| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Michael Smith Laboratories, Department of Microbiology and Immunology, and Faculty of Land and Food Systems, University of British Columbia, Vancouver BC V6T 1Z4, Canada
Correspondence
James W. Kronstad
kronstad{at}msl.ubc.ca
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
A list of primers used in the study, and an amino acid alignment of the Sit1 protein from the serotype D strain B3501A of C. neoformans with fungal orthologues, are available as supplementary data with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Five serotypes (A, B, C, D and AD) are recognized, based on the antigenicity of the polysaccharide capsule, and three varieties have been described: neoformans (D), grubii (A) and gattii (B and C). Several virulence factors have been identified for the fungus, including the polysaccharide capsule, production of melanin by the enzyme laccase, the ability to grow at 37 °C, and survival within macrophages (Casadevall & Perfect, 1998). The polysaccharide capsule is antiphagocytic and suppresses the immune response, while the expression of laccase and melanin formation are necessary for survival within alveolar macrophages, resistance to oxidative stress, and extrapulmonary dissemination to the brain (Bose et al., 2003; Casadevall & Perfect, 1998; Gomez & Nosanchuk, 2003; Janbon, 2004; Liu et al., 1999; Noverr et al., 2004; Perfect, 2005; Williamson, 1997). Capsule and melanin production are regulated by several factors. For example, capsule size is influenced by iron and CO2 levels, serum, and the location of the fungus in host tissue (Bose et al., 2003; Janbon, 2004; Vartivarian et al., 1993; Zaragoza et al., 2003). Melanin synthesis is regulated by iron and copper, and by low glucose levels (Alspaugh et al., 1997; Jacobson & Compton, 1996; Polacheck et al., 1982; Salas et al., 1996; Zhu et al., 2001; Zhu & Williamson, 2004). The cAMP pathway is known to regulate both capsule and melanin, and the PKC1/MAP kinase pathway has also been implicated in melanin production, because loss of the C1 domain of PKC1 leads to reduced laccase activity (Alspaugh et al., 1997; D'Souza et al., 2001; Heung et al., 2005; Hicks et al., 2004).
We are interested in the mechanisms of iron regulation and uptake in C. neoformans. Physiological and genetic studies indicate that iron acquisition is mediated by both high- and low-affinity uptake systems, with a role for cell surface ferric reductase activity, secreted reductants such as 3-hydroxyanthranilic acid, and melanin (Jacobson et al., 1998; Lian et al., 2005; Nyhus et al., 1997; Nyhus & Jacobson, 1999). Based on similarities with Saccharomyces cerevisiae (Stearman et al., 1996), the high-affinity uptake system is believed to be composed of an iron permease, Ftr1, and a multicopper oxidase, Fet3, and iron regulation of these components has been confirmed by transcriptome analysis (Lian et al., 2005). Iron uptake is also potentially mediated by siderophores that are exported from cells to bind ferric iron, with subsequent import. This strategy is common for both fungi and bacteria, and siderophores may allow pathogens to steal iron from host proteins such as transferrin or ferritin, or from nutritional competitors in their environment (Barasch & Mori, 2004; Haas, 2003). However, Jacobson & Petro (1987) were unable to identify extracellular iron chelators and hydroxamate-type siderophores in C. neoformans, although they did demonstrate that the fungus can use the siderophore deferoxamine in the presence of FeCl3.
Specific transporters are required for non-reductive uptake of ferric–siderophore complexes. Interestingly, some fungi, such as the pathogen Candida albicans and S. cerevisiae, have homologues for siderophore transporters, even though they apparently do not possess the enzymes for the synthesis of siderophores (reviewed by Haas, 2003). It is presumed that the transporters allow these fungi to utilize siderophores from competitors. In S. cerevisiae, there are four siderophore transporters: ARN1, ARN2/TAF1, ARN3/SIT1 and ARN4 (Lesuisse et al., 2001; Haas, 2003; Yun et al., 2000a, b). The specificity of some of these transporters has been examined and, although issues of redundancy exist, Arn3p/Sit1p has high affinity for the hydroxamate siderophore ferrioxamine B (FOB; feroxamine), and lesser affinity for ferrichromes and ferricrocin (Kosman, 2003; Lesuisse et al., 2001; Yun et al., 2000a, b). C. albicans contains a similar set of transporters to those found in S. cerevisiae, and one of these, CaArn1/CaSit1, is required for epithelial cell invasion but not for systemic infection (Ardon et al., 2001; Haas, 2003; Heymann et al., 2002; Hu et al., 2002; Kosman, 2003). Aspergillus fumigatus is also thought to have siderophore transporters, and the loss of ability to synthesize siderophores compromises virulence in this fungus (Haas, 2003; Hissen et al., 2005; Schrettl et al., 2004). Recently, we have identified the transcript for a putative siderophore transporter (SIT1) by serial analysis of gene expression (SAGE) in C. neoformans, and have found that the message is elevated in cells grown in low-iron medium (LIM) (Lian et al., 2005). This discovery suggests that C. neoformans may be able to utilize siderophores via a specific transporter or receptor, as predicted by Jacobson & Petro (1987).
