03.

subset: commoncrawl


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May 22, 2018. <a href="http://acnegone.wpengine.com/does-alcohol-cause-acne" target=" _blank". Congeners in white wine are known as tannins.
Dec 12, 2015. Best Alcoholic Drinks if You Have Acne. in the nasolabial folds; Red wine (and some white wine) DOES contain antioxidants like resveratrol,
Dec 12, 2017. But there is a way to catch a buzz without ruining your face. it can go on to cause inflamed skin papules (lesion-like bumps) and cystic acne. Omarosa Secretly Recorded Her Firing in the White House Situation Room. (note that we aren't saying "the best for you") is probably red wine, says Spizuoco.
Collagen Cream For Acne – Natural Skin Care Products Philippines Collagen Cream For Acne Best Skin Care Product For Asian Skin Best.
May 25, 2017. See how drinking wine and alcohol affects skin and what you can do about it!. some types can result in visible blood vessels, swelling, acne-like breakouts, It's been widely understood that red wine can cause flare-ups in.
However, modern research has revealed that it does indeed contain important compounds that can benefit your health. Polypheno.
Ever heard that drinking alcohol can result in ruddy skin splattered with pimples? The rumors aren't all they're cracked up to be – that glass of wine isn't going to.
Apr 26, 2017. But now I can't even have six glasses a glass of wine anymore. that it can also cause face swelling, "acne-like breakouts", bumpy skin, and.
The food you eat—from wrinkle-fighting antioxidants in fruits and vegetables to hydrating healthy fats in fish—may matter to your skin almost as much as it does to your waistline. Olive oil, tomato.
White wine came out the worst — they found that just a couple of glasses per month was associated with an 14 percent increased risk of developing the skin condition by 14 percent. Kick that number up to five or more glasses of Moscato a month and you're looking at a 49 percent increased chance of developing rosacea.
Apr 21, 2017. Researchers found that consumption of white wine and liquor posed the. Rosacea is a chronic disease that causes redness and flushing of the skin, Science has identified many factors that may potentially cause rosacea, and. Acne may be caused by an imbalance of skin bacteria Researchers may be.
White wine may increase risk for rosacea in women, study says. "In the past, people thought red wine would cause more flushing than white wine," she said.
A ton of research has been done on what exactly causes acne but the findings are somewhat unclear. However, while white bread, white pasta,
Not only does it act as a skin brightener, but its powerful anti-inflammatory and antioxidant properties are useful in soothing acne. helps remove plaque which causes discolouration and can prevent.
Nov 24, 2017. Of course, I always gleefully click on any story about how red wine actually. of course, I started whining as if abstaining from alcohol had caused my acne. He did tell me that a sudden change in my gut bacteria (ew) could.
Negative and unpleasant effects that may be caused by the common food: allergy, bad breath, body odor, constipation, heartburn, stress, acne, diarrhea and more.
Oct 4, 2017. There are so many ways alcohol can affect nearly every part of your body, your life and ultimately your appearance. Often the effects of alcohol.
The Best And Worst Drinks For Your Skin. substances as well as the toxins they cause — and most of. a bottle of water and a glass of red wine.
Acne is a skin condition that is known to affect teenagers, though it can in fact occur at any age. The primary symptoms of the condition include red lumps on the skin that are filled with pus and can cause severe irritation.
Natural treatment for four types of PCOS including insulin resistance, post-Pill PCOS, inflammatory, and hidden cause PCOS.
Mar 4, 2018. White wine's high sugar content can lead to inflammation and. and laying a breeding ground for acne-causing bacteria to clog your pores.
Red Wine and Acne. As. regardless of the cause of your acne, red wine can aggravate them. Red wine does not automatically cause a person to develop acne.
White vinegar works in treating acne because it is highly acidic. Once applied to the skin, it makes it acidic as well, which is something the bacteria that cause acne dislike. Because the pH balance of your face changes, the.
