Combined inactivation of the Candida albicans GPR1 and TPS2 genes results in avirulence in a mouse model for systemic infection

Inhibition of the biosynthesis of trehalose, a well-known stress protectant in pathogens, is an interesting approach for antifungal or antibacterial therapy. Deletion of TPS2, encoding trehalose-6-phosphate (T6P) phosphatase, results in strongly reduced virulence of Candida albicans due to accumulation of T6P instead of trehalose in response to stress. To further aggravate the deregulation in the pathogen, we have additionally deleted the GPR1 gene, encoding the nutrient receptor that activates the cyclic AMP-protein kinase A signaling pathway, which negatively regulates trehalose accumulation in yeasts. A gpr1 mutant is strongly affected in morphogenesis on solid media as well as in vivo in a mouse model but has only a slightly decreased virulence. The gpr1 tps2 double mutant, on the other hand, is completely avirulent in a mouse model for systemic infection. This strain accumulates very high T6P levels under stress conditions and has a growth defect at higher temperatures. We also show that a tps2 mutant is more sensitive to being killed by macrophages than the wild type or the gpr1 mutant. A double mutant has susceptibility similar to that of the single tps2 mutant. For morphogenesis on solid media, on the other hand, the gpr1 tps2 mutant shows a phenotype similar to that of the single gpr1 mutant. Taken together these results show that there is synergism between Gpr1 and Tps2 and that their combined inactivation results in complete avirulence. Combination therapy targeting both proteins may prove highly effective against pathogenic fungi with increased resistance to the currently used antifungal drugs.     

Trehalose hydrolysis is not required for human serum-induced dimorphic transition in Candida albicans: evidence from a tps1/tps1 mutant deficient in trehalose synthesis

Exponential yeast-like cells of a Candida albicans wild-type strain exhibited strong capacity for germ tube formation in a glucose-containing medium (YPD) after induction with human serum at 37 degrees C, whereas the isogenic double disruptant tps1/tps1 mutant, which is deficient in trehalose synthesis, failed to produce germ tubes. In a medium without glucose (YP), the morphological transition fraction was roughly equivalent in both strains. Substitution of glucose by galactose or glycerol increased the number of wild-type proliferating cells able to enter the dimorphic program with no noticeable change in their trehalose content, while stationary cells, which accumulate a large amount of trehalose, did not form germ tubes. When fresh medium was added, a high proportion of these resting cells recovered their ability to carry out dimorphic transition. The tps1/tps1 mutant followed the same pattern of hyphae formation, despite the fact that it was unable to accumulate trehalose either during dimorphism induction or after several stress challenges. Furthermore, trehalose-6-phosphate synthase activity was barely detectable in the mutant. These results strongly suggest that serum-induced dimorphic transition does not require trehalose mobilization; they also support the idea that TPS1 is the only activity involved in trehalose biosynthesis in C. albicans.     

Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis

The GGS1/TPS1 gene of the yeast Saccharomyces cerevisiae encodes the trehalose-6-phosphate synthase subunit of the trehalose synthase complex. Mutants defective in GGS1/TPS1 have been isolated repeatedly and they showed variable pleiotropic phenotypes, in particular with respect to trehalose content, ability to grow on fermentable sugars, glucose-induced signaling and sporulation capacity. We have introduced the fdp1, cif1, byp1 and glc6 alleles and the ggs1/tps1 deletion into three different wild-type strains, M5, SP1 and W303-1A. This set of strains will aid further studies on the molecular basis of the complex pleiotropic phenotypes of ggs1/tps1 mutants. The phenotypes conferred by specific alleles were clearly dependent on the genetic background and also differed for some of the alleles. Our results show that the lethality caused by single gene deletion in one genetic background can become undetectable in another background. The sporulation defect of ggs1/tps1 diploids was neither due to a deficiency in G₁ arrest, nor to the inability to accumulate trehalose. Ggs1/tps1 mutants were very sensitive to glucose and fructose, even in the presence of a 100-fold higher galactose concentration. Fifty-percent inhibition occurred at concentrations similar to the K_m values of glucose and fructose transport. The inhibitory effect of glucose in the presence of a large excess of galactose argues against an overactive glycolytic flux as the cause of the growth defect. Deletion of genes of the glucose carrier family shifted the 50% growth inhibition to higher sugar concentrations. This finding allows for a novel approach to estimate the relevance of the many putative glucose carrier genes in S. cerevisiae. We also show that the GGS1/TPS1 gene product is not only required for the transition from respirative to fermentative metabolism but continuously during logarithmic growth on glucose, in spite of the absence of trehalose under such conditions.     