In this study, we constructed null mutants for SIT1 in the serotype D strain B3501A and the serotype A strain H99 of C. neoformans, and established that the gene is required by both strains for siderophore (FOB) utilization and growth in a low-iron environment. We also found that SIT1 influences melanization and cell wall structure, and that this role is more pronounced in the serotype D strain B3501A. As has been observed in other studies (Barchiesi et al., 2005; Hicks et al., 2004), our analysis revealed differences in sit1 phenotypes for the strains of the A and D serotypes. We also extended our studies to show that the cAMP pathway influences SIT1 transcript levels. Finally, we found that SIT1 is not required for virulence in a mouse model of cryptococcosis.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Strain B3501A was provided by Dr J. Kwon-Chung (National Institutes of Health, Bethesda. MD). Strains JEC21, JEC43 and H99, and all of the mutants with defects in cAMP signalling components, were provided by Dr J. Heitman (Duke University, Durham, NC). The ura5 mutant of B3501A was isolated by selection on 5-fluoroorotic acid plates. LIM was prepared with 20mM HEPES and 20 mM NaHCO3, as described by Vartivarian et al. (1993). The water for LIM was treated with Chelex-100 resin (Bio-Rad) to chelate iron. LIM+Fe was prepared by addition of 100 µM Fe3+/EDTA (Sigma catalogue ref. EDFS). LIM+bathophenanthroline disulfonic acid (BPDA)+deferoxamine was prepared by addition of 550 µM BPDA (Sigma) and 100 µM deferoxamine (deferrioxamine; Sigma). To measure growth in liquid media, 5 ml yeast extract peptone dextrose (YPD) medium was inoculated with a single colony, and grown overnight at 30 °C in a gyratory shaker at 250 r.p.m. One hundred microlitres of culture was transferred to 5 ml yeast nitrogen base medium, and grown overnight at 30 °C. Cells were washed four times with sterile water, and transferred to flasks containing 50 ml LIM, LIM+Fe or LIM+BPDA+deferoxamine at a concentration of 1x106 cells ml–. The cultures were shaken at 250 r.p.m. at 30 °C, and growth was monitored by measuring OD600 on a Beckman DU530 UV/VIS spectrophotometer with a 1 cm path length.
|
; Yun et al., 2000a, b) to search the C. neoformans genome sequence databases for strains JEC21 (http://www.tigr.org/tdb/e2k1/cna1/), B3501A (http://www-sequence.stanford.edu/group/C.neoformans/index.html) and H99 (http://cneo.genetics.duke.edu). The gene identifications assigned to SIT1 in JEC21 and H99 are CNA07920 and GLEAN05672, respectively. Transmembrane regions for the Sit1 sequence were predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM).
RNA isolation and analysis.
Cells were grown in LIM and LIM+Fe, as described above, harvested by centrifugation, flash-frozen in a dry ice/ethanol bath, and lyophilized overnight at –20 °C. Cell pellets were pulverized with glass beads for 10 min, and the cell powder was resuspended in 15 ml Trizol extraction buffer (Invitrogen). RNA was isolated according to the manufacturer's recommendations, with the additional step of LiCl precipitation at 4 °C after ethanol precipitation. Northern blot preparation and hybridization were performed as described by Sambrook et al. (1989), and all hybridization experiments were performed with two independent preparations of RNA from cells grown in LIM or LIM+Fe. The hybridization probe was a PCR-amplified 409 bp DNA fragment from exon 6 of the SIT1 gene, using primers SITEXON6F and SITEXON6R (all primer sequences are listed in Supplementary Table S1). The probe was labelled with 32P using an oligolabelling kit (Amersham Pharmacia Biotech).
Primers for real-time RT-PCR analysis were designed for each serotype using Primer Express software 3.0 (Applied Biosystems) (Supplementary Table S1). Cell cultures were prepared as described below for the laccase assay. Total RNA was purified with the RNeasy kit (Qiagen), treated with DNase (Qiagen), and cDNA was generated using the SuperScript First-Strand Synthesis system (Invitrogen). PCR reactions contained 750 nM of each primer, 1xPower SYBR Green PCR Master Mix (Applied Biosystems) and 100 ng cDNA in a final volume of 20 µl. PCR reactions were performed on the Applied Biosystems 7500 Fast Real-Time PCR system, with 50 °C incubation for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative gene expression was quantified using the SDS software 1.3.1 (Applied Biosystems), based on the 2–
CT method (Livak & Schmittgen, 2001), and ACT1 was used as an endogenous control for normalization.
Construction of sit1 : : URA5 (serotype D) and sit1 : : NEO (serotype A) alleles.
A PCR overlap strategy was used to prepare a deletion allele for strain B3501A (Davidson et al., 2002). DNA fragments containing the 5' (742 bp) and 3' (853 bp) regions flanking the SIT1 gene were amplified from genomic DNA from strain JEC21. Note that JEC21 and B3501A are closely related strains (Loftus et al., 2005). The primers adjacent to the 5' and 3' regions of the gene were SIT1A and SIT1C, and SIT1D and SIT1F, respectively. The URA5 marker was amplified from plasmid pJHM973 using hybrid primers SIT1B and SIT1E for SIT1 and URA5, respectively. The three fragments were joined by overlapping PCR (Davidson et al., 2002) using primers SIT1A and SIT1F. For strain H99, a modified overlap strategy was employed that increased the yield of the final construct (Yu et al., 2004). DNA fragments containing the 5' (1151 bp) and 3' (1162 bp) regions flanking the SIT1 gene were amplified from genomic DNA of H99. The primers adjacent to the 5' and 3' regions of the gene were SITA1 and SITA3, and SITA4 and SITA6, respectively. The neomycin cassette was amplified from pJAF1 using hybrid primers SITA2 and SITA5 for SIT1 and NEO, respectively. Fragments were used in second- and third-round PCR reactions, as described by Yu et al., 2004. The nested primers used in the third round of PCRs were SITANF and SITANR. The DNA constructs were introduced into the appropriate parent strains by biolistic transformation (Toffaletti et al., 1993). Transformants were screened by colony PCR using primers SIT1UP and 6328 for strain B3501A, and SITAUP and NEOPS for strain H99. Transformants in which the wild-type (wt) allele was replaced were confirmed by Southern analysis using the 5' fragment (generated with primers SIT1A and SIT1C) as a probe; the absence of SIT1 transcripts in the mutants was confirmed by quantitative real-time PCR. Two mutants for each strain were retained for analysis. To complement the mutations, the wt SIT1 gene was reintroduced by biolistic transformation into one mutant of each strain background, on the vector pCH233 that confers resistance to nourseothricin (NAT).