10 Myths and Facts About Adult Acne. "The idea that chocolate and caffeine cause acne has never really panned out.". WebMD Feature Reviewed by Brunilda.
Causes Of Pimples On Neck And Chest Infected hair follicles or pimples are clogged with bacteria, white blood cells and pus. The mixture of white blood cells, bacteria and pus is called plaque. Sep 6, 2017. Acne is a common and treatable skin condition that causes pimples or "zits". back, and chest; however, you can also have acne on your neck, Acne
Stress is hard on your body and can cause puffy eyes and acne, so if you need a beauty motivation to manage. Focus on what makes you feel beautiful. Does getting your eyebrows shaped and waxed make.
Browse the WebMD Questions and Answers A-Z library for insights and advice for better health.
Alcohol does inflict a bit of damage to our skin. There is a surprising indirect connection between alcohol and breakouts, and drinking copious amounts of alcohol will lend itself to sallow, unhealthy skin with a large side of pimples. Why do alcoholic beverages affect our skin so much? Alcohol and Acne. Your diet and acne are related.
There is a growing question: Does alcohol cause acne outbreaks? Like all things related to acne, alcohol will affect different people in a different ways. Drinking.
Red Wine Found Top Alcohol Trigger for Conspicuous Facial Condition
Acne and rosacea. Acne; 10 things to try. Does drinking cause rosacea?. Women who drank white wine or hard liquor had a greater risk of developing rosacea than.
White Vinegar for Acne. In This Article. Acne bacteria cause deep tissue damage in the skin, White vinegar does not produce immediate results in acne treatment.
Oct 24, 2013. White wine, like mixed cocktails and beer, contains sugar, in addition. "Red wine can also cause histamine release in some people, leading to.
Acne and rosacea. Acne; 10 things to try. Does drinking cause rosacea?. Women who drank white wine or hard liquor had a greater risk of developing rosacea than.
For example, a National Rosacea Society survey found that red wine was a trigger for more than 72 percent of people asked, white wine for 49 percent, and beer for 42 percent.
Jul 18, 2016. Does alcohol cause acne and pimples?. White wine – widely considered to be inferior to red wine, and the research backs it up; red wine.
Experts conclude white wine and alcohol lack any anti-inflammatory components. Rosacea (an inflammatory condition) might benefit from the antioxidants in red wine over the long term. (Hmmm, I like that.) On the reverse side, red wine has more histamine and inflammatory factors which might lead to flushing. It looks like chard, pinot, cab, wine.
Feb 21, 2018. How alcohol can indirectly cause or worsen acne. White wine may not cause hangovers as severe as its red counterpart, but it can still.
Feb 12, 2018. There is a lot of confusion in regards to benefits of red wine. Is it or. Red Wine Vs White Wine: Which Is Healthier?. The resveratrol in red wine can slow down the growth of bacteria that cause acne, as per a recent study.
More Acne Causes Articles ...
- List Of Dairy Products That Cause Acne: Mar 3, 2015. Glycemic load food chart: foods that cause acne. a plenty of other important stuffs to pay attention to, so why bother to add one more item to that list?. Particularly, the role of milk or dairy products requires a closer scrutiny. Milk ...
- Can Soy Lecithin Cause Acne: Soy (The Meat of the Issue) • Performing Woman – Feb 25, 2013. When you turn it into tofu, you can use the flavorless block in so many different ways–from stir-fries to smoothies!. The third was the acne. In other words, with every scoop of soy prote...
- Does Lithium Carbonate Cause Acne: Feb 25, 2011. Smoking may cause cancer development in the mouth alone, on the throat alone. How Much Does Lithium Carbonate 90 300 mg 68.69$ In Oklahoma. vitamins needed to fight acne causing agents Lithium Carbonate 300. Do mood stabilizers have sid...
- Does Protein Powder Causes Pimples: Taken daily as part of a total body wellness plan, protein powder can be a. This can help to repair the damage caused by ailments or a low protein diet and. taking a protein powder supplement, making it ideal for those who suffer from acne. Aug 26, 2...


