Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 200347616811694Original ArticleCn var. gatti sod1S. D. Narasipura et al.
Molecular Microbiology (2003) 47(6), 1681–1694
Characterization of Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant of Cryptococcus neoformans var. gattii: role in biology and virulence Srinivas D. Narasipura,1 Jeffrey G. Ault,2 Melissa J. Behr,3 Vishnu Chaturvedi1,4 and Sudha Chaturvedi1* 1 Mycology Laboratory, 2Electron Microscopy Core, 3 Anatomic and Pathology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Ave., Albany, NY 12208-2002, USA. 4 Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY, USA. Summary The pathogenic yeast Cryptococcus neoformans (Cn) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans, which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii. The present study focused on the characterization of a Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphurcontaining amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance
Accepted 26 November, 2002. *For correspondence. E-mail
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in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen. Introduction Cryptococcus neoformans (Cn) var. gattii is an encapsulated pathogenic yeast found in tropical climates, most commonly on Eucalyptus trees. Cn var. gattii differs from two other well-known Cn varieties, grubii and neoformans, in phenotypic characters, natural habitat, epidemiology, clinical manifestations of disease and response to antifungal therapy (Kwon-Chung and Bennett, 1992; Fisher et al., 1993; Speed and Dunt, 1995; Casadevall and Perfect, 1998; Chen et al., 2000; Sorrell, 2001). Cn var. gattii predominantly infects immunocompetent individuals, whereas varieties grubii and neoformans are common in immunocompromised individuals. The mechanism of differences in host predilection remain largely unknown, except for two experimental studies that reported that Cn var. gattii inhibits phagocyte response, whereas the other two varieties are readily killed by phagocyte oxidants (Church and Washburn, 1992; Dong and Murphy, 1995). It was hypothesized that this differential phagocyte response could result from innate differences among the antioxidants of these varieties. Support for this observation comes from our earlier work on comparative analysis of Cu,Zn superoxide dismutase (SOD; encoded by SOD1) from three Cn varieties, which revealed significant differences in the physical, biochemical, molecular and structural properties of this enzyme (Hamilton and Holdom, 1997; Chaturvedi et al., 2001). These results are important, as SOD is one of the essential elements of the primary antioxidant defence system. SOD protects cells from the toxic effects of reactive oxygen intermediates (ROIs) by dismutation of superoxide radicals (O2–) into hydrogen peroxide (H2O2) and oxygen (O2), thereby preventing O2–-mediated reduction of iron and subsequent generation of highly toxic hydroxyl radicals (OH•; Miller and Britigan, 1997). Cu,Zn SOD has been extensively investigated in the eukaryotic model organism Saccharomyces cerevisiae. Yeast lacking Cu,Zn SOD shows severe growth defects, auxotrophies for sulphur-containing amino acids, a higher rate of spontaneous mutation under aerobic conditions and rapid loss of viability under nutrient deprivation (Gralla
1682 S. D. Narasipura et al. and Valentine, 1991; Liu et al., 1992; Longo et al., 1996). Cu,Zn SOD has also been isolated from other fungi, including Neurospora crassa, Schizosaccharomyces pombe, Candida albicans and Aspergillus fumigatus (Goscin and Fridovich, 1972; Misra and Fridovich, 1972; Hwang et al., 1999; Mutoh et al., 2002). The association of A. fumigatus Cu,Zn SOD with the cell wall and its secretion into the medium suggest that this enzyme may protect cells from externally generated free radicals (Hamilton et al., 1996). Significant inhibition in human neutrophil (PMN)-induced killing of Cn by exogenous addition of SOD also suggests a protective role for this enzyme (Chaturvedi et al., 1996). However, none of the data described above provide direct evidence for any role for Cu,Zn SOD in fungal virulence. Virulence determinants of Cn are currently the subject of intensive investigation in Cn varieties neoformans and grubii. Several important virulence traits were recognized early by biochemical and classical genetics, including polysaccharide capsule, laccase production and ability to grow at 37∞C. With the availability of DNA transformation systems since 1992, many more factors contributing to virulence in these two varieties have been identified, i.e. NMT1, CNA1, GPA1, URE1 and phospholipase (Lodge et al., 1994; Alspaugh et al., 1997; Odom et al., 1997; Cox et al., 2000; 2001). These molecular pathogenesis studies have also revealed previously unknown differences in virulence for var. grubii and var. neoformans. For example, var. grubii transcription factor STE12a was shown to be essential for haploid fruiting but not necessary for mating and virulence, whereas STE12a in var. neoformans was found to be essential for virulence and haploid fruiting but dispensable for mating (Yue et al., 1999; Chang et al., 2000). Similarly, calcineurin was essential for cation resistance in var. grubii, although it was not essential for cation resistance in Cn var. neoformans, suggesting a divergent role for this gene in these two varieties (Cruz et al., 2000). There are no studies yet on mechanisms of virulence in Cn var. gattii, presumably because an efficient DNA transformation and gene disruption system has not yet been described. The present study describes the development of a transformation system and the successful disruption of Cu,Zn SOD (SOD1) in Cn var. gattii. Results Disruption and reintroduction of the SOD1 gene in Cn var. gattii The SOD1 gene was disrupted by replacing 907 bp of the 957 bp open reading frame (ORF) of the gene with the URA5 selectable marker, which resulted in the Cu,Zn SOD null mutant sod1::URA5 (Fig. 1A). Our initial attempts to use the URA5 selectable marker from Cn var. neoformans for transformation of var. gattii were unsuc-