Plate assays.
Siderophore utilization assays were performed as described by Heymann et al. (2002). Plates of ferrous-iron-free medium (IFM) were prepared in the same manner as for LIM, with the addition of 20 g Bacto-agar l–1 and 550 µM BPDA chelator. Two hundred microlitres of cell suspension containing 107 c.f.u. ml–1 were spread on IFM, and a disk of Whatman paper saturated with 10 µl 100 µM deferoxamine or ferrichrome (Sigma) was placed in the centre of the plate. Plates were incubated for 2 days at 30 °C, and zones of growth were recorded by digital photography. Melanin assays were performed on 3,4-dihydroxyphenylalanine (DOPA; Sigma) medium that was prepared with 20 g Bacto agar l–1 in 900 ml distilled water. Separately, 1 g L-asparagine (Sigma), 1 g glucose, 3 g KH2PO4, 0.25 g MgSO4.7H2O and 200 mg DOPA were dissolved in 100 ml distilled H2O and the pH was adjusted to 5.6. One milligram of thiamine/HCl (Sigma) and 5 µg biotin (Sigma) were added, the mixture was filter-sterilized, and then added to the autoclaved agar after cooling to 50 °C. The following modifications were made to DOPA medium prior to filter-sterilization to test various parameters: 4x DOPA contained 4 mM instead of 1 mM DOPA (Sigma), and 4xDOPA+glucose contained 4 mM DOPA and 1.0 % glucose (instead of 0.1 %). To test for melanin production, 10 µl 107 c.f.u. ml–1 cell suspension was spotted on the plates and pigment was assessed after 2 days incubation at 30 °C. Temperature sensitivity assays were performed on low-glucose asparagine (LGA) medium and on DOPA medium. Tenfold serial dilutions of cells (5 µl) were spotted on either LGA or DOPA plates, with an initial inoculum of 107 c.f.u. ml–1. Plates were incubated for 2 days at 30 or 37 °C. Cell wall integrity assays were performed as above, except that 0.015 % SDS, 300 µg Congo red ml–1 or 30 µg calcofluor white ml–1 was added to the medium. All experiments were repeated at least three times.
Laccase assays.
Laccase assays were performed by inoculating 50 ml LGA (asparagine medium with 0.1 % glucose) with a single colony and growing the cells for 2 days at 30 °C. Cells were washed three times with 50 ml sterile deionized water, resuspended in 50 ml asparagine medium with no glucose, and grown for 4 h at 30 °C. In experiments to examine the effect of metal availability on laccase activity or LAC1 transcript levels, 100 µM CuSO4 or 100 µM Fe3+/EDTA was included during the 4 h incubation. Cells were pelleted and washed three times with 50 ml 0.05 M NaPO4 and resuspended at 108 c.f.u. ml–1. To measure activity, 108 (1 ml) cells were incubated with 1 mM DOPA for 30 min at 30 °C, the cells were pelleted, and A475 of the supernatant was recorded (A475 0.001 corresponds to 1 U laccase). The laccase assays were repeated three times.
Microscopy.
Transmission electron microscopy (TEM) was performed on a Hitachi H7600 microscope. The cells were grown, as described for the DOPA plate assays, on DOPA or LGA medium for 4 days at 30 °C. The full 10 µl spot of cell growth from DOPA or LGA medium was resuspended in 1 ml water, vortexed for 10 min, and then centrifuged for 5 min at 13 200 r.p.m. Cells were fixed in 2.5 % glutaraldehyde in 0.05 M cacodylate buffer at 28 °C under vacuum. Microwave (Ted Pella Microwave) processing was employed at 100 W, with 2 min on and 2 min off (repeated twice). Samples were washed twice with cacodylate buffer at 28 °C for 40 s, using power level 2. Cells were fixed in osmium tetroxide at 28 °C under vacuum, by microwave processing at 100 W, 2 min on and 2 min off (repeated twice). Samples were dehydrated in increasing concentrations of 50, 70, 90 and 100 % ethanol (the final step was repeated three times). Samples were infiltrated with Spurrs resin in increasing ratios of Spurrs : acetone, 1 : 3, 1 : 1 and 3 : 1, at vacuum power level 3 for 3 min. Resin was polymerized by baking the sample overnight at 60 °C. Samples were then cut into 70 nm sections on a Leica Ultracut T Ultramicrotome, and stained with 2 % uranyl acetate for 14 min and lead citrate for 7 min.
Virulence assays.