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Mar 7, 2020
On SATURDAY, MARCH 7, 2020 we invite you to join at Rentschler Field in East Hartford at the 3rd Annual Divinely Fit Summit from 8:30 am to 3:00 p.m. for an epic experience with our ambitious movement. This is a full day event (8:30 am -9:00 am registration) dedicated to empowering women and men to discover a transparent spiritual, mental and physical connection with themselves and others through fitness. With Special Guest and Presenter, Billy Blanks the creator of Tae Bo® and Dr. T. Charles Brantley the Relationship Trainer.
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I posted some comments on a recent climate-related study but for some ignorant reason put in the "engineering and applied sciences" category (which most posters seem to ignore). I never even noticed his forum, which would have been much more appropriate.
It's the kind of topic that usually generates some "interest" (often of the highly emotionally charged variety), so I thought I'd call some attention to it here, too, just in case anybody else has some comments.
Edited by Moronium, 03 February 2019 - 08:51 PM.


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How NOT to talk to women on Fetlife
Ok, before I give you a giggle at some of the more ridiculous private messages I've got over the past month on Fetlife, I think it's important to note that my profile clearly says in HUGE and clear writing that I'm in a monogamous relationship with my submissive and that I'm on Fetlife to share ideas and thoughts, not to engage in intimate conversations or seek any more submissives. You can see that I've replied to some in a desperate attempt to at least make them think before PMing, and others I've just… well, they're probably lost causes.
I've blocked any names and images for privacy reasons.
I'm here to educate, not to mock. I think my next blog should most definitely be about "How to engage in a conversation with a female dominant". …. I could even change that to "How to engage in conversation with any sane female"..? Surely it can't be so difficult? I guess it is, considering that probably 9 out of 10 messages I get on Fetlife are like this. No I'm not joking.
If you have some suggestions for social interaction via BDSM sites, please feel free to send them to me.
Pampering my sub
I wanted to do something special for my sub last night. I spent the whole day planning something nice for him to eat – he'd chosen to have his favourite food: chicken breast stuffed with cream cheese and pesto and wrapped in bacon and garlic fries. I had that and a cup of tea (he is a major tea addict!) and a nice warm fluffy pink dressing gown ready for him when he got home.
It was a really miserable wet day outside, he came in looking a bit damp and tired. I helped him out of his clothes and into his cosy dressing gown and sat him down at the table. We had our candlelit dinner and listened to some Opeth. It was pretty perfect. He seemed to thoroughly enjoy the meal.
After that I gave him some time to relax – we watched some scifi and played some videogames together. I went down to have a shower and let him have some time to himself.
About an hour before bed I asked if he'd like to go downstairs. He agreed. I lay him down on the bed and gave him a nice sensual massage. This was rudely interrupted by some terrible songs on his random playlist on Spotify which was pumping out some of the most unrelaxing music … almost ruining the mood! He flopped about a bit and eventually changed it to Marilyn Manson's "Mechanical Animals"…. which surprisingly was perfect for my intention. It's kinda slow and sexy.
I massaged each part of his body with scented oils and teased his cock a little – not too much. Just enough to keep him on edge for a little bit…. the teasing only lasted about 20 minutes. I could see him tiring and aching so I let him cum and he shivered around the bed for a little bit. He ended up lying there, with his eyes closed. Huge smile on his face. I felt a deep feeling of satisfaction warming my body as I looked down at him. I cleaned him up and we cuddled for a little bit. Then it was time for sleep.


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Find out more
For more than 20 years SportBusiness has been delivering the data, information, insight and analysis that make the world's most successful sports sector businesses tick.
The Ligue Nationale de Rugby last week secured an increase of 4.2 per cent in the value of the media rights of the top-tier domestic league, the Top 14, despite limited competition.
Canal Plus and Eurosport are battling it out over the pay-television rights to French rugby union's Pro D2, with the existing rights fee set to treble.


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The Guardian
- Details
- Hits: 4185
The Guardian
Our Master Muhammad [s] was 8 years of age when his grandfather Abdul Muttalib died. So, his uncle, Abu Talib took care of him.
Abu Talib's name was Abdul Munaf. His mother's name was Fatima bint Amru. She belonged to the Bani Makhzum tribe. Abu Talib was named Shaikh al-Bat-ha.
Our Master Muhammad [s] lived with his uncle. His uncle treated him kindly. Fatima bint Asad, his uncle's wife, treated him kindly, too. She preferred him to all of her sons. So, our Master Muhammad [s] grew with a good family.
Abu Talib's love for his nephew increased day by day because of his good manners and great politeness.
For example, when the food was ready, the orphan boy reached it politely. He said:
In the Name of Allah!
When he finished, he said:
Thank Allah!
One day, Abu Talib missed his nephew Muhammad. So, he raised his hand from the food and said:
"Till my son comes!"
When Muhammad came, Abu Talib gave him a cup of yogurt. Muhammad drank out of the cup. Abu Talib's sons drank out of it one by one, too. They were all full. The uncle admired that. So, he said to his nephew:
"Muhammad you're blessed!"