cessful. The comparative analysis of URA5 from Cn var. neoformans and var. gattii revealed that this gene is ª9% divergent at the intragenic region and 30% divergent at the promoter region. These results suggested that URA5 from Cn var. neoformans might not be functional in var. gattii. Therefore, to increase the transformation efficiency as well as the homologous recombination, we used URA5 and SOD1 from Cn var. gattii type strain NIH 444. A NotIlinearized sod1::URA5 disruption allele was transformed into a ura5 strain of var. gattii. Sixty transformants were obtained on synthetic medium lacking uracil. These transformants were analysed initially for a presumptive nogrowth phenotype on YPD menadione broth. This screening procedure yielded four suspected sod1 clones. Next, genomic DNA was isolated from these clones and analysed by polymerase chain reaction (PCR) for the absence of the 500 bp fragment of the SOD1 ORF using primer pair V523 and V524 (details in Experimental procedures). If the sod1::URA5 allele has been integrated at the homologous site, then the primer pair V523 and V524 should not give the 500 bp product; however, if it is integrated at the ectopic site, then the genomic DNA should give one 500 bp product of SOD1 wild type. Out of four isolates, two lacked the SOD1 wild-type locus, as revealed by the absence of 500 bp amplicons (Fig. 1B). Southern hybridization confirmed that the SOD1 wild-type locus had been replaced by the sod1::URA5 disruption allele by homologous recombination, as no signal for SOD1 was detected in the mutant strain when it was probed with the 500 bp fragment of SOD1 used from the 907 bp deleted region of wild-type SOD1 (Fig. 1C). The percentage homologous recombination achieved was 3.4 (two positive out of 60 transformants). Next, we generated a reconstituted strain in which a wild-type copy of SOD1 with its own promoter was reintegrated in the genome. To make a reconstituted strain, we reverted the ura+ phenotype by plating the sod1::URA5 mutant onto medium with 5-fluoroorotic acid (5-FOA) to select for ura– cells. This revertant was used as the recipient for transformation with a pCR6.1 plasmid containing full-length SOD1 (2.8 kb) and URA5 selectable marker. PCR analysis confirmed the restoration of the wild-type gene in these two reconstituted strains. Interestingly, restriction digestion of genomic DNA with XbaI and BstXI produced an identical band size to that of the wild-type strain, which suggested homologous integration of SOD1 to the sod1::URA5 locus. These reconstituted strains were designated as sod1 + SOD1-A and B. To examine the expression of the SOD1 gene, we performed reverse transcription (RT)-PCR on total RNA isolated from SOD1 wild type, the sod1 mutant and the sod1 + SOD1 reconstituted strains. In contrast to the SOD1 wild-type strain, in which a 400 bp amplicon was readily detected, no message was detected in the sod1 © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
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Fig. 1. Disruption and reconstitution of Cn var. gattii SOD1 gene. A. A 2.8 kb fragment containing the SOD1 gene (solid thick line and dashed line outside 2.8 kb) indicates the genomic locus based on restriction enzyme digestion. The ‘probe’ indicates the 500 bp sequence within the ORF used for PCR, Southern hybridization and RT-PCR to confirm gene replacement. B. PCR for SOD1 ORF. Primers designed from the SOD1 ORF (indicated by an arrow in A) amplified a 500 bp product from genomic DNA of SOD1 wild-type and sod1 + SOD1 reconstituted strains, but not from genomic DNA of the sod1 mutant strain. Negative control is PCR without genomic DNA. C. Southern blot of genomic DNA from the SOD1 wild-type, sod1 mutant and sod1 + SOD1 reconstituted strains. Genomic DNA was digested with XbaI and BstXI and probed with the 500 bp PCR product depicted in (A). No hybridization signal was detected in the sod1 mutant, whereas both SOD1 wild-type and sod1 + SOD1 (A and B) reconstituted strains produced ≈8 kb, 4 kb and 0.8 kb bands. D. RT-PCR to examine the expression of the SOD1 gene. Total RNA was isolated, reverse transcribed to cDNA and amplified with primers directed to SOD1 or actin. RT-PCR products were fractionated by electrophoresis in a 1% agarose gel and stained with ethidium bromide. Positive control is RT-PCR with RNA provided in the kit to confirm the efficacy of the kit, and negative control is RT-PCR in the absence of RNA.
mutant strain. This result supports the conclusion that SOD1 has been functionally deleted in the sod1 mutant strain. After reintroduction of the SOD1 wild-type gene by transformation, SOD1 expression was restored in the sod1 + SOD1 strains A and B (Fig. 1D). Further genotypic and phenotypic analyses confirmed that both reconstituted strains exhibited identical properties; therefore, results from sod1 + SOD1-A are shown in all other experiments. © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
Characterization of Cn var. gattii sod1 phenotypes To address whether Cu,Zn SOD is essential for maintaining normal growth of Cn in laboratory medium, we determined the doubling time of exponentially dividing SOD1 wild type, sod1 mutant and sod1 + SOD1 reconstituted strains in YPD broth at both 30∞C and 37∞C. The doubling time of the sod1 mutant was slightly longer at 30∞C and 37∞C (3.3 ± 0.2 h and 3.4 ± 0.1 h) than that of the SOD1
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Fig. 2. sod1 mutant is viable in stationary phase culture. Cells were allowed to grow in flasks for 48 h in YPD broth, washed three times with sterile distilled water and reincubated in water. Viability was monitored on the indicated days by plating aliquots onto YPD plates, and cfu were counted. The experiment was repeated twice with similar results; and a representative experiment is shown.
