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Infection and Immunity, February 2007, p. 714-722, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.01351-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cell Wall Targeting of Laccase of Cryptococcus neoformans during Infection of Mice

Scott R. Waterman,¹ Moshe Hacham,¹ John Panepinto,¹ Guowu Hu,¹ Soowan Shin,¹ and Peter R. Williamson^1,2*

Section of Infectious Diseases, Department of Medicine, University of Illinois at Chicago College of Medicine, Chicago, Illinois,¹ Jesse Brown VA Medical Center, Chicago, Illinois²

Received 22 August 2006/ Returned for modification 16 September 2006/ Accepted 1 November 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Laccase is a major virulence factor of the pathogenic fungus Cryptococcus neoformans, which afflicts both immunocompetent and immunocompromised individuals. In the present study, laccase was expressed in C. neoformans lac1 cells as a fusion protein with an N-terminal green fluorescent protein (GFP) using C. neoformans codon usage. The fusion protein was robustly localized to the cell wall at physiological pH, but it was mislocalized at low pH. Structural analysis of the laccase identified a C-terminal region unique to C. neoformans, and expression studies showed that the region was required for efficient transport to the cell wall both in vitro and during infection of mouse lungs. During infection of mice, adherence to alveolar macrophages was also associated with a partial mislocalization of GFP-laccase within cytosolic vesicles. In addition, recovery of cryptococcal cells from lungs of two strains of mice (CBA/J and Swiss Albino) later in infection was also associated with cytosolic mislocalization, but cells from the brain showed almost exclusive localization to cell walls, suggesting that there was more efficient cell wall targeting during infection of the brain. These data suggest that host cell antifungal defenses may reduce effective cell wall targeting of laccase during infection of the lung but not during infection of the brain, which may contribute to a more predominant role for the enzyme during infection of the brain.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cryptococcus neoformans is a basidiomycete fungal pathogen that infects both immunocompetent and immunocompromised individuals (2, 34). This fungus has several characteristics that allow it to survive during pathogenesis, and it produces numerous factors that cause host cell damage. These characteristics and factors include the ability to grow at 37°C, production of a polysaccharide capsule, expression of laccase, urease, and phospholipase, and production of mannitol (4, 5, 10, 27, 32, 35). The relative importance of each of the characteristics is not fully understood and may be strain dependent; however, the presence of capsule and the presence of laccase are considered major virulence factors.

Laccase expression has been correlated with virulence in numerous studies using multiple strains of the fungus (15, 29, 32). Laccase has been proposed to contribute to virulence through production of melanin pigments and prevention of iron-dependent Fenton products, with resultant accentuation of extrapulmonary dissemination to the brain (14, 19, 26, 36). However, laccase plays little role in pulmonary persistence (26). C. neoformans has two laccase genes in its genome, LAC1 and LAC2, but only LAC1 is expressed significantly under most conditions and deletion of LAC2 results in no reduction in virulence in mice (31, 41). Laccase from C. neoformans is a member of a class of multicopper oxidases that are primarily extracellular proteins expressed in fungi, plants, and insects (24). The main functions in these organisms are polymerization of monolignols to produce the lignin structure of the cell wall of plants, decomposition of lignin during tree rotting by lignolytic mushrooms, and protection from microbial pathogens and insects. In C. neoformans, laccase presumably breaks down lignin from the rotting trees in the environment that comprise its ecological niche (16), which may explain its location in the cell wall, which allows easy access to lignin polymers (39). This peripheral cell wall location is unique for a laccase and may have also evolved to facilitate protection against killing by environmental amoebae. It has been proposed that intracellular residence in amoebae was an intermediate reservoir that facilitated evolutionary pressures leading to macrophage subterfuge and eventual success as an opportunistic pathogen (25, 33). Since properties leading to cell wall targeting of laccase are poorly understood, we studied laccase targeting to the cell wall in vitro and during infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fungal strains, plasmids, and media. C. neoformans ATCC 208821 (=H99) was a generous gift from J. Perfect. Strain H99 lac1 ura5 (41) was employed as a recipient strain for expression studies. The strains were grown in YPD medium (2% glucose, 1% yeast extract, 2% Bacto peptone[Difco]) or YPD agar; this was followed by incubation in asparagine media without glucose (1 g/liter asparagine, 10 mM sodium phosphate [pH 6.5 or the pH indicated below], 0.25 g/liter MgSO₄, 10 µM CuSO₄) for laccase expression. Asparagine minimum selective media for transformant selection and for detection of laccase production have been described previously (40). Plasmid pCIP containing the URA5 gene was a kind gift from K.J. Kwon-Chung.