Disruption of the Candida albicans TPS2 gene encoding trehalose-6-phosphate phosphatase decreases infectivity without affecting hypha formation

Deletion of trehalose-6-phosphate phosphatase, encoded by TPS2, in Saccharomyces cerevisiae results in accumulation of trehalose-6-phosphate (Tre6P) instead of trehalose under stress conditions. Since trehalose is an important stress protectant and Tre6P accumulation is toxic, we have investigated whether Tre6P phosphatase could be a useful target for antifungals in Candida albicans. We have cloned the C. albicans TPS2 (CaTPS2) gene and constructed heterozygous and homozygous deletion strains. As in S. cerevisiae, complete inactivation of Tre6P phosphatase in C. albicans results in 50-fold hyperaccumulation of Tre6P, thermosensitivity, and rapid death of the cells after a few hours at 44 degrees C. As opposed to inactivation of Tre6P synthase by deletion of CaTPS1, deletion of CaTPS2 does not affect hypha formation on a solid glucose-containing medium. In spite of this, virulence of the homozygous deletion mutant is strongly reduced in a mouse model of systemic infection. The pathogenicity of the heterozygous deletion mutant is similar to that of the wild-type strain. CaTPS2 is a new example of a gene not required for growth under standard conditions but required for pathogenicity in a host. Our results suggest that Tre6P phosphatase may serve as a potential target for antifungal drugs. Neither Tre6P phosphatase nor its substrate is present in mammals, and assay of the enzymes is simple and easily automated for high-throughput screening.     

Role of Trehalose Biosynthesis in Aspergillus fumigatus Development,Stress Response,and Virulence