wild type (2.8 ± 0.1 h and 2.9 ± 0.2 h) or the sod1 + SOD1 reconstituted strain (2.7 ± 0.3 h and 2.8 ± 0.1 h). However, these differences in growth between the sod1 mutant and SOD1 wild type were not statistically significant (P = 0.4). These results indicate that Cu,Zn SOD is not essential for Cn growth. Similarly, Cu,Zn SOD was seen not to be required for Cn growth under nutrient deprivation, as no difference in growth between the sod1 mutant and the SOD1 wild type was observed when they were assessed in terms of colony-forming units (cfu) of cells cultured for 15 days in water (Fig. 2). An initial drop in cfu, noted after 3 days of incubation in water, might result from a shift from a very rich medium such as YPD to water, and this drop in cfu was consistent among SOD1 wild-type,
sod1 mutant and sod1 + SOD1 reconstituted strains. Also, the sod1 mutant did not show auxotrophies for the sulphur-containing amino acids lysine and methionine, as the growth (OD600) determined for the sod1 mutant in complete CSM medium or in CSM lacking either lysine or methionine was similar (data not shown). It was also interesting to note that Cu,Zn SOD was not required for Cn mating. The sod1 mutant produced abundant filaments and basidiospores within 7 days at 24∞C, similar to the behaviour of SOD1 wild type, when mixed with var. neoformans MATa type strain NIH430 (data not shown). Such successful mating reactions between var. neoformans and var. gattii tester strains have been reported in the literature (Kwon-Chung et al., 1982). We did not isolate basidiospores from these crossings to test for the viability of the progeny. However, neither SOD1 wild-type nor sod1 mutant strains produced any basidiospores and filaments when mixed with var. gattii MATa type strain NIH191. Although it is not known why SOD1 wild type and the sod1 mutant were unable to mate with tester strain NIH191, one possibility is that NIH191 might no longer be mating competent. Sod1 mutant is highly sensitive to menadione The cytosolic localization of Cu,Zn SOD implies that its main function is to protect cells against reactive oxygen intermediates (ROI; Jamieson, 1998). It is also reasonable to suspect that this localization may also enhance the efficiency of Cn in coping with oxidative stresses in the host. To address this question, we grew Cn strains in the presence of menadione (a redox cycling agent that generates superoxide ions in the cytosol) to an initial OD600 of 0.1. The sod1 mutant was extremely sensitive to menadione, and its growth was completely inhibited at 3 mg ml-1 at 30∞C. This effect was more pronounced at 37∞C, as even a 1 mg ml-1 concentration of menadione was enough to inhibit the sod1 mutant completely,
Fig. 3. sod1 mutant is highly sensitive to menadione. Cells were grown in YPD broth containing menadione (0–3 mg ml-1) at initial OD600 of 0.1 under shaking conditions (180 r.p.m.). The data shown were acquired 16 h after incubation. Experiments were repeated three times with similar results; a representative experiment is shown.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
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Fig. 4. sod1 mutant shows fragmented vacuoles. Cn cells were grown in YPD broth overnight at 30∞C and then processed for electron microscopic analysis as described in Experimental procedures. N, nucleus, VS, small vacuole, VL, large vacuole; arrow points to fragmentation of large vacuole in the sod1 mutant. Bar corresponds to 120 nm.
whereas the SOD1 wild-type strain was relatively resistant. Introduction of the SOD1 gene restored the menadione resistance of the sod1 mutant to the wild-type level. These results strongly suggest that cytosolic Cu,Zn SOD protects Cn against oxidative stress (Fig. 3). Sod1 mutant shows fragmented vacuoles Ultrastructural examination of the sod1 mutant revealed fragmentation of its large vacuole into aberrant shapes (Fig. 4). In general, the wild-type strain had two kinds of vacuoles: one large, loosely packed vacuole (VL) and two to six small, densely packed vacuoles (VS). It was noted that VL in sod1 mutant was fragmented, as the internal contents of the fragmented vacuole appeared to be similar to that of the VL seen in SOD1 wild type. The analysis of 30 individual electron microscopic (EM) sections revealed that >75% of the sod1 mutant cells had fragmented VL (12 ± 4.6 VL per cell) compared with one intact VL in SOD1 wild type (P < 0.0001). Reconstitution of the sod1 mutant with SOD1 wild type restored vacuolar morphology, suggesting that Cu,Zn SOD is essential for vacuolar integrity, and possibly for vacuolar function. Sod1 is defective in the production of several virulence factors We next examined whether SOD1 deletion in Cn influences other known virulence factors. The virulence traits tested included production of melanin, laccase, urease, phospholipase B, polysaccharide capsule and mannitol. The sod1 mutant exhibited a marked defect in melanization (Fig. 5A). Melanin production was restored in a sod1 + SOD1 reconstituted strain. As laccase is the sole enzyme responsible for melanin production in Cn (Salas © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
et al., 1996), laccase activity was measured from the equal number of sod1 mutant and SOD1 wild-type cells as described previously (Zhang et al., 1999). SOD1 deletion resulted in significant reduction in laccase activity in the sod1 mutant compared with SOD1 wild type (P < 0.01; Fig. 5B). Similarly, the sod1 mutant was severely defective in the production of phospholipase B on egg yolk agar, and in the production of urease on urea agar media (Fig. 5C and D). Reconstitution of the sod1 mutant with SOD1 wild type restored all the virulence-associated phenotypes to a large extent. On the other hand, SOD1 deletion had no influence on capsule synthesis, as revealed by India ink stains of SOD1 wild-type, sod1 mutant and sod1 + SOD1 reconstituted strains grown in low-iron medium for 3 days (Fig. 6A). Interestingly, the sod1 mutant produced significantly more mannitol (P < 0.05) in the medium than the SOD1 wild-type strain (Fig. 6B). Sod1 mutant is attenuated in virulence in the mouse meningoencephalitis model We tested whether Cu,Zn SOD is required for virulence of Cn var. gattii in a mouse meningoencephalitis model. As shown in Fig. 7, after injection of 1 ¥ 106 yeast cells, median survival of mice inoculated with the SOD1 wildtype strain was 6 days, whereas the median survival of mice inoculated with the sod1 mutant was 23 days (P < 0.0001). Reconstitution of the SOD1 gene into the sod1 mutant restored virulence, with a median survival of mice injected with sod1 + SOD1 of 9 days (P < 0.0003). These results suggested that Cu,Zn SOD plays an important role in Cn var. gattii pathogenesis. Next, we examined the in vivo tissue reaction induced by Cn strains in infected mice. As Cn has a tropism for
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Fig. 5. Defective production of virulence factors in sod1 mutant. A. Cells were grown in YPD broth overnight, then washed three times with PBS, and a 5 ml suspension of 107 cells ml-1 was placed on Niger seed agar for the determination of melanin synthesis and incubated at 30∞C for 4 days. B. Phenoloxidase activity from an equal number of glucose-starved cells was determined by measuring the oxidation of the diphenolic substrate ABTS (IU of activity = 0.01 A420 absorbance unit in 30 min). The results are the mean ± SD of three individual experiments. Asterisk denotes P £ 0.05 compared with SOD1 wild type. C and D. An aliquot of 5 ml of Cn cells (described in detail in A) were placed on Christensen’s agar for the determination of urease synthesis, and on egg yolk agar for the determination of phospholipase synthesis.
