Cryptococcus gattii isolates from the British Columbia cryptococcosis outbreak induce less protective inflammation in a murine model of infection than Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans causes approximately one million cases of cryptococcosis per year in people with AIDS. In contrast, the related species C. gattii is responsible for a much smaller number of cases, but these often occur in immunocompetent people. In fact, C. gattii has emerged in the last decade as the frequent cause of cryptococcosis in otherwise healthy people in British Columbia. We analyzed the immune responses elicited by three C. gattii strains and one C. neoformans strain in mice as a first step toward understanding why C. gattii is able to cause disease in immunocompetent hosts. The C. gattii strains all induced a less protective inflammatory response in C57BL/6 mice by inhibiting or failing to provoke the migration of neutrophils to sites of infection. The C. gattii strains also failed to elicit the production of protective cytokines, such as tumor necrosis factor alpha, compared to the ability of the C. neoformans strain. Despite these differences, the strain representing the major outbreak genotype from British Columbia showed a virulence equivalent to that of the C. neoformans strain, while two other C. gattii strains had reduced virulence. Taken together, our results indicate that C. gattii strains thrive in immunocompetent hosts by evading or suppressing the protective immune responses that normally limit the progression of disease caused by C. neoformans.

Metabolic adaptation in Cryptococcus neoformans during early murine pulmonary infection.

SUMMARY: The pathogenic fungus Cryptococcus neoformans generally initiates infection in mammalian lung tissue and subsequently disseminates to the brain. We performed serial analysis of gene expression (SAGE) on C. neoformans cells recovered from the lungs of mice and found elevated expression of genes for central carbon metabolism including functions for acetyl-CoA production and utilization. Deletion of the highly expressed ACS1 gene encoding acetyl-CoA synthetase revealed a requirement for growth on acetate and for full virulence. Transcripts for transporters (e.g. for monosaccharides, iron, copper and acetate) and for stress-response proteins were also elevated thus indicating a nutrient-limited and hostile host environment. The pattern of regulation was reminiscent of the control of alternative carbon source utilization and stress response by the Snf1 protein kinase in Saccharomyces cerevisiae. A snf1 mutant of C. neoformans showed defects in alternative carbon source utilization, the response to nitrosative stress, melanin production and virulence. However, loss of Snf1 did not influence the expression of a set of genes for carbon metabolism that were elevated upon lung infection. Taken together, the results reveal specific metabolic adaptations of C. neoformans during pulmonary infection and indicate a role for ACS1 and SNF1 in virulence.

Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans.

BACKGROUND: Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy. RESULTS: Initially, CGH was used to compare the approximately 100 kilobase MATa and MATalpha mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MATa) and the clinical isolate NIH12 (MATalpha) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains. CONCLUSION: The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids.

Iron source preference and regulation of iron uptake in Cryptococcus neoformans.

The level of available iron in the mammalian host is extremely low, and pathogenic microbes must compete with host proteins such as transferrin for iron. Iron regulation of gene expression, including genes encoding iron uptake functions and virulence factors, is critical for the pathogenesis of the fungus Cryptococcus neoformans. In this study, we characterized the roles of the CFT1 and CFT2 genes that encode C. neoformans orthologs of the Saccharomyces cerevisiae high-affinity iron permease FTR1. Deletion of CFT1 reduced growth and iron uptake with ferric chloride and holo-transferrin as the in vitro iron sources, and the cft1 mutant was attenuated for virulence in a mouse model of infection. A reduction in the fungal burden in the brains of mice infected with the cft1 mutant was observed, thus suggesting a requirement for reductive iron acquisition during cryptococcal meningitis. CFT2 played no apparent role in iron acquisition but did influence virulence. The expression of both CFT1 and CFT2 was influenced by cAMP-dependent protein kinase, and the iron-regulatory transcription factor Cir1 positively regulated CFT1 and negatively regulated CFT2. Overall, these results indicate that C. neoformans utilizes iron sources within the host (e.g., holo-transferrin) that require Cft1 and a reductive iron uptake system.

Role of homoserine transacetylase as a new target for antifungal agents.