Cells were prepared by inoculating 5 ml YPD with a single isolated colony, followed by overnight growth on a gyratory shaker (250 r.p.m) at 30 °C. Cells were washed three times with sterile PBS and resuspended in 5 ml PBS. Cells were counted with a haemocytometer, and counts were confirmed by plating serial dilutions of strains on YPD agar, with subsequent growth for 2 days at 30 °C to determine c.f.u. The serotype D strain B3501A and the corresponding sit1 mutant were tested initially using DBA1 mice (16–20 g; Jackson Laboratories) with a defect in C3 complement. Five mice were used for wt B3501A, and four mice were inoculated with the sit1 mutant. The mice were inoculated by intranasal inhalation of 5x106 cells in 50 µl PBS per mouse. For serotype A, A/Jcr mice (16–20 g; NIH Program) were infected intranasally with an inoculum of 5x104 fungal cells in 50 µl PBS per mouse. Ten mice were used for each of the wt, sit1 and sit1+SIT1 strains. An additional experiment was performed using a 10-fold smaller inoculum of 5x103 cells in 50 µl PBS, and five mice per strain. Animals that appeared moribund or in pain were euthanized.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Examination of the gene indicated that it contained five introns and six exons, and encoded a predicted polypeptide of 604 aa with 13 putative transmembrane regions. We compared Sit1 to related sequences in other fungi and found that the closest orthologue, Arn3/Sit1 of S. cerevisiae, showed 39 % identity and 55 % similarity, resulting in an e value of 10–112 using the BLASTP algorithm (Altschul et al., 1990). SIT1 was also the best match with two of the three other siderophore transporter genes (ARN1 and ARN2) of S. cerevisiae. This was true in searches of all three C. neoformans genomes (strains JEC21, B3501A and H99), although we did identify three other candidates for siderophore transporter genes in each genome. Alignments of the Sit1 amino acid sequence with Arn3/Sit1 and orthologues from other fungi are presented in Supplementary Fig. S1. To begin a functional analysis of SIT1, we used RNA blot analysis to confirm that the transcript was elevated in cells from LIM in strains B3501A and H99 (serotype A) (Fig. 1).
|
) (Davidson et al., 2002; Varma et al., 1992; Yu et al., 2004; Methods). Two independent transformants were obtained and characterized for both B3501A and H99. Replacement of the wt allele by the appropriate marker in the mutants was confirmed by colony PCR and Southern analysis (Fig. 2). The sit1 mutants were reconstituted by transformation with the wt SIT1 gene from strain JEC21 cloned into a vector containing an NAT-resistance gene.
|
) to establish conditions in which the cells must use ferric iron sequestered from the medium by deferoxamine. Under these conditions, zones of growth were apparent for all wt and reconstituted strains, but not for any of the sit1 mutants at 2 or 7 days (Fig. 3). These assays indicate that the sit1 mutants were unable to use FOB as a sole source of iron. In S. cerevisiae, Arn3/Sit1 can mediate the uptake of FOB or ferrichrome (Lesuisse et al., 1998; Yun et al., 2000a, b). We therefore tested our strains in the same plate assays with ferrichrome in place of deferoxamine, and we observed growth for all of the strains (data not shown). This result indicates that SIT1 is not required for ferrichrome utilization, although it is not possible to rule it out as a substrate from this experiment.
|
). None of the strains (wt or mutant) was able to grow in LIM with the BPDA chelator (data not shown), and all strains showed similar growth patterns in LIM+Fe, although growth was slightly delayed for sit1 mutants (wt levels were achieved by 36 h; Fig. 4). The wt H99 strain also showed reduced growth in LIM (OD600 
5 at 54 h) compared with that of strain B3501A (OD600 8 at 54 h), suggesting that this strain may be more sensitive to low-iron conditions. Overall, these results indicate that Sit1 plays a role in iron acquisition in a low-iron environment, and in the use of FOB as a sole iron source.
|
; Jacobson & Compton, 1996; Jacobson, 2000). Therefore, we tested the ability of the sit1 mutants to produce melanin, and found enhanced melanization in the B3501A mutant grown on agar medium containing the substrate DOPA (Fig. 5a). In particular, the mutant produced melanin more rapidly, and the surface of the colony appeared rougher, with a higher sheen compared to that of the wt (Fig. 5a). In addition, we noted that B3501A wt cells were able to invade the agar, whereas the sit1 mutant could not (data not shown). No obvious change in melanization was observed for the H99 sit1 mutants compared to that for the wt (Fig. 5a). We also tested a higher substrate concentration to determine if wt melanization would be enhanced to match the sit1 mutant phenotype (Fig. 5a). We found that a fourfold higher level of DOPA enhanced melanin formation in strain B3501A but not in H99, suggesting that substrate availability may have been a factor in the melanization difference between the B3501A sit1 mutant and the wt. We also found that 1.0 % glucose could repress melanin production in all strains, indicating that the sit1 mutation did not affect the known negative regulatory influence of glucose (Fig. 5a) (Nurudeen & Ahearn, 1979; Pukkila-Worley et al., 2005).
|
). These results indicated that laccase activity was affected by the loss of SIT1 in each strain, even though no difference in melanization was noted for the sit1 mutant of strain H99 grown on solid medium. It is possible that the variation in laccase activity was the result of differences in metal availability to the enzyme between mutant and wt cells. That is, loss of SIT1 may have influenced metal homeostasis in the cells, resulting in a change in laccase activity due to metal loading on the copper-requiring enzyme. To test this hypothesis, we repeated the assays with cells incubated in the presence of added copper, and found that laccase activity was elevated in the wt and reconstituted strains, matching the level in the mutants (Fig. 5c). These results suggest that loss of SIT1 influences copper loading on laccase, perhaps through altered metal homeostasis and/or changes in laccase expression or localization (normally in the cell wall) between mutant and wt strains.