Aspergillus fumigatus is a pathogenic mold which causes invasive, often fatal, pulmonary disease in immunocompromised individuals. Recently, proteins involved in the biosynthesis of trehalose have been linked with virulence in other pathogenic fungi. We found that the trehalose content increased during the developmental life cycle of A. fumigatus, throughout which putative trehalose synthase genes tpsA and tpsB were significantly expressed. The trehalose content of A. fumigatus hyphae also increased after heat shock but not in response to other stressors. This increase in trehalose directly correlated with an increase in expression of tpsB but not tpsA. However, deletion of both tpsA and tpsB was required to block trehalose accumulation during development and heat shock. The ΔtpsAB double mutant had delayed germination at 37°C, suggesting a developmental defect. At 50°C, the majority of ΔtpsAB spores were found to be nonviable, and those that were viable had severely delayed germination, growth, and subsequent sporulation. ΔtpsAB spores were also susceptible to oxidative stress. Surprisingly, the ΔtpsAB double mutant was hypervirulent in a murine model of invasive aspergillosis, and this increased virulence was associated with alterations in the cell wall and resistance to macrophage phagocytosis. Thus, while trehalose biosynthesis is required for a number of biological processes that both promote and inhibit virulence, in A. fumigatus the predominant effect is a reduction in pathogenicity. This finding contrasts sharply with those for other fungi, in which trehalose biosynthesis acts to enhance virulence.Aspergillus fumigatus is a ubiquitous thermotolerant mold which plays an important role in the recycling of environmental carbon and nitrogen (32, 38). A. fumigatus is also an important opportunistic human pathogen that invades the lungs of immunocompromised patients, causing a progressive pneumonia that can disseminate to the heart, brain, and other organs (21). Invasive aspergillosis is a leading cause of death in transplant and leukemic patients, with a mortality rate exceeding 50% despite the best available antifungal therapy (19). This poor response to existing antifungal therapy has led to a great interest in novel therapeutic approaches directed against this organism.In both the environment and the host, A. fumigatus is exposed to a variety of stressors. While growing in compost, A. fumigatus is commonly found at temperatures exceeding 50°C (5). Similarly, in human tissues, hyphae are subjected to nutrient deprivation and oxidative stress from host immune cells (24, 31). Little is known about the mechanisms by which A. fumigatus survives under these conditions. One possible mechanism by which A. fumigatus adapts to environmental stress is via the biosynthesis of the carbohydrate trehalose, which is involved in mediating the stress response and virulence of other pathogenic fungi such as Candida albicans and Cryptococcus neoformans (1, 22, 30, 48). The trehalose content of these fungal cells may increase up to 50-fold in response to oxidative stress, osmotic stress, and heat shock (27).Trehalose is a nonreducing disaccharide of glucose that is synthesized by bacteria, plants, insects, and fungi. In fungi, trehalose functions both as a reserve carbohydrate and as a stress metabolite (37, 39, 40). As a reserve carbohydrate, trehalose is found in vegetative resting cells and spores, where it can constitute up to 15% of the dry weight of these structures (46). It is an important source of energy in fungal development, as it is utilized in cell processes such as glycolysis, sporulation, and germination. In addition, trehalose helps the cell withstand environmental stress and nutrient limitation. Trehalose molecules protect the cell by preventing aggregation of denatured proteins and scavenging free radicals (37). Interestingly, trehalose is absent from mammalian cells, and therefore enzymes involved in trehalose biosynthesis have been considered as potential targets for antifungal therapy.In medically relevant fungi, trehalose is synthesized from glucose. First, the enzyme hexokinase converts a molecule of glucose into glucose-6-phosphate. Then, trehalose-6-phosphate synthase (Tps) catalyzes the production of trehalose-6-phosphate (T6P) from glucose-6-phosphate and a molecule of UDP-glucose. Finally, a phosphate group is removed from trehalose-6-phosphate by trehalose-6-phosphate phosphatase (Tpp) to yield trehalose (29, 42). When trehalose is required for energy, it can be recycled back to glucose through the universal enzyme trehalase (20). The enzymes involved in fungal trehalose biosynthesis have been well characterized in Saccharomyces cerevisiae. These proteins are encoded by four genes: TPS1, which encodes trehalose-6-phosphate synthase; TPS2, which encodes trehalose-6-phosphate phosphatase; and TPS3 and TSL1, redundant genes which encode a large regulatory subunit of the synthase complex (6, 15, 28, 29, 42, 44). All four proteins are tightly regulated both at the genetic level and at the protein level, and they form a protein complex to catalyze the synthesis of trehalose (6, 45).In other fungi, trehalose biosynthesis appears to play similar but not identical roles in governing growth, stress response, and virulence. In the pathogenic yeasts C. albicans and C. neoformans, the single-copy trehalose-6-phosphate synthase Tps1 is required for normal oxidative stress response and hyphal development and has a role in mediating virulence (1, 22, 30, 48). In Aspergillus nidulans, the trehalose-6-phosphate synthase TpsA is necessary for normal fungal development, thermosensitive growth and response to sublethal exposure to oxidative stress (16). In Aspergillus niger, two putative trehalose-6-phosphate synthases, TpsA and TpsB, have been identified. Inactivation of tpsA resulted in a reduction in T6P activity; however, the role of TpsA and TpsB in trehalose metabolism has not been examined (47). Importantly, trehalose biosynthesis has not been studied in A. fumigatus, which is the most important filamentous fungus causing human disease.We undertook the study of the role of trehalose biosynthesis in A. fumigatus development, stress response, and virulence. We found that the trehalose content increased in A. fumigatus hyphae throughout development and when actively growing hyphae were exposed to heat shock, but not when they were exposed to other environmental stresses. Increases in trehalose content directly correlated with elevated expression levels of two putative trehalose-6-phosphate synthase genes, tpsA and tpsB. Disruption of both tpsA and tpsB was required to prevent trehalose biosynthesis. Abolition of trehalose biosynthesis via disruption of both tpsA and tpsB affected conidial germination, thermotolerance, and high-level oxidative stress response in vitro. Surprisingly, disruption of tpsA and tpsB resulted in a strain that was hypervirulent by several measures in a murine model of invasive aspergillosis.     