Microbial amino acid biosynthesis is a proven yet underexploited target of antibiotics. The biosynthesis of methionine in particular has been shown to be susceptible to small-molecule inhibition in fungi. The first committed step in Met biosynthesis is the acylation of homoserine (Hse) by the enzyme homoserine transacetylase (HTA). We have identified the MET2 gene of Cryptococcus neoformans H99 that encodes HTA (CnHTA) by complementation of an Escherichia coli metA mutant that lacks the gene encoding homoserine transsuccinylase (HTS). We cloned, expressed, and purified CnHTA and determined its steady-state kinetic parameters for the acetylation of L-Hse by acetyl coenzyme A. We next constructed a MET2 mutant in C. neoformans H99 and tested its growth behavior in Met-deficient media, confirming the expected Met auxotrophy. Furthermore, we used this mutant in a mouse inhalation model of infection and determined that MET2 is required for virulence. This makes fungal HTA a viable target for new antibiotic discovery. We screened a 1,000-compound library of small molecules for HTA inhibitors and report the identification of the first inhibitor of fungal HTA. This work validates HTA as an attractive drug-susceptible target for new antifungal agent design.

Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for anti-cryptococcal therapy.

The iron- and cAMP-regulated gene SIT1 influences ferrioxamine B utilization, melanization and cell wall structure in Cryptococcus neoformans.

The mechanisms by which pathogens sense and transport iron are important during infection, because of the low availability of free iron in the mammalian host. Iron is a key nutritional cue for the pathogen Cryptococcus neoformans, because it influences expression of the polysaccharide capsule that is the major virulence factor of the fungus. In this study, C. neoformans mutants were constructed with a defect in the iron-regulated gene SIT1 that encodes a putative siderophore iron transporter. Analysis of mutants in serotype A and D strains demonstrated that SIT1 is required for the use of siderophore-bound iron, and for growth in a low-iron environment. The sit1 mutants also showed changes in melanin formation and cell wall density, and it was found that mutants defective in protein kinase A, which is known to influence melanization and capsule formation, showed elevated SIT1 transcripts in both the serotype A and the serotype D backgrounds. Finally, the mutants were tested for virulence in a murine model of cryptococcosis, and it was found that SIT1 was not required for virulence. Overall, these studies establish links between iron acquisition, melanin formation and cAMP signalling in C. neoformans.

Iron regulation of the major virulence factors in the AIDS-associated pathogen Cryptococcus neoformans.

Iron overload is known to exacerbate many infectious diseases, and conversely, iron withholding is an important defense strategy for mammalian hosts. Iron is a critical cue for Cryptococcus neoformans because the fungus senses iron to regulate elaboration of the polysaccharide capsule that is the major virulence factor during infection. Excess iron exacerbates experimental cryptococcosis and the prevalence of this disease in Sub-Saharan Africa has been associated with nutritional and genetic aspects of iron loading in the background of the HIV/AIDS epidemic. We demonstrate that the iron-responsive transcription factor Cir1 in Cr. neoformans controls the regulon of genes for iron acquisition such that cir1 mutants are "blind" to changes in external iron levels. Cir1 also controls the known major virulence factors of the pathogen including the capsule, the formation of the anti-oxidant melanin in the cell wall, and the ability to grow at host body temperature. Thus, the fungus is remarkably tuned to perceive iron as part of the disease process, as confirmed by the avirulence of the cir1 mutant; this characteristic of the pathogen may provide opportunities for antifungal treatment.

SHIP represses the generation of alternatively activated macrophages.

We recently reported that SHIP restrains LPS-induced classical (M1) activation of in vitro differentiated, bone marrow-derived macrophages (BMMPhis) and that SHIP upregulation is essential for endotoxin tolerance. Herein, we show that in vivo differentiated SHIP-/- peritoneal (PMPhis) and alveolar (AMPhis) macrophages, unlike their wild-type counterparts, are profoundly M2 skewed (alternatively activated), possessing constitutively high arginase I (ArgI) and Ym1 levels and impaired LPS-induced NO production. Consistent with this, SHIP-/- mice display M2-mediated lung pathology and enhanced tumor implant growth. Interestingly, BMMPhis from SHIP-/- mice do not display this M2 phenotype unless exposed to TGFbeta within normal mouse plasma (MP) during in vitro differentiation. Our results suggest that SHIP functions in vivo to repress M2 skewing and that macrophage polarization can occur during differentiation in response to TGFbeta if progenitors have elevated PIP3.