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Today is the day I have decided to take charge of my bad habits and begin down the road to good credit. I am a full-time student and a part-time worker and I'm graduating this May. I have a job lined up and will be making 4x what I make now. I've made some silly mistakes, basically spending way more than I make, etc. I have no derogatory marks on my report so I think that means the process of rebuilding my credit might not be so bad afterall. I have found the advice on these forums very helpful and will be using this community now and in the future for advice and as a source of information!
Here's what I'm up against:
American Express Costco TrueEarnings: $ 904/1000
Discover It Student Card: $ 941/1200
Capital One QuickSilver: $ 747/750
Capital One Platinum Mastercard: $ 805/1000
Chase Slate: $ 989/1000
Amazon Store Card: $ 680/1700
TU: 597, Experian: 602, Equifax: 579
My goal is 680+ 90 days after the start of my new job. I'll be paying these down ASAP.
Credit Repair Tyler, TX – Fast Credit Repair Across America


text:
Office of the Governor
State Capitol, 1900 Kanawha Blvd. E
Charleston, WV 25305
Office Phone:
304.558.2000 or 1.888.438.2731
Governor's Mansion:
Contact the Governor's Office
By clicking "submit" you agree that any information provided is considered a public record and is subject to disclosure.
Banner Images Courtesy of the West Virginia Department of Commerce