Construction and expression of an N-terminal GFP-laccase fusion protein. The cryptococcal shuttle vector pORA-KUT, containing the URA5 transformation marker, was used to express a fusion between the LAC1 protein and a synthetic green fluorescent protein (Cneo-GFP), utilizing C. neoformans codon usage (21). First, pORA-KUT containing the sequence of the EF-1 terminator was digested with BglII, and a PCR-amplified fragment of genomic DNA from H99 (obtained using primers LacProL4-BglII and LacProR4-BglII) was digested with BglII and inserted into compatible sites to produce pORA-KULP. The plasmid was recovered, verified by sequencing, and digested with PstI, and a PCR-amplified fragment of Cneo-GFP DNA (obtained using primers GFPmyc-PstIL and GFPmyc-PstIBamHIR) was digested with PstI and inserted into compatible sites. The plasmid was recovered, the sequence was verified, the plasmid was digested with BamHI, and a PCR-amplified fragment of the H99 laccase gene (obtained using primers LacTerL-BamHI and LacTerR-BamHI) was digested and ligated into compatible sites to produce pORA-KULP-624. Sequential deletion of the C terminus of laccase was performed by digesting pORA-KULP-624 with BamHI and replacing the full-length laccase sequence with PCR-amplified truncated fragments of H99 LAC1 using primer LacTerL-BamHI and the following five primers: LacH99+200, LacH99+271, LacH99+424, LacH99+471, and LacH99+570 (Table 1). Amino acid assignments were based on the H99-derived cDNA sequence of LAC1 (GenBank accession no. DQ897640), which was obtained by automated sequencing of a clone from a cDNA library of H99 described previously (8). The plasmids were recovered, the sequences were verified, and the plasmids were linearized with SceI and transformed into C. neoformans H99 Mat lac1 ura5 cells by electroporation using standard methods (8). An H99 Mat ura5 strain transformed with a pORA-KUT plasmid without GFP was used as a control for examination of epifluorescence under in vivo conditions. Transformants were selected on the basis of equivalent copy number demonstrated by uncut Southern analysis, as described previously (38). For in vitro expression, cells were incubated in asparagine media for different times and examined by deconvolution microscopy using an IX-70 Olympus microscope and the soft agar embedding technique described previously (21).

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TABLE 1. Primers used

DNA in nuclei of cells was visualized by epifluorescence with 1 µg/ml of 4',6-diaminidino-2-phenylindole dilactate (DAPI) (Sigma-Aldrich) in phosphate-buffered saline (pH 7.0), and cell walls were visualized by a similar method using calcofluor white stain according to the manufacturer's directions (Sigma-Aldrich).

Preparation of mouse bronchoalveolar lavage macrophages. Bronchoalveolar lavage macrophages were prepared as previously described (19), with a slight modification. Briefly, macrophages were collected by bronchial lavage with 0.5 ml Hanks' balanced salt solution (Invitrogen) containing 10 U/ml heparin and 50 U/ml penicillin/streptomycin. A total of 2 x 10³ to 5 x 10³ cells were harvested from each mouse and were examined to determine epifluorescence.

Mice. Female CBA/J or Swiss Albino mice (18 ± 2 g) were purchased from Frederic Cancer Research and housed in specific-pathogen-free conditions in enclosed filter top cages. Food and sterile water were given ad libitum. The mice were maintained by the Biological Research Laboratory at the University of Illinois, and protocols were approved by an animal institutional review board.

Intratracheal and intravenous inoculation. Infections were established by intranasal inoculation of 10⁵ CFU of the C. neoformans strains as described previously (21). Three animals per group per time were infected. Mice were anesthetized by intraperitoneal injection of 150 mg/kg pentabarbital per mouse and restrained, and a 25-µl inoculum was delivered intranasally. Aliquots of the inoculum were analyzed to determine the number of CFU in order to monitor the amounts delivered. Infection by the intravenous route was performed with 10⁶ CFU of a C. neoformans strain; each group contained three mice, as described previously (32). All fungal preparations were grown in media containing 2% glucose (asparagine media) prior to inoculation to repress laccase expression, so that any laccase expression observed occurred during infection. Mice were warmed under a heating lamp for 15 min prior to intravenous inoculation. A tuberculin syringe was filled with a dilute fungal culture, and a 30-guage needle was attached. The needle was inserted into the lateral tail vein, and a 100-µl inoculum was delivered. Aliquots of the inoculum were analyzed to determine the number of CFU in order to monitor the amount delivered.

Harvesting of tissue. Brains were collected after sacrifice at the times indicated below by removing the top of the cranium and excising the brain from the brain stem. Lungs were removed from the chest cavity by excision. Organs were placed in tubes containing 2 ml of sterile water and homogenized mechanically using a glass mortar and pestle.

Statistics. Vesicle numbers of fungal cells were recorded for 10 cells in the largest-diameter plane for each cell, and numbers for each cell were compared using a two-tailed Student t test as described previously (22). Errors were expressed as standard errors of the means (SEM) or 95% confidence intervals as indicated below.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of an N-terminal GFP-laccase fusion protein is robust and is targeted to the cell wall at physiological pH. As shown in Fig. 1A, an episomal expression plasmid was constructed using PCR-amplified H99 DNA and primers described in Table 1 to insert an N-terminal green fluorescent protein after Thr-41 just downstream of the LAC1 consensus leader sequence, resulting in expression under the LAC1 native promoter. The construct was then transformed into H99 lac1 ura