We also examined whether the changes in laccase activity could have been due in part to differences in transcript levels between mutant and wt cells. Real-time PCR was used to measure laccase (LAC1) transcript levels in wt, mutant and reconstituted cells grown in low-glucose medium, with or without the addition of iron or copper (see Methods). As shown in Fig. 5(d), no differences in LAC1 transcript levels were found for the H99 strains grown under any conditions, or for the B3501A strains grown in medium without metal supplementation. However, higher transcript levels were found in the B3501A mutant grown in medium with iron or copper compared with those in the wt or reconstituted strains. These findings suggest that loss of SIT1 influences laccase expression at the transcript level in the serotype D strain, and that metal repletion is important for this effect.
Loss of SIT1 in the serotype D background results in temperature sensitivity and altered cell wall structure
We were intrigued by the idea that loss of SIT1 might influence the localization of laccase, as suggested by the influence of substrate concentration and metal availability described above. Given the location of laccase in the cell wall, we investigated whether the mutants showed differences in wall integrity. First, we tested growth in the presence of calcofluor white, SDS and Congo red on low-glucose or DOPA plates. Only slight sensitivity to these compounds was observed for the B3501A sit1 mutant compared with that for the wt, and no difference was noted for the H99 strains (data not shown). The cells were also tested for temperature sensitivity (a phenotype associated with cell wall integrity) by spotting serial dilutions of cells on DOPA plates (Fig. 6). The sit1 mutants showed slightly less growth than wt at 30 °C, and reduced growth at 37 °C for the B3501A mutant (Fig. 6). The temperature sensitivity was particularly apparent in the B3501A background, and no difference was noted for the H99 strains (Fig. 6). Similar results were obtained in medium without DOPA (data not shown). The temperature sensitivity of the sit1 mutant in the B3501A background was confirmed in growth assays in liquid LGA medium (data not shown).
|
). The cell walls of the wt and reconstituted cells contained distinct and dense concentric rings, and ordered polysaccharide fibrils, consistent with the observations of Eisenman et al. (2005). In contrast, the mutant walls appeared to be less dense, and they varied in thickness, with less ordered fibrils. The mutant cells in the B3501A strain were also easily distorted by the TEM processing (Fig. 7). For example, among a sample of 40 cells examined for each strain, 89 % of sit1 mutant cells from strain B3501A were distorted (not spherical) versus 7.4 % of the wt cells (data not shown). An independent preparation for TEM with prolonged dehydration steps reduced the distortion in the mutant cells, but the defect was still more apparent in the mutant cells (data not shown). These results suggest that loss of sit1 caused an altered organization of the cell wall in strain B3501A. Although the mutant cells in the H99 background appeared to have a less dense wall, they did not show the same distortion upon processing; this is consistent with the lack of changes in melanization, and temperature and cell wall sensitivity, in the H99 mutants. Finally, the differences in the cell wall noted for the sit1 mutants were apparent by TEM even in the non-melanized cells grown on LGA plates (data not shown). Taken as a whole, these results suggest that cell wall changes were present in the sit1 mutants, and that these were particularly apparent in the serotype D strain B3501A.
|
). We were therefore interested in the effect of the cAMP pathway on siderophore utilization in C. neoformans, especially given the influence of the pathway on melanin. We obtained mutants defective in the genes encoding the catalytic subunits of PKA in both serotype A and serotype D strains (D'Souza et al., 2001; Hicks et al., 2004), and used RT-PCR to measure SIT1 transcript levels. We found that a defect in pka1, but not pka2, resulted in elevated SIT1 transcripts in strain JEC21 (a close relative of B3501A), while both the pka1 and the pka2 mutants showed elevated transcripts in strain H99 (Fig. 8). These results suggest that similarities exist between S. cerevisiae and C. neoformans in the regulation of siderophore transport expression by PKA. The possible connection between siderophore utilization (perhaps involving SIT1) and the cAMP pathway prompted us to examine the sit1 mutants for defects in other traits associated with cAMP signalling, including capsule formation (D'Souza et al., 2001) and mating (Alspaugh et al., 2002). No difference in capsule size or induction was noted between any of the wt or mutant cells grown in LIM and LIM+Fe conditions, suggesting that SIT1 is not required for this trait (data not shown). Similarly, mating tests with serotype A or D MATa strains and the corresponding MAT
sit1 mutants indicated that no unilateral mating defect was caused by the loss of SIT1 (data not shown).