text: COVID-19 Economic Recovery Resource Portal for Business Alaska and the Nation are pulling together to face this health and economic challenge. On this page, you will find updates and links to resources to help weather these hard economic times and prepare for the recovery thereafter. Updates will be made as additional information and resources become available. Latest News AK CARES Grants to Small Businesses The Alaska Department of Commerce, Community, and Economic Development (DCCED) and the Alaska Industrial Development and Export Authority (AIDEA) have partnered with Credit Union 1 to provide relief to small Alaskan businesses impacted by the COVID-19 pandemic through the AK CARES Grant program. AK CARES will be operated on a first-come, first-served basis. The application will become available on June 1st; the same date Credit Union 1 will begin accepting applications. Prior to that date, businesses interested in applying are encouraged to review the checklist and FAQs provided on the AK CARES Grant website, as they list the information necessary for the application package. For more information, and to view the checklist and FAQs, please visit the AK CARES Grant website. Paycheck Protection Program "Safe Harbor" Provision The Small Business Administration and Department of the Treasury have recently determined that any borrower, together with their affiliates, receiving a Paycheck Protection Program loan whose principal amount is less than $2 million will be deemed as having met the PPP's good faith certification requirement. Borrowers with loans whose principal is greater than $2 million may still meet the PPP's good-faith certification requirement; however, such loans will be subject to review by the Small Business Administration. For more information this new "safe harbor" provision in the PPP, please see questions 46 and 47 of Small Business Administration's May 13 PPP FAQ Release. SBA Making Economic Injury Disaster Loans Available SBA to Make Economic Injury Disaster Loans Available to U.S. Agricultural Businesses Impacted by COVID-19 Pandemic The Small Business Administration's Economic Injury Disaster Loan (EIDL) portal has reopened with an additional $10 billion in funding which is now also available to Agricultural Business Concerns. Apply now if you are a small business in need of up to $10,000 in 100% forgivable loan advances available through the Emergency EIDL Advance program. Coronavirus Aid, Relief and Economic Security Act The Department of Commerce, Community, and Economic Development is working to understand the fast paced and ever-changing effects of COVID-19 on Alaska and our communities. We have endeavored to provide up to date information via our Commerce web site. On April 2, 2020 the Small Business Administration released the interim final rule for the Small Business Administration Business Loan Program Temporary Changes: Paycheck Protection Program (PPP). Please see for details Paycheck Protection Program SBA provides guidance on how small businesses can apply for the Paycheck Protection Program Alaska Approved Lenders AIDEA Creates Billion Dollar Loan Guaranty Program (Anchorage) – The Alaska Industrial Development and Export Authority (AIDEA) Board on Friday unanimously approved a resolution that creates in AIDEA the Sustaining Alaska's Future Economy Guaranty Program (AK SAFE). This program will confront the economic crisis resulting from the COVID-19 pandemic, which threatens Alaska businesses and the people who work in these businesses. Read full press release here SBA Guide to CARES Act The Small Business Administration has prepared a Guide for Small Business Owners to the Coronavirus Aid, Relief, and Economic Security (CARES) Act. The guide provides information on SBA programs including the Paycheck Protection Program, the Small Business Debt Relief Program, Economic Injury Disaster Loans, and Emergency Economic Injury Grants. To read the final enabling language click here Alaska Critical Workforce Infrastructure The Alaska Critical Workforce Infrastructure page contains a listing of Alaska's critical workforce infrastructure as first published in Attachment A to Health Mandate 10.1 - International and Interstate Travel - Order for Self-Quarantine. If you would like to review this mandate, or any other health man-dates issued by the State of Alaska, they can be found on the Governor's website or the COVID-19 Response website for the Alaska Department of Health and Social Services. Governor's Health Mandate 10 All people arriving in Alaska from international locations or travel from other states, whether resident, worker or visitor, are required to selfquarantine for 14 days and monitor for illness. Arriving residents and workers in self-quarantine, should work from home, unless you support critical infrastructure. Please see for details SBA Implements Automatic Deferment on Existing SBA Disaster Loans through the End of 2020 Borrowers of home and business disaster loans do not have to contact SBA to request deferment. Deferments through December 31, 2020 will be automatic for existing Disaster Loans. For more information visit the SBA website. Resources for Business In addition to the working capital loans now available through the Department of Commerce, Community, & Economic Development, a myriad of State and Federal resources are available to support commerce as Alaskans continue to do what needs to be done to contain the spread of COVID-19 and begin the process of recovery. A directory of business-focused organizations able to assist in this recovery effort is available on the Business Response Directory. Below are links to listings of available State and Federal resources for businesses navigating their way through the COVID-19 landscape. Updates will be provided to these listings as access to new resources are made accessible. If you have any recommendations on resources not already listed, please contact the Alaska Development Team at email@example.com. Emergency & General Assistance Grants Emergency & Business Assistance Loans Tax Incentives Benefits Business & Travel Alerts / Documentation In accordance with Health Mandate 10, all travelers to Alaska, resident or otherwise, are required to undergo a 14-day self-quarantine to monitor for illness upon arrival into the state. Additionally, businesses identified as being part of critical infrastructure industries are required to submit protocols to the Department of Commerce, Community, & Economic Development as to how they intend to contain the potential spread of the COVID-19 virus as well as keep their employees and the communities in which they operate safe during this public health emergency. Below are a series of links to documents and templates to help individuals and businesses comply with these requirements. Governor's Health Mandate 10 Alaska Critical Workforce Infrastructure - Attachment A to Health Mandate 10 Travel Quarantine Alert Mandatory Travel Declaration Template COVID-19 Pandemic Action Plan Template For information on the steps being taken by the State to combat the spread of COVID-19, please visit the COVID-19 Update page available through the Department of Health & Social Services. If you would like to see information on the status the COVID-19 pandemic in Alaska or beyond, please refer to the sites listed under the COVID-19 Updates tab. Please email firstname.lastname@example.com if you have a recommendation on links to relevant state and/or federal business recovery program information.