The repression of trehalose transport and metabolism in Escherichia coli by high osmolarity is mediated by trehalose-6-phosphate phosphatase.

Trehalose transport and metabolism in Escherichia coli are induced by trehalose in the growth medium but only at low osmolarity. In contrast, synthesis of internal trehalose as an osmoprotectant occurs only at high osmolarity, independent of the carbon source. The synthesis of internal trehalose proceeds via the UDP-glucose-mediated transfer of glucose to glucose-6-phosphate, forming trehalose-6-phosphate, which is then hydrolysed to trehalose. We demonstrate that the inducer for the synthesis of the trehalose transport system as well as of amylotrehalase, the key enzyme in trehalose metabolism at low osmolarity, is trehalose-6-phosphate. We found that the inability to induce these proteins at high osmolarity is primarily due to activity of trehalose-6-phosphate phosphatase, the enzyme responsible for the final step in the synthesis of internal trehalose under these conditions. A gene, otsP, necessary for the synthesis of the biosynthetic trehalose-6-phosphate phosphatase, is located at min 42 closely linked to otsA/B the structural genes for the trehalose-6-phosphate synthase. There is another gene locus near 84 min on the chromosome, that we termed otsR, which is involved in the regulation of otsA/B and possibly otsP. The nature of this regulatory gene is unclear at present.     

Isolation and Biochemical Activities of Trehalose-6-Monomycolate of Mycobacterium tuberculosis

A monoester of trehalose linked at the 6-position with mycolic acids (trehalose-6-monomycolate) was isolated from the wax D fraction of virulent human Mycobacterium tuberculosis, and its biochemical action on host-cell mitochondria was studied. Trehalose-6-monomycolate showed a delayed toxicity for mice. The 50% lethal dose at 2 weeks was 452 mug. It induced in vitro a swelling of mouse liver mitochondria and uncoupled respiration and phosphorylation in the nicotinamide adenine dinucleotide pathway of the electron transport chain. The site of functional damage was located specifically at coupling site II. Mitochondrial adenosine triphosphatase was slightly stimulated by trehalose-6-monomycolate. These findings indicate that trehalose-6-monomycolate affects mitochondrial oxidative phosphorylation in a similar manner to, but to a lesser extent than, trehalose-6, 6'-dimycolate (cord factor) of M. tuberculosis.     

Role of trehalose in resistance to macrophage killing: study with a tps1/tps1 trehalose-deficient mutant of Candida albicans