brain, we collected brain tissues from infected mice 7 days after infection. SOD1 wild type induced severe infection, with the formation of 17 ± 7.0 lesions per brain section (each brain section comprised rostral cortex, caudal cortex, mid-brain and cerebellum with brainstem) compared with the sod1 mutant with 2.0 ± 1.2 lesions per brain section (P < 0.0001). Infection with the sod1 + SOD1 reconstituted strain was intermediate in severity, resulting in an average of 8 ± 4.0 lesions per brain section. The number of yeasts per lesion for SOD1 wild-type, sod1 mutant and
Fig. 6. Capsule and mannitol synthesis in sod1 mutant. A. Cn cells were grown in low-iron medium for 3 days at 30∞C. Cells were mixed with India ink, and the capsule, which excludes the ink particles, was photographed. Final magnification ¥ 400. B. Extracellular mannitol produced from Cn cells was determined by a colorimetric method with absorbance at 412 nm. The results are the mean ± SD of three individual experiments. Asterisk denotes P < 0.05 compared with SOD1 wild type.
sod1 + SOD1 reconstituted strains ranged from 2 to 54, 1 to 3 and 1 to 37 respectively (Fig. 8). These results suggested that the sod1 mutant is defective in brain colonization and persistence. No lesions were found in sections from control brain (Fig. 8).
Fig. 7. Virulence of sod1 mutant in a mouse model. SOD1 wild-type, sod1 mutant and sod1 + SOD1 reconstituted strains were grown in YPD broth for 16 h; a suspension of 1 ¥ 106 cells was then injected intravenously into 6-week-old male BALB/c mice, which were observed daily until moribund. © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
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Fig. 8. Defective brain colonization and persistence of sod1 mutant. Three mice were infected with 1 × 105 Cn cells intravenously, then euthanized on day 7, and the brain sections were stained with Mayer's mucicarmine. A and B. Sections from SOD1 wild-type infection reveal a large lesion (arrow) filled with fluid and SOD1 wild-type cells in the thalamus. Organisms were also present in smaller numbers in the lateral ventricles and meninges (double arrow) (A at 20×). Higher magnification of the large cyst revealed numerous variable sized SOD1 wild-type cells, some with budding (B at 200×). C and D. A section from the sod1 mutant infection shows a very small cyst (arrow) (C at 20×), and higher magnification reveals a cyst with one budding sod1 mutant cell (D at 200×). E and F. A section from the sod1 + SOD1 reconstituted strain infection reveals one large (arrow) and a few small cysts (double arrow) (E at 20×), and higher magnification of the large cyst reveals many Cn cells (F at 200×). © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
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Fig. 9. sod1 susceptibility to human PMN killing. Human PMNs at an effector-to-target ratio of 10:1 were incubated with opsonized Cn cells (5 ¥ 103) for 4 h at 37∞C in 5% CO2-95% air. The percent Cn killed was determined from cfu counts as described in Experimental procedures. Results are mean ± SEM from PMNs of three individual donors. Asterisk denotes P £ 0.05 compared with SOD1 wild type. To determine whether exogenous SOD protects the sod1 mutant from PMN killing, we preincubated PMNs with 10 mg ml-1 SOD for 1 h, followed by incubation with opsonized Cn cells as shown above.
Sod1 mutant is susceptible to PMN killing Human PMNs were incubated with opsonized SOD1 wildtype, sod1 mutant and sod1 + SOD1 reconstituted strains in an effector-to-target ratio of 10:1 for 4 h, and then Cn were cultured on YPD medium to determine survivability. The sod1 mutant was significantly more susceptible to the antifungal activity of PMN than SOD1 wild type (percentage cells killed 51.00 ± 4.20 versus 23.00 ± 2.30; P < 0.05). The exogenous addition of the O2– inhibitor SOD inhibited PMN-induced killing of the sod1 mutant, whereas killing was minimal for SOD1 wild-type and sod1 + SOD1 reconstituted strains. These results confirm that Cu,Zn SOD protects against PMN-mediated killing of Cn. var. gattii (Fig. 9). It was interesting to note that overall PMN-induced killing of Cn var. gattii wild type was quite low, 23.00 ± 2.30%, compared with the 80–85% killing of Cn var. grubii reported earlier (Chaturvedi et al., 1996). Increasing the PMN-to-Cn ratio from 10:1 to 100:1 did not enhance killing of Cn var. gattii (data not shown).
Discussion The successful construction of a gene deletion mutant, sod1, constitutes the first instance of the disruption of a functional gene in Cn var. gattii. These results suggest that the absence of the SOD1 gene in Cn var. gattii (i) abrogates protection against oxidative killing by human PMN and by the redox cycling agent menadione; (ii)
causes reduced expression of the well-known virulence factors, laccase, urease and phospholipase; and (iii) causes attenuation of virulence in a mouse model of cryptococcal meningoencephalitis. Reintroduction of the wildtype SOD1 gene complemented these defects and largely restored virulence. Thus, SOD1 is required for the primary pathogen Cn var. gattii to express a number of other virulence factors, especially the metalloproteins, for its survival under high oxidative stress conditions and for its virulence in an infected host. This is believed to be the first report in which superoxide dismutase has been implicated directly in the virulence of a fungal pathogen. The SOD1 gene has been well characterized in a model eukaryotic organism, S. cerevisiae, in which it is required for mating, aerobic growth, biosynthesis of sulphur-containing amino acids and long-term survival under nutrient deprivation (Bilinski et al., 1985; Gralla and Valentine, 1991; Liu et al., 1992; Longo et al., 1996). In general, SODs are important in maintaining an optimal intracellular redox environment in the cytoplasm that is needed to prevent lipid peroxidation, DNA cross-linking and cysteine–cysteine bonding of essential enzymes (Powis et al., 1995). In a Cn sod1 mutant, the functional properties of laccase, urease and phospholipase were abrogated. Superoxide radicals are known to oxidize iron– sulphur cluster proteins liberating iron (Keyer and Imlay, 1996). Thus, Cu,Zn SOD-deficient Cn may contain increased pools of reactive iron, as has been shown for SOD1-deficient S. cerevisiae and Escherichia coli (Liochev and Fridovich, 1994; Keyer and Imlay, 1996). This free iron may react with hydrogen peroxide and, possibly, other reactive oxygen species to generate hydroxyl radical, which is highly