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- 22 December 2013 / Ethnos – Andreas Dracopoulos: "It is our vision to help our country get back on its feet again"
Andreas Dracopoulos, Co-President of the Stavros Niarchos Foundation, spoke to Magda Liveri and Ethnos Sunday Edition about the Foundation's grantmaking activities, the Stavros Niarchos Foundation Cultural Center, and the importance of philanthropic giving.
- 20 December 2013 / Collaboration of the National Library of Greece with Future Library
The Ministry of Education and Religious Affairs announced the collaboration of the National Library of Greece (NLG) with the Future Library organization, which is supported exclusively by the Stavros Niarchos Foundation (SNF).
- 18 December 2013 / SNF Members at the "MED Confederation" International Conference
Members of the Stavros Niarchos Foundation will participate at the international conference examining the launch of the "MED Confederation" organized by la Caixa Foundation on December 19th and December 20th in Barcelona.
- 18 December 2013 / Greek National Opera Programming for the Period 2014-2019
On December 17th the Greek National Opera (GNO) presented its programming for the period 2014-2019. The Minister of Culture and Sports, Mr. Panos Panagiotopoulos lauded the GNO for its work and social contribution and announced the extension of the tenure of the GNO's Artistic Director, Mr. Myron Michailidis. The press conference was attended by the Mr. George Agouridis, board member of the Stavros Niarchos Foundation and Mr. Ioannis Trohopoulos, Managing Director of the Stavros Niarchos Foundation Cultural Center.
- 17 December 2013 / "Journey to the SNFCC" Event at the National Library of Greece
On the day of the Winter Solstice, Saturday, December 21st, at 12:00 pm, the Stavros Niarchos Foundation Cultural Center (SNFCC) is hosting an open event of literature and art at the main reading room of the National Library of Greece (NLG), in collaboration with renowned Greek artists.
- 17 December 2013 / The Prefecture of Lakonia and DIAZOMA honor the Stavros Niarchos Foundation
On Wednesday December 18th the Prefecture of Lakonia and DIAZOMA are holding a special event in Sparti in honor of the Stavros Niarchos Foundation. The SNF supported the study DIAZOMA carried out for the restoration of the ancient theater of Sparta.
- 13 December 2013 / CTY Presentation in Larisa
On December 17th, 2013 the Center for Talented Youth (CTY) will hold a presentation for the local community of Larisa.
- 13 December 2013 / CSIS Event – Greece's European Council Presidency: An Opportunity to Expand Transatlantic Dialogue
On Friday, December 13th, the Center for Strategic and International Studies (CSIS) organized an open discussion on the topic, "2014 – Greece's European Council Presidency: An Opportunity to expand Transatlantic Dialogue."
- 10 December 2013 / 1st Crisis Observatory Annual Conference
On December 13th, 2013 the Crisis Observatory, an initiative of the Hellenic Foundation for European and Foreign Policy (ELIAMEP) supported exclusively by the Stavros Niarchos Foundation, is organizing its 1st Annual Conference on "Austerity and Beyond: Politics and Policies for the Crisis in Greece and the Eurozone".


text:
Hardware components
Software apps and online services
So Lets Start
You Will Need Some Components
You Can Buy all Components From Amazon
Utsource is Very Popular Electronics Components Company..
- Arduino Nano
- 16×2 LCD module
- Resistance 10k
- Adapter 5V
About This Project
This is an automatic Transistor Tester, to identify pinout and characteristics of various discrete semiconductors ( Transistors NPN, PNP, ). It's also evaluating resistors, capacitors, inductances, etc... The original project was written in a mix of C and AVR Assembly Code for AVR gcc tooling AVR Studio.
Most Arduino UNO Starter Kits contain the necessary components ( UNO R3 board, 1602 LCD, push buttons, Breadboard, Jumper wires, resistors ).This project makes also Arduino open source, open hardware philosophy look better, because the previously.Hex files for Arduino UNO and MEGA 2560 provided from the Transistor Tester original project was not putting Arduino in a good light ( UNO version was provided without LCD,
How to Use ?Connect component to test to any TP1, TP2, TP3 (component can be Transistors NPN, PNP, FET, Resistors, Capacitors, Diodes, LED and many more.)Warning: TEST only capacitor you have discharged before !
NextPCB is reputed as one of the most popular PCB manufacturers in china. Situated at the heart of Shenzhen, NextPCB has been in the PCB manufacturing and assembly industry for over 15 years, providing some of the most innovative printed circuit boards, with assembly technologies of the highest quality standards, fastest delivery turnaround (as fast as 24hours), lowest manufacturer direct prices, and support via dedicated customer service professionals.
Using some of the latest machines and equipment, NextPCB provides two major services; PCB Manufacturing and PCB Assembly.
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