Accumulation of trehalose by yeast is an important protective mechanism against different stress conditions. This study examined the effect of trehalose on several growth features, as well as its association with the intracellular survival of yeasts exposed to macrophages. A tps1/tps1 mutant and its parental counterpart, CAI4, exhibited similar growth rates and preserved their dimorphic conversion and agglutination ability. However, electron-microscopy of cell-wall architecture showed a partial loss of material from the outer cell-wall layer in the tps1/tps1 mutant. Flow-cytometry revealed that the mutant had lower auto-fluorescence levels and a higher fluorescein isothiocynate staining efficiency. When co-cultured with macrophages, a slight reduction in binding to macrophages and slower ingestion kinetics were revealed for the tps1/tps1 mutant, but these did not interfere significantly with the amount of yeast ingested by macrophages after co-incubation for 2 h. Under the same conditions, CAI4 cells were more resistant to macrophage killing than was the tps1 null mutant, provided that the macrophages had been stimulated previously with interferon-gamma. Measurement of trehalose content and the anti-oxidant activities of yeast cells recovered after phagocytosis revealed that the trehalose content and the glutathione reductase activity were increased only in CAI4 cells, whereas levels of catalase activity were increased similarly in both strains. These results suggest that the presence of trehalose in Candida albicans is a contributory factor that protects the cell from injury caused by macrophages.     

The byp1-3 allele of the Saccharomyces cerevisiae GGS1/TPS1 gene and its multi-copy suppressor tRNAGLN (CAG): Ggs1/Tps1 protein levels restraining growth on fermentable sugars and trehalose accumulation

Byp1-3 is an amber nonsense allele of the Sacchromyces cerevisiae GGS1/TPS1 gene which encodes the small subunit of the trehalose synthase complex. Mutations in this gene confer an inability to grow on glucose or fructose but the phenotype of byp1-3 mutants is leaky in a strain-dependent manner. Overexpression of the isolated byp1-3 allele suppressed the growth defect of a ggs1/tps1 mutant. Expression of an in-vitro-generated mutant allele of GGS1/TPS1 that lacks all the coding sequences downstream from the byp1-3 mutation led to the production of a shortened protein that did not complement the ggs1/tps1 mutant. We have isolated, as an allele-specific multi-copy suppressor of the growth defect of the byp1-3 mutant on fructose, the gene for tRNA^GLN (CAG). Thus the leaky phenotype of byp1-3 mutants is due to a low level of read through of the internal nonsense codon by tRNA^GLN (CAG). Using overexpression of the isolated byp1-3 allele, as well as of the tRNA^GLN (CAG) gene, we were able to demonstrate that as little as about 10% of the normal Ggs1/Tps1 protein level is sufficient for slow growth on fructose. We also show a correlation between the level of Ggs1/Tps1, the ability to accumulate trehalose in stationary phase and the ability to grow on fermentable sugars. Sequence analysis of the cloned tRNA^GLN (CAG) gene showed that it is located 700 bp upstream of URA10. However, we found considerable differences to the reported sequence of URA10, in particular in the non-coding region.Communicated by K. Wolf     

Effects of Plagiorchis elegans (Trematoda: Plagiorchiidae) infection on the carbohydrate metabolism of fourth instar Aedes aegypti (Diptera: Culicidae)

Infection of fourth-instar Aedes aegypti (L.) with the entomopathogenic digenean Plagiorchis elegans (Rudolphi) alters the carbohydrate metabolism of the insect. Within 24 h of cercarial penetration, total body extracts of infected fourth instars exhibited decreased trehalase activity, increased trehalose-6-phosphatase activity, and a concomitant accumulation of trehalose when compared with uninfected larvae. The amounts of glucose, glycogen and lipids, and the activity of glycogen phosphorylase a were similar in extracts of infected and control larvae. The predominant fatty acids, in both control and infected larvae, were C 18:0, C 18:1, and C 18:3. There were no significant differences in the types or proportions of fatty acids found in control and infected larvae. Parasitic infection is discussed in terms of impaired trehalose metabolism.     

Influence of phospholipids from Listeria monocytogenes and Aspergillus fumigatus on the bactericidal and fungicidal activity of mouse peritoneal macrophages

Investigations were carried out on an influence of phospholipids from A. fumigatus and L. monocytogenes on the bactericidal and fungicidal activity of mouse peritoneal macrophages. L. monocytogenes bacilli and C. neoformans fungi served as the experimental microorganisms. Phospholipids from A. fumigatus and L. monocytogenes were shown to significantly increase macrophage activity under in vivo and in vitro conditions. This was accompanied by an intensified bactericidal and fungicidal activity of extracts from the lysosomal fraction of phagocyte examined. Phospholipid-stimulated macrophages were observed to liberate bactericidal factors into the culture fluid. Lymphocytes had no influence upon this process.     