toxic for biomolecules. Another possibility is that a SOD-induced reductive environment in the cytosol may be critical for the metal ion insertion in metalloenzymes. Support for this possibility comes from another study, in which SOD was shown to protect the Fe–Zn active centre of calcineurin in a mammalian system (Wang et al., 1996). Reintroduction of the Cn SOD1 wild-type gene restored laccase, urease and phospholipase functions to a large extent in the sod1 mutant, thus confirming the role of Cn Cu,Zn SOD in sustaining the function of other metalloenzymes. It was interesting to note that SOD1 deletion in Cn did not influence the synthesis of capsular proteins: both the sod1 mutant and the SOD1 wild type produced a large capsule when grown in low-iron medium. On the other hand, mannitol synthesis was upregulated in the sod1 mutant compared with the wild-type parent strain. This is not surprising considering the fact that Cu,Zn SOD, being an important antioxidant, plays a crucial role not only in eliminating O2– but also in preventing the subsequent generation of highly toxic OH. Under the influence of distal ROIs (perhaps found throughout the cytosol of the sod1 © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
Cn var. gatti sod1 mutant), the mannitol upregulation may be a strategy for coping with the loss of Cu,Zn SOD by the sod1 mutant. There is also ample indirect evidence that mannitol protects Cn by scavenging distal ROIs such as OH• and HOCl (Chaturvedi et al., 1996). Recently, a vesicular (H+)-ATPase proton pump (Vph1p) has been identified in Cn var. grubii, and disruption of the vph1 gene resulted in a growth defect at 37∞C and also defects in the expression of many virulence factors including the metalloenzymes laccase and urease. These results provide the first evidence for the critical role of vacuoles in Cn survival and pathogenesis (Erickson et al., 2001). We have observed the fragmentation of large vacuoles (VL) into aberrant shapes in a sod1 mutant (12 ± 4.6 VL per cell), which might also be the cause of defective expression of many secreted virulence factors. Vacuolar fragmentations were also observed in the S. cerevisiae sod1 mutant. Vacuoles play a critical role in iron homeostasis in S. cerevisiae. Subcellular fractionation of iron-loaded cells suggests that the vacuole is the major site of iron sequestration (Raguzzi et al., 1988). Under the influence of increased superoxide levels, vacuolar iron pools may be converted to a more accessible and active Fe2+ state that can catalyse OH• production and can damage macromolecules in the immediate vicinity of the vacuoles. It is also possible that the primary damage occurs to cytosolic components, resulting in defective vacuolar structure and function. Future experiments may confirm these possibilities. A second explanation for the attenuation of virulence in the sod1 mutant could be its enhanced susceptibility to ROIs. This possibility is suggested by the (i) increased killing of the sod1 mutant by human PMN and (ii) enhanced sensitivity to the redox cycling agent menadione. PMN killing of the sod1 mutant was significantly abrogated by pretreatment with exogenous SOD, which indicated that the O2– was critical for PMN-induced killing. Thus, Cn Cu,Zn SOD may be important for in vivo survival of the fungus. Further support for this conclusion comes from the results of experimental infection in mice. The median survival of mice infected with the sod1 mutant was 23 days, versus 6 days for mice infected with the SOD1 wild-type parent strain. Introduction of SOD1 wild type into the sod1 mutant restored virulence, with a median survival of 9 days. These results indicate that SOD1 deletion significantly delays mortality in experimental cryptococcosis. A related experimental study on brain infection and colonization indicated that the sod1 mutant caused significantly smaller and fewer lesions compared with parent and reconstituted strains. These results suggest that Cn colonization in the brain is greatly reduced by SOD1 deletion. However, we cannot determine from these experiments whether oxidative conditions prohibit the survival of the sod1 mutant before it can establish residency in the © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
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brain tissues, or whether it is more sensitive and falls prey to the oxidative burst of a host immune response while residing in the brain tissues. Overall, Cu,Zn SOD seems to be critical for the ability of Cn var. gattii to survive and multiply in the host. It is important to recall that SODdeficient mutants of Helicobacter pylori, Campylobacter coli and Mycobacterium tuberculosis were also shown to be hypersensitive to oxidative stress and defective in host colonization (Purdy et al., 1999; Edwards et al., 2001; Seyler et al., 2001). It was noteworthy that SOD1 deletion in general had no influence on Cn var. gattii mating, sporulation, growth, stationary phase survival or sulphur metabolism. These results are in contrast to phenotypes observed in the S. cerevisiae sod1 mutant, in which the loss of Cu,Zn SOD was associated with 80–90% growth inhibition, mating and sporulation defects, dramatic loss of viability under nutrient deprivation and a defect in sulphur-containing amino acid metabolism (Liu et al., 1992; Longo et al., 1996). These differences between S. cerevisiae sod1 and Cn var. gattii sod1 could result from the fact that the latter fungus is highly adapted as a human and animal pathogen and contains additional antioxidant systems that are absent in S. cerevisiae. There is ample evidence that laccase (the enzyme involved in melanin synthesis) serves as an antioxidant in Cn (Salas et al., 1996). Although both laccase and melanin production was significantly reduced in the sod1 mutant, the minuscule amount of antioxidant remaining in the mutant might be sufficient to sustain life under saprobic conditions. Cn also produce large amounts of the hexitol mannitol in culture, which was shown indirectly to act as an antioxidant (Chaturvedi et al., 1996). Recently, an atypical cytosolic manganese-containing superoxide dismutase (SOD3) has been identified in C. albicans, and Sod3p was shown functionally to complement the defects of the S. cerevisiae sod1 but not sod2 (MnSOD2) mutant (Lamarre et al., 2001). The Sod3p enzyme was reported to protect C. albicans against nutrient-limiting conditions and during the induction of oxidative stress. At this time, the search of the Cn var. neoformans and Cn var. grubii EST databases (http:// www.genome.ou.edu/cneo.html) using the amino acid sequence of C. albicans SOD3 as a query produced a single hit that showed ª63% homology to MnSOD (SOD2), suggesting that the existing Cn database does not have a putative sequence for SOD3. However, this finding must be interpreted with caution as Cn EST databases are not yet complete. An important technical advance in our study was the successful disruption of a gene by homologous recombination in Cn var. gattii. The frequency of homologous recombination was 3.4%, which was comparable with that reported for Cn varieties grubii and neoformans. To avoid mismatches during homologous recombination, we used