|
20±2 days) were noted between these strains (data not shown). Fungal cell counts from brain and lung tissue were performed for two mice for each of the wt and the mutant strains, and one mouse for the sit1+SIT1 reconstituted strain. Cell counts were equivalent for the brains [105 cells (g tissue)–1] and lungs [106 cells (g tissue)–1] for all of the infections, further indicating that SIT1 is not necessary for extrapulmonary dissemination in this model. The sit1 mutant cells isolated from the brain were retested in the siderophore utilization assay to confirm that the strain was unable to use deferoxamine (data not shown). In a second trial, a 10-fold lower inoculum of 5x103 cells was used with five mice per strain. Again, there was no difference in virulence between sit1 mutants and wt strains, although the mice survived slightly longer with the lower inoculum (26–27 days; data not shown). Overall, these results indicated that SIT1 is not required for virulence in this model. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Siderophore production by C. neoformans has not been detected (Jacobson & Petro, 1987), but the fungus may use transporters to capitalize on siderophores produced by nutritional competitors in the environment. We therefore characterized the putative siderophore transporter gene SIT1, and found that the gene is essential for the use of FOB, but not ferrichrome, as a sole source of iron. In addition, the lack of growth of sit1 mutants in LIM may indicate a connection to components of the high- or low-affinity uptake systems, such as the iron permease FTR1 or the multicopper oxidase FET3 (Lian et al., 2005). With regard to the use of FOB, our observations suggest that none of the other candidate genes for siderophore transporters is functionally redundant with SIT1 under the conditions tested. However, the interpretation of redundancy can be complicated, as demonstrated by attempts to examine the specificity of the four siderophore transporters in S. cerevisiae (discussed by Kosman, 2003; Lesuisse et al., 2001; Yun et al., 2000a, b). In brief, it appears that there is considerable redundancy among the four transporters, although there is some specificity of Sit1 for FOB and Taf1/Arn2 for triacylfusarinine. In addition, variation is observed when the influence of transporter deletions is assessed in different strain backgrounds or laboratories. This suggests that transporter specificity may be a multigenic trait (Kosman, 2003). Given the variation that we have already observed between serotype A and D strains, the evaluation of specificity for Sit1 in C. neoformans and for the other candidates for siderophore transporters will likely require careful analysis of single and multiple deletion mutants in a single strain background.
Connections between Sit1, melanin, cell wall and signalling pathways
Loss of Sit1 in the B3501A background resulted in enhanced melanin formation, and increased laccase activity was seen for the sit1 mutants in both B3501A and H99. Our results suggest that loss of SIT1 results in increased laccase activity by multiple mechanisms, including transcription of the LAC1 gene, access of the enzyme to substrate (perhaps due to altered localization or cell wall structure), and copper loading of the enzyme. LAC1 transcription is known to be responsive to iron and copper (Zhu et al., 2003), and our observations are consistent with a role for Sit1 in metal homeostasis. Additionally, Walton et al. (2005) have demonstrated the importance of copper and iron homeostasis for melanization, through an insertional mutagenesis approach to identify mutants with reduced melanin production. Specifically, they have identified mutations in C. neoformans homologues of the copper transporter Ccc2 and the copper chaperone Atx1, along with a chitin synthase gene and two transcriptional regulators. The mutants defective in CCC2 or ATX1 fail to grow on medium with an iron chelator, perhaps due to a role in providing copper to iron-uptake functions, such as the Fet3 multicopper oxidase. The role of the chitin synthase gene is interesting and provides a link between cell wall defects and laccase localization. Mutants with a defect in the chitin synthase gene give a ‘leaky’ melanin phenotype, likely due to a failure of laccase retention in the wall (Walton et al., 2005).
Our TEM observations suggest a reduced density for the cell wall in the sit1 mutants in both strain backgrounds. A recent description of the microstructure of the wall of C. neoformans has revealed that melanin is present in concentric layers of spherical granular particles with a diameter of 40–130 nm (Eisenman et al., 2005). In light of this description, it is possible that SIT1 contributes to laccase localization and therefore to melanin deposition in the wall. However, changes in the cell wall were noted even in the absence of the melanin substrate, and were also observed in strain H99, in which loss of SIT1 did not noticeably affect melanin production (although laccase activity was slightly increased). Therefore, it seems that melanin localization is not the sole cause of the cell wall changes. Although further analysis is needed, it is possible that Sit1 may play a role in the exocytosis or endocytosis of components such as laccase, or factors required for laccase activity, to the cell wall, particularly in the serotype D strain. Connections between siderophore transporters, trafficking and the cell wall have been established in S. cerevisiae. For example, Arn1 cycles from the plasma membrane to endosomes, depending on the concentration of siderophore (Kim et al., 2002). Additionally, Arn3 has been shown recently to interact with a cell wall protein, Sed1, and a role for the cell wall Fit proteins in siderophore transport has been established in yeast (Park et al., 2005; Protchenko et al., 2001).
Our observations on the influence of copper addition on LAC1 transcription and laccase activity in the sit1 mutants are similar to the results reported by Zhu et al. (2003). They have found that a defect in vacuolar acidification, due to deletion of the VPH1 gene (encoding a vacuolar H+-ATPase), leads to a defect in copper assembly into laccase. The mutants properly translate, secrete and localize laccase, but the enzyme is inactive. Copper addition restores laccase activity and results in increased transcription of the LAC1 gene. They discuss the possibility that insertion of copper into laccase, perhaps in the outer Golgi network where the ATPase is located, might require acidification. The similarities with the influence of Sit1 on laccase support the hypothesis that Sit1 is important for metal homeostasis, perhaps through a role in trafficking or storing iron.
The influence of Sit1 on melanin production suggests possible connections with known regulators of this process in C. neoformans. For example, the PKC1/MAPK pathway has been recently implicated in melanization because the loss of the C1 domain (for diacylglycerol binding) of PKC1 leads to loss of laccase activity and melanin production (Heung et al., 2005). The C1 mutants display a similar phenotype to the serotype D sit1 mutant, in that they have disorganized melanization (due to displacement of laccase) and reduced cell wall integrity. The cAMP pathway also controls laccase expression (D'Souza et al., 2001; Pukkila-Worley et al., 2005), and we found that mutations in the genes encoding the catalytic subunits of PKA influenced SIT1 transcript levels. These results suggest that, as in S. cerevisiae, PKA negatively regulates some iron-uptake functions (Robertson et al., 2000). In this regard, SIT1 will be a useful target gene for future analysis of gene expression, including transcription factor characterization, because of the co-regulation of transcript levels by the cAMP pathway and iron.