Differential host susceptibility to intracerebral infections with Candida albicans and Cryptococcus neoformans.

To investigate the immune defense mechanisms employed against fungi in the brain, mice were experimentally infected by intracerebral inoculation of Candida albicans or Cryptococcus neoformans. Parameters such as median survival time and numbers of yeast cells in the brains were assessed for naive and immunomodulated mice. We found that no mice survived either C. albicans or C. neoformans challenge at doses of > or = 10(6) yeast cells per mouse. However, when the inoculum size was decreased (< or = 10(5) yeast cells per mouse), C. albicans was no longer lethal (100% survival), whereas 100 and 70% of the mice still succumbed to challenge doses of 10(4) and 10(3) C. neoformans yeast cells, respectively. Pharmacological manipulation and transfer experiments revealed that the myelomonocytic compartment had a minor role against C. neoformans but was deeply involved in the control of intracerebral C. albicans infection. By counting the number of yeast cells in the brains of naive and immunomodulated animals, we established that, unlike C. albicans, C. neoformans remained essentially in the brain, where massive colonization and damage occurred whether naive or immunomodulated defense mechanisms were employed by the host. Overall, these data suggest that the differential role of the myelomonocytic compartment, together with the diverse tropisms of the two fungi, can explain the different development and outcome of intracerebral C. albicans and C. neoformans infections.     

The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII

Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1, hxk2 grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1, hxk2 double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucoseinduced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the delection of HXK2. However, both the ggs1 and the ggs1, hk2 mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1 strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex. We discuss a possible requirement of trehalose synthesis for a metabolic balance of sugar phosphates and free inorganic phosphate during the transition from derepressed to fermentative metabolism.     

An Os-1 family histidine kinase from a filamentous fungus confers fungicide-sensitivity to yeast

Three groups of fungicides (phenylpyrroles, dicarboximides, aromatic hydrocarbons) are effective against filamentous fungi. The target of these fungicides is the osmotic stress signal transduction pathway, which is dependent on the Os-1 family of two-component histidine kinases. These fungicides usually have no fungicidal effect on the yeast Saccharomyces cerevisiae. In this report, we found that expression of Hik1, an Os-1 orthologue from rice blast fungus, can confer fungicide-sensitivity to yeast. This requires both the histidine kinase and the response regulator domains of Hik1. Analysis of yeast mutants indicated that this sensitivity is Hog1- and Ssk1-dependent. In addition, our studies revealed an interaction between Hik1 and Ypd1. These observations suggest that Hik1 is a direct target of the fungicides or is a mediator of fungicide action and that the fungicidal effect is transmitted to the Hog1 pathway via Ypd1.     

The cryptococcal enzyme inositol phosphosphingolipid-phospholipase C confers resistance to the antifungal effects of macrophages and promotes fungal dissemination to the central nervous system

In recent years, sphingolipids have emerged as critical molecules in the regulation of microbial pathogenesis. In fungi, the synthesis of complex sphingolipids is important for the regulation of pathogenicity, but the role of sphingolipid degradation in fungal virulence is not known. Here, we isolated and characterized the inositol phosphosphingolipid-phospholipase C1 (ISC1) gene from the fungal pathogen Cryptococcus neoformans and showed that it encodes an enzyme that metabolizes fungal inositol sphingolipids. Isc1 protects C. neoformans from acidic, oxidative, and nitrosative stresses, which are encountered by the fungus in the phagolysosomes of activated macrophages, through a Pma1-dependent mechanism(s). In an immunocompetent mouse model, the C. neoformans Deltaisc1 mutant strain is almost exclusively found extracellularly and in a hyperencapsulated form, and its dissemination to the brain is remarkably reduced compared to that of control strains. Interestingly, the dissemination of the C. neoformans Deltaisc1 strain to the brain is promptly restored in these mice when alveolar macrophages are pharmacologically depleted or when infecting an immunodeficient mouse in which macrophages are not efficiently activated. These studies suggest that Isc1 plays a key role in protecting C. neoformans from the intracellular environment of macrophages, whose activation is important for preventing fungal dissemination of the Deltaisc1 strain to the central nervous system and the development of meningoencephalitis.     