1690 S. D. Narasipura et al. Table 1. Strains and plasmids used in this study. Strains and plasmids
Parent
Genotype
Source
NIH 430 (ATCC 28958)
Wild-type MATa (serotype D)
NIH 191 (ATCC 32608) NIH 444 (ATCC 32609) NIH 444 ura5 Cnsod1 Cnsod1u– Cnsod1 + SOD1 pCIP-3
Wild type MATa (serotype C)
American Type Culture Collection (ATCC), Manassas, VA ATCC
Wild type MATa (serotype B)
ATCC
NIH 444 ura5 Cnsod1 Cnsod1u– pBluescript
pCR3.1
pCR2.1
This study This study This study This study J. Edman Edman and Kwon-Chung (1990) This study
pCR4.1
pCR2.1
pCR5.1
pCR4.1
pCR6.1
pCR4.1
MATa ura5 MATa ura5 sod1::URA5 MATa ura5 sod1::ura5 MATa ura5 sod1::ura5 SOD1::URA5 2 kb fragment containing the URA5 gene from Cn var. neoformans in Bluescript vector 1.4 kb PCR insert containing URA5 ofCn var. gattii in pCR2.1vector 2.8 kb PCR insert containing the SOD1gene from Cn var. gattii in pCR2.1 vector 1.4 kb URA5 subcloned into ClaI of sod1in pCR 4.1 vector (sod1 disruption cassette) 1.4 kb PCR insert of URA5 gene subcloned into NotI of PCR4.1 (SOD1 complementation cassette)
the SOD1 genomic locus from Cn var. gattii. We demonstrated earlier a 20–29% difference in deduced amino acids of SOD1 among the three Cn varieties (Chaturvedi et al., 2001). Strict sequence identity is required for highefficiency homologous recombination in Cn (Davidson et al., 1999). To increase the transformation efficiency in Cn var. gattii, we used the URA5 gene from the same variety. It is important to point out here that the URA5 gene from var. gattii is 91% identical to the URA5 gene from var. neoformans. However, when the upstream region containing the promoter sequence was compared, there was only 71% identity between these two varieties, indicating a possibility of low transformation of var. gattii with the var. neoformans URA5 gene. Our findings indicate that it should now be feasible to disrupt any nonessential gene in Cn var. gattii. In conclusion, we have shown that Cu,Zn SOD is critical for the pathogenesis of Cn var. gattii but is not required for the saprobic life of the fungus. The mechanism of action of Cu,Zn SOD could be protection against oxidative killing by phagocytes, combined with a reduction in the activity of a number of virulence-related factors.
Experimental procedures Strains, media and plasmids All strains and plasmids used in this study are listed in Table 1. Standard yeast media, e.g. YPD (yeast extract peptone dextrose) and CSM (complete synthetic medium) with relevant amino acid dropouts were made as described previously (Guthrie and Fink, 1991). Niger seed agar (Salkin, 1979), urea agar (McGinnis, 1980), egg yolk agar (Chen et al., 1997) and limited iron medium with EDTA and batho-
This study This study This study
phenanthroline disulphonic acid (Pierini and Doering, 2001) were used for the determination of melanin production, urease activity, phospholipase B activity and capsule formation respectively. Cn var. gattii transformation was done using biolistic DNA delivery as described previously (Toffaletti et al., 1993). For biolistic transformation, ª 5 mg of linear DNA was used, and transformants were selected on CSM lacking uracil.
Preparation of probes Probes used for Southern hybridization were prepared from restriction fragments or PCR-amplified fragments, as described in the text. Probes were labelled with 32P as described previously (Ausubel et al., 1998).
Isolation of ura5 auxotrophs and cloning of the URA5 gene A ura5 auxotrophic derivative of NIH 444 of Cn var. gattii was isolated by selection for 5-FOA resistance as reported in another study (Kwon-Chung et al., 1992). Our initial attempt to transform Cn var. gattii with pCIP-3 (2 kb URA5 gene from Cn var. neoformans in Bluescript vector; Edman and Kwon-Chung, 1990) was unsuccessful. Therefore, the URA5 gene was PCR amplified from genomic DNA of NIH 444 from var. gattii with primer sets V479 (5¢GGCATGGGTGATATACGTTTG-3¢) and V480 (5¢-TAGATACGATGAAGATTGACAGCC-3¢). The resulting 1.4 kb URA5 fragment was cloned into pCR2.1 to yield pCR3.1 and sequenced. A BLAST search confirmed the closest match with the Cn URA5 gene. The GenBank accession number for the var. gattii URA5 sequence is AF536328. The alignment of URA5 from var. gattii with that of var. neoformans showed 92% and 71% identity at the intragene and promoter regions respectively. Approximately 20 transformants mg-1 linear DNA (pCR3.1) were obtained on CSM minus uracil medium. © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
Cn var. gatti sod1
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Isolation and disruption of the Cn var. gattii SOD1 gene
Reintroduction of the wild-type Cn var. gattii SOD1 gene
Full-length SOD1 was PCR amplified from genomic DNA of NIH 444 using primer sets 5¢-TACGAGGAA GAGAAATGGGG-3¢ (V483) and 5¢-ATGTTGATCTGT GAACGCCC-3¢ (V484). The 2.8 kb SOD1 PCR product was cloned into pCR2.1 vector to yield pCR4.1. pCR4.1 vector was digested with ClaI, which resulted in the removal of a total of 907 bp of the 957 bp SOD1 ORF. A 1.4 kb PCR fragment of the URA5 gene obtained from genomic DNA of NIH 444 strain was subcloned into ClaI sites to yield pCR5.1 as shown in Fig. 1. A total of 5 mg of NotI-linearized pCR5.1 (sod1::URA5) disruption plasmid was precipitated on 0.6 mg gold microcarrier beads (Bio-Rad) and biolistically transformed into the NIH 444 ura5 strain (Toffaletti et al., 1993). Ura+ transformants were selected on CSM lacking uracil. These clones were initially analysed for no-growth phenotypes on YPD broth containing 3 mg ml-1 menadione (superoxide-generating compound). Genomic DNA was prepared from suspected sod1 clones using the DNeasy Plant Mini kit (Qiagen). PCR was performed according to the manufacturer’s directions (Life Technologies) with the following SOD1 specific primers designed from the deleted region of the gene: 5¢-TACCGGAAATGTTGACGGA (V-527) and 5¢CACAACTCCGCCATCAAAV (V-524).