SIT1 and fungal infection
We found that SIT1 was not required for virulence in a murine inhalation model of cryptococcosis, even though the sit1 mutants showed poor growth in LIM. We were prompted to test the virulence of the sit1 mutants because of observations in bacterial (Ratledge & Dover, 2000) and other fungal pathogens. Specifically, a siderophore transporter is not required for the virulence of C. albicans in a murine systemic infection model, but is required for epithelial cell invasion (Heymann et al., 2002). In contrast, the high-affinity iron permease Ftr1 is required for systemic infection with C. albicans (Ramanan & Wang, 2000). Iron removal from holotransferrin may be siderophore-mediated in A. fumigatus, and the SidA gene that encodes an ornithine monooxygenase involved in siderophore synthesis is essential for virulence of A. fumigatus (Hissen et al., 2004, 2005; Schrettl et al., 2004). However, reductive iron assimilation by the high-affinity uptake system through FtrA is not required for virulence. There are a number of explanations for why SIT1 may not be required for virulence in C. neoformans. First, C. neoformans does not appear to produce its own siderophores (Jacobson & Petro, 1987), so it is unclear whether a transporter would be useful during infection. Second, even if siderophores were available in the host, other siderophore transporters might contribute to iron acquisition in place of Sit1. That is, there are issues of specificity and redundancy to consider, and we have so far only found that Sit1 can promote use of the bacterial siderophore FOB (not expected to be present in the host). Finally, it has recently been shown in C. albicans that the reductive iron-uptake system is responsible for the acquisition of iron that is tightly bound to ferritin (Knight et al., 2005). If the same were true for C. neoformans, then the functional FTR1/FET3 system in the sit1 mutants might effectively acquire iron from ferritin in the mouse. Generally, it is possible that sufficient iron is available in vivo to allow survival of the fungus by other iron-uptake mechanisms, even in the absence of Sit1. An exploration of the role of other uptake systems (e.g. Ftr1/Fet3) in virulence is currently under way.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: N. L. Glass
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alspaugh, J. A., Pukkila-Worley, R., Harashima, T., Cavallo, L. M., Funnell, D., Cox, G. M., Perfect, J. R., Kronstad, J. W. & Heitman, J. (2002). Adenylyl cyclase functions downstream of the G alpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans. Eukaryot Cell 1, 75–84.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef][Medline]
Ardon, O., Bussey, H., Philpott, C., Ward, D. M., Davis-Kaplan, S., Verroneau, S., Jiang, B. & Kaplan, J. (2001). Identification of a Candida albicans ferrichrome transporter and its characterization by expression in Saccharomyces cerevisiae. J Biol Chem 276, 43049–43055.
Barasch, J. & Mori, K. (2004). Cell biology: iron thievery. Nature 432, 811–813.[CrossRef][Medline]
Barchiesi, F., Cogliati, M., Esposto, M. C., Spreghini, E., Schimizzi, A. M., Wickes, B. L., Scalise, G. & Viviani, M. A. (2005). Comparative analysis of pathogenicity of Cryptococcus neoformans serotypes A, D and AD in murine cryptococcosis. J Infect 51, 10–16.[CrossRef][Medline]
Bose, I., Reese, A. J., Ory, J. J., Janbon, G. & Doering, T. L. (2003). A yeast under cover: the capsule of Cryptococcus neoformans. Eukaryot Cell 2, 655–663.
Casadevall, A. & Perfect, J. R. (1998). Cryptococcus neoformans. Washington, DC: American Society for Microbiology.
Cowart, R. E., Singleton, F. L. & Hind, J. S. (1993). A comparison of bathophenanthrolinedisulfonic acid and ferrozine as chelators of iron(II) in reduction reactions. Anal Biochem 211, 151–155.[CrossRef][Medline]
Davidson, R. C., Blankenship, J. R., Kraus, P. R., de Jesus Berrios, M., Hull, C. M., D'Souza, C., Wang, P. & Heitman, J. (2002). A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148, 2607–2615.
D'Souza, C. A., Alspaugh, J. A., Yue, C., Harashima, T., Cox, G. M., Perfect, J. R. & Heitman, J. (2001). Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans. Mol Cell Biol 21, 3179–3191.
Eisenman, H. C., Nosanchuk, J. D., Webber, J. B., Emerson, R. J., Camesano, T. A. & Casadevall, A. (2005). Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans. Biochemistry 44, 3683–3693.[CrossRef][Medline]
Gomez, B. L. & Nosanchuk, J. D. (2003). Melanin and fungi. Curr Opin Infect Dis 16, 91–96.[Medline]
Haas, H. (2003). Molecular genetics of fungal siderophore biosynthesis and uptake: the role of siderophore in iron uptake and storage. Appl Microbiol Biotechnol 62, 316–330.[CrossRef][Medline]
Heung, L. J., Kaiser, A. E., Luberto, C. & Del Poeta, M. (2005). The role and mechanism of diacylglycerol-protein kinase C1 signaling in melanogenesis by Cryptococcus neoformans. J Biol Chem 280, 28547–28555.
Heymann, P., Gerads, M., Schaller, M., Dromer, F., Winkelmann, G. & Ernst, J. F. (2002). The siderophore iron transporter of Candida albicans (Sit1p/Arn1p) mediates uptake of ferrichrome-type siderophores and is required for epithelial invasion. Infect Immun 70, 5246–5255.