Encapsulation of Cryptococcus neoformans regulates fungicidal activity and the antigen presentation process in human alveolar macrophages.

Our previous studies have shown that unstimulated alveolar macrophages (AM) play a predominant role as antigen-presenting cells in Cryptococcus neoformans infections, while the function as effector cells seems to be of minor relevance. The present study focuses on the role of encapsulation of C. neoformans on fungicidal activity and the antigen presentation process of AM. Fungicidal activity in unstimulated AM occurs to a higher degree when the acapsular strain is employed, but this is impaired compared with other natural effectors, such as peripheral blood monocytes (PBM) and polymorphonuclear (PMN) cells. Cryptococcus-laden AM also induce a higher proliferative response in autologous CD4+ lymphocytes when the acapsular strain is used compared with encapsulated yeast. The enhanced blastogenic response is, in part, ascribed to an augmented IL-2 production by T cells. In addition, higher levels of interferon-gamma (IFN-gamma), but not IL-4, are produced by the responding T cells, when the acapsular strain is used compared with the encapsulated yeast. Moreover, IFN-gamma is able to induce fungicidal activity in AM against the encapsulated yeast and augments killing activity of the acapsular strain. This phenomenon is not mediated by nitric oxide production, but is correlated with an enhancement of fungicidal activity of cytoplasmic cationic proteases. We speculate that encapsulation of C. neoformans could down-regulate the development of the immune response mediated by Cryptococcus-laden AM at lung level.     

Macrophage Autophagy in Immunity to Cryptococcus neoformans and Candida albicans

Autophagy is used by eukaryotes in bulk cellular material recycling and in immunity to intracellular pathogens. We evaluated the role of macrophage autophagy in the response to Cryptococcus neoformans and Candida albicans, two important opportunistic fungal pathogens. The autophagosome marker LC3 (microtubule-associated protein 1 light chain 3 alpha) was present in most macrophage vacuoles containing C. albicans. In contrast, LC3 was found in only a few vacuoles containing C. neoformans previously opsonized with antibody but never after complement-mediated phagocytosis. Disruption of host autophagy in vitro by RNA interference against ATG5 (autophagy-related 5) decreased the phagocytosis of C. albicans and the fungistatic activity of J774.16 macrophage-like cells against both fungi, independent of the opsonin used. ATG5-knockout bone marrow-derived macrophages (BMMs) also had decreased fungistatic activity against C. neoformans when activated. In contrast, nonactivated ATG5-knockout BMMs actually restricted C. neoformans growth more efficiently, suggesting that macrophage autophagy plays different roles against C. neoformans, depending on the macrophage type and activation. Interference with autophagy in J774.16 cells also decreased nonlytic exocytosis of C. neoformans, increased interleukin-6 secretion, and decreased gamma interferon-induced protein 10 secretion. Mice with a conditionally knocked out ATG5 gene in myeloid cells showed increased susceptibility to intravenous C. albicans infection. In contrast, these mice manifested no increased susceptibility to C. neoformans, as measured by survival, but had fewer alternatively activated macrophages and less inflammation in the lungs after intratracheal infection than control mice. These results demonstrate the complex roles of macrophage autophagy in restricting intracellular parasitism by fungi and reveal connections with nonlytic exocytosis, humoral immunity, and cytokine signaling.