A sod1::URA5 prototrophic revertant, sod1::ura5, was obtained by counterselection using 5-FOA medium (KwonChung et al., 1992). This strain was transformed biolistically with BamHI-linearized pCR6.1 plasmid, and clones were selected on CSM lacking uracil. The growth rates at 30∞C of the 100 randomly selected reconstituted strains were compared with the wild-type strain in YPD broth containing 3 mg ml-1 menadione. Five of these strains with a more or less similar growth pattern to the wild-type strain were chosen for further analysis by PCR, Southern hybridization and RTPCR. Southern analysis confirmed that, in two of the reconstituted strains, the sod1::URA5 locus was replaced by SOD1 by homologous integration. These reconstituted strains were designated sod1 + SOD1 A and B.
Southern hybridization and RT-PCR Genomic DNA (10 mg) was digested with various restriction enzymes and elecrophoresed in 0.8% agarose gel. The DNA was then transferred to nylon membranes (Boehringer Mannheim) using standard protocols (Sambrook et al., 1989). Hybridization, washing and detection of hybridized bands were performed according to the instructions from the manufacturer (Boehringer Mannheim). For RT-PCR, total RNA from the Cn var. gattii wild-type SOD1 strain, the sod1 mutant strain and the sod1 + SOD1 reconstituted strain grown in YPD broth was isolated using the RNeasy mini kit (Ambion) after the mechanical disruption of cells with glass beads. First-strand cDNA synthesis was performed with 0.5 mg of total RNA and oligo (dT) primers as per the protocol described by the Gene Amp RNA PCR kit (Perkin-Elmer). The second-strand cDNA synthesis was performed with the primer sets 5¢-GCTGTTGCTGTC CTAAAGGG (V-548) and 5¢-GGAGATACCAATGACACCGC3¢ (V549), yielding a 413 bp SOD1 PCR amplicon. These primers were designed from the two separate exonic regions of SOD1. If there was any genomic DNA contamination in the RNA mixture, this primer set would yield 513 bp, because of the presence of two introns of 50 bp each. RT-PCR was also carried out with actin as a loading control. The actin primer sets were 5¢-CAGCTGGAAGGTAGACAAAGAGGC-3¢ (V-649) and 5¢-CGCTATTCCTCCGTATCGATCTTGC-3¢ (V650); these were again designed from two exonic regions of the actin gene to yield a 545 bp product from RNA and a 600 bp product from DNA, resulting from the presence of one intron of 51 bp. RT-PCR of control RNA provided in the kit was also tested to confirm the efficacy of the kit, which yielded an expected amplicon of 308 bp. © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
Characterization of growth and survival of the sod1 mutant In vitro growth rates of SOD1 wild type, sod1 mutant and sod1 + SOD1 reconstituted strains were determined by growing them in YPD broth at 30∞C and 37∞C at 180 r.p.m. with an initial OD600 of 0.1. Growth was followed up to 32 h by monitoring the turbidity every 4 h. Cn generation time was determined by removing an aliquot every 1 h from the exponential phase of growth and counting cells in a haemocytometer. For the long-term stationary phase survival experiment, Cn cells grown for 48 h in YPD broth were removed, washed with sterile distilled water and resuspended in water, and incubation with shaking was continued for another 15 days. Cell viability was determined by plating aliquots from cultures onto YPD agar, and colony-forming units (cfu) were counted.
Sensitivity to oxidative conditions The sensitivity of SOD1 wild-type, sod1 mutant and sod1 + SOD1 reconstituted strains to oxidative stress was determined as described previously (Gralla and Valentine, 1991). Cn cultures were inoculated at OD600 = 0.1 in YPD broth containing menadione ranging from 0 to 3 mg ml-1. Growth was measured at both 30∞C and 37∞C 16 h after incubation by monitoring the turbidity at OD600.
Ultrastructure characterization For ultrastructural analysis of Cn by electron microscopy, strains were grown for 16 h before harvesting. Cells were pelleted, washed in phosphate-buffered saline (PBS, pH 7.4), and fixed with 2% paraformaldehyde and 0.2% gluteraldehyde in 0.1 M PBS for 3 h at room temperature. Cells were washed three times with PBS and frozen using a Balzers HPM010, then freeze-substituted in 1% osmium in acetone for 72 h at -90∞C, followed by 48 h incubation at -60∞C. Cells were slowly brought to room temperature, washed twice in 100% acetone for 1 h each, infiltrated with 1:1 acetone– araldite epon and polymerized overnight at 65∞C. Semi-thick (80–120 nm) sections were cut and stained with uranyl acetate and lead, and examined using a Zeiss 910 electron microscope.
1692 S. D. Narasipura et al. Virulence factor expression
Mating and haploid filamentation assays
Laccase and melanin production. The activity of laccase, the rate-limiting enzyme in melanin production, was assessed by measuring the oxidation of 2,2¢-azinobis (3ethylbenzthiazolin-6-sulphonate) (ABTS) as described previously (Zhang et al., 1999). SOD1 wild-type, sod1 mutant and sod1 + SOD1 reconstituted strains were incubated in minimal asparagine medium with 0.1% glucose for 16 h with shaking at 30∞C. Cells were pelleted, washed twice with water, resuspended in minimal asparagine medium without glucose and incubated with shaking for 5 h at 30∞C. Cells were pelleted and then resuspended in 0.1 M sodium acetate buffer (pH 5.0) at 2 ¥ 108 cells ml1 . The oxidation of ABTS was assessed spectrophotometrically by measuring the A420 of the supernatant of the cell suspension 30 min after the addition of ABTS. One unit of enzyme activity was defined as 0.01 absorbance units at 30 min. Melanin production by the SOD1 wild-type, sod1 mutant and sod1 + SOD1 reconstituted strains was assayed on Niger seed agar. A 5 ml suspension of 107 cells ml-1 from each strain was placed on the medium and incubated at 30∞C for 4 days.