Hicks, J. K., D'Souza, C. A., Cox, G. M. & Heitman, J. (2004). Cyclic AMP-dependent protein kinase catalytic subunits have divergent roles in virulence factor prodution in two varieties of the fungal pathogen Cryptococcus neoformans. Eukaryot Cell 3, 14–26.
Hissen, A. H., Chow, J. M., Pinto, L. J. & Moore, M. M. (2004). Survival of Aspergillus fumigatus in serum involves removal of iron from transferrin: the role of siderophores. Infect Immun 72, 1402–1408.
Hissen, A. H., Wan, A. N., Warwas, M. L., Pinto, L. J. & Moore, M. M. (2005). The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence. Infect Immun 73, 5493–5503.
Hu, C. J., Bai, C., Zheng, X. D., Wang, Y. M. & Wang, Y. (2002). Characterization and functional analysis of the siderophore-Fe transporter CaArn1p in Candida albicans. J Biol Chem 277, 30598–30605.
Jacobson, E. S. (2000). Pathogenic roles for fungal melanins. Clin Microbiol Rev 13, 708–717.
Jacobson, E. S. & Compton, G. M. (1996). Discordant regulation of phenoloxidase and capsular polysaccharide in Cryptococcus neoformans. J Med Vet Mycol 34, 289–291.[Medline]
Jacobson, E. S. & Petro, M. J. (1987). Extracellular iron chelation in Cryptococcus neoformans. J Med Vet Mycol 25, 415–418.[Medline]
Jacobson, E. S., Goodner, A. P. & Nyhus, K. J. (1998). Ferrous iron uptake in Cryptococcus neoformans. Infect Immun 66, 4169–4175.
Janbon, G. (2004). Cryptococcus neoformans capsule biosynthesis and regulation. FEMS Yeast Res 4, 765–771.[CrossRef][Medline]
Kim, Y., Yun, C.-W. & Philpott, C. C. (2002). Ferrichrome induces endosome to plasma membrane cycling of the ferrichrome tranporter, Arn1p, in Saccharomyces cerevisiae. EMBO J 21, 3632–3642.[CrossRef][Medline]
Knight, S. A., Vilaire, G., Lesuisse, E. & Dancis, A. (2005). Iron acquisition from transferrin by Candida albicans depends on the reductive pathway. Infect Immun 73, 5482–5492.
Kosman, D. J. (2003). Molecular mechanisms of iron uptake in fungi. Mol Microbiol 47, 1185–1197.[CrossRef][Medline]
Lesuisse, E., Simon-Casteras, M. & Labbe, P. (1998). Siderophore-mediated iron uptake in Saccharomyces cerevisiae: the SIT1 gene encodes a ferrioxamine B permease that belongs to the major facilitator superfamily. Microbiology 144, 3455–3462.[Abstract]
Lesuisse, E., Blaiseau, P. L., Dancis, A. & Camadro, J. M. (2001). Siderophore uptake and use by the yeast Saccharomyces cerevisiae. Microbiology 147, 289–298.
Lian, T. S., Simmer, M. I., D'Souza, C. A., Steen, B. R., Zuyderduyn, S. D., Jones, S. J., Marra, M. A. & Kronstad, J. W. (2005). Iron-regulated transcription and capsule formation in the fungal pathogen Cryptococcus neoformans. Mol Microbiol 55, 1452–1472.[CrossRef][Medline]
Liu, L., Tewari, R. P. & Williamson, P. R. (1999). Laccase protects Cryptococcus neoformans from antifungal activity of alveolar macrophages. Infect Immun 11, 6034–6039.
Livak, K. J. & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(–delta delta C(T)) method. Methods 25, 402–408.[CrossRef][Medline]
Loftus, B. J., Fung, E., Roncaglia, P., Rowley, D., Amedeo, P., Bruno, D., Vamathevan, J., Miranda, M., Anderson, I. J. & other authors (2005). The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307, 1321–1324.
Noverr, M. C., Williamson, P. R., Fajardo, R. S. & Huffnagle, G. B. (2004). CNLAC1 is required for extrapulmonary dissemination of Cryptococcus neoformans but not pulmonary persistence. Infect Immun 3, 1693–1699.
Nurudeen, T. A. & Ahearn, D. G. (1979). Regulation of melanin production by Cryptococcus neoformans. J Clin Microbiol 10, 724–729.
Nyhus, K. J. & Jacobson, E. S. (1999). Genetic and physiologic characterization of ferric/cupric reductase constitutive mutants of Cryptococcus neoformans. Infect Immun 5, 2357–2365.
Nyhus, K. J., Wilborn, A. T. & Jacobson, E. S. (1997). Ferric iron reduction by Cryptococcus neoformans. Infect Immun 65, 434–438.[Abstract]
Park, Y. S., Jeong, H. S., Sung, H. C. & Yun, C. W. (2005). Sed1p interacts with Arn3p physically and mediates ferrioxamine B uptake in Saccharomyces cerevisiae. Curr Genet 47, 150–155.[CrossRef][Medline]
Perfect, J. R. (2005). Cryptococcus neoformans: a sugar-coated killer with designer genes. FEMS Immunol Med Microbiol
), two sit1 : : URA5 disruption mutants of B3501A (b-s10, 
; b-s42, 
), and two reconstituted strains of B3501A (sit1 : : URA5+SIT1) (b-s42R1 and b-s42R2, x). Comparison is also shown in (b, d, f) with strain H99 (wt, 