Cn var. gattii SOD1 wild-type, sod1 mutant and sod1 + SOD1 reconstituted MATa strains and NIH 430 and NIH 191 MATa strains were grown on YPD agar for 48 h at 30∞C, co-cultured on V8 mating medium and incubated at room temperature in the dark for 2–5 days. Haploid filamentation was assayed by incubating spotted suspension of cells on filament agar at 24∞C for up to 4 weeks (Wickes et al., 1996; Yue et al., 1999).
Urease and phospholipase B production. Urease and phospholipase B activities of SOD1 wild-type, sod1 mutant and sod1 + SOD1 reconstituted strains were tested in Christensen’s urea agar and egg yolk agar media as reported in the literature (Chen et al., 1997; Cox et al., 2000). Capsule induction. All strains assayed for capsule production were incubated in YPD medium at 30∞C for 48 h and subsequently inoculated into 10 ml of low-iron medium. After 72 h of shaking (250 r.p.m.) at 30∞C, the cells were mixed with standard India ink and photographed at 400¥ magnification on an Olympus AX70 compound microscope equipped with an Olympus PM-C350 digital camera. Mannitol production. Extracellular mannitol produced by Cn was determined by a published calorimetric method with some modifications (Sanchez, 1998). Cn cells were grown overnight in YPD broth to mid-log phase at 30 ∞C at 180 r.p.m. Approximately 2 ¥ 108 cells were withdrawn and pelleted at 5000 r.p.m. for 2 min. The supernatant (100 ml) was used for the measurement of extracellular mannitol, and we then back-calculated for total extracellular mannitol secreted by 2 ¥ 108 cells. Fresh YPD broth was used as a blank. The assay was performed by mixing 100 ml of sample with 500 ml of 500 mM formate (pH 3.0) and 300 ml of 5 mM sodium periodate in a 1.5 ml microcentrifuge tube. The mixture was allowed to stand at room temperature for 15 s, and then 300 ml of a solution consisting of 100 mM acetyl acetone, 2000 mM acetate and 20 mM sodium thiosulphate was added. This mixture was heated in a boiling water bath for 2 min and then cooled to room temperature, and absorbance was read at 412 nm. D-Mannitol (Sigma) was used for preparation of the standard curve. Each experiment was performed in triplicate on two separate occasions.
Pathogenicity test The pathogenic potential of the sod1 mutant strain, compared with that of SOD1 wild-type and sod1 + SOD1 reconstituted strains, was tested in a mouse model of meningoencephalitis. Briefly, Cn strains were grown in YPD broth for 16 h, washed and resuspended in sterile saline. Cells were counted by trypan blue exclusion using a haemocytometer, and by cfu on YPD agar. Six male BALB/c mice (ª 6 weeks old, 15–20 g) in each group were injected with a suspension of 10 6 cells intravenously. The injected animals were observed for any overt sign of illness, and all sick animals were promptly sacrificed by CO2 inhalation to minimize pain and suffering. Brain tissues from dead mice were cultured on Niger seed agar for Cn recovery. Additionally, the histological lesions in the brain were examined in detail from mice infected with one log less Cn cells (1 ¥ 105) as described for the survival curve experiments, except that all infected animals were euthanized by day 7 (Chaturvedi et al., 2002). Whole brain from two mice from each group was fixed entire in 10% neutral-buffered formalin along with one control brain. Four transverse brain slices containing rostral cortex, caudal cortex, mid-brain and cerebellum with brainstem were processed into paraffin blocks using routine histological methods. The tissue sections were stained with haematoxylin and eosin (H&E) and Mayer’s mucicarmine (Luna, 1992) to stain the capsule bright magenta. Lesions were counted in six brain sections of four slices each from two brain tissues. The values were represented as mean number of lesions ± SD per brain section.
Neutrophil (PMN) fungicidal activity PMNs were isolated from the peripheral blood of normal human volunteers by Ficoll-Paque (Pharmacia LKB Biotechnology) centrifugation. PMNs containing red blood cells (RBCs) were harvested from the bottom of the tube, mixed with 50 ml of lysing solution (0.8% NH4CL, 0.37% disodium EDTA) and incubated at room temperature for 10 min to lyse RBCs. PMNs were washed twice in RPMI (tissue culture medium) and adjusted to the appropriate concentration in the same medium. PMN-induced killing of Cn var. gattii SOD1 wild-type, sod1 mutant and sod1 + SOD1 reconstituted strains was determined as described earlier (Chaturvedi et al., 1996). Aliquots (100 ml) of PMNs, 100 ml of viable Cn cells (5 ¥ 103) and 20 ml of pooled human serum (PHS) were added to the wells of a 96-well tissue culture plate and incubated at 37∞C in 5% CO2-95% air. After incubation for 4 h, the plates were centrifuged at 2000 r.p.m. for 10 min, and the supernatants were carefully aspirated through 27-gauge needles. The effector cells were lysed by the addition of 100 ml © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1681–1694
Cn var. gatti sod1 of 0.05% Triton X-100, and the Cn cells were serially diluted and plated on YPD agar. The YPD plates were incubated at 30∞C for 2–3 days for quantification of cfu. Equivalent amounts of Cn grown in the culture medium without PMNs were treated identically with Triton X-100, and cfu were recorded as a control. The results were expressed as percentage of Cn killed = 1–(cfu experiment/cfu control) ¥ 100. To determine the significance of Cu,Zn SOD in PMN fungicidal activity, we preincubated PMNs for 1 h with SOD enzyme (10 mg ml-1). The controls included PMNs treated with SOD inactivated by autoclaving at 121∞C for 15 min.
Statistical analysis Statistical analysis of the data was performed using INSTAT software for MacIntosh. Student’s t-test was used for comparison of two groups. Results were considered significant at a P-value < 0.05.
Acknowledgements We thank Adriana Verschoor for editorial comments. We also thank three anonymous reviewers for their valuable comments. Nucleotide sequencing was performed at the Molecular Genetics Core, Wadsworth Center. The study was supported financially in part by NIH grants RO1- A48462 (S.C.) and R29-AI41968 (V.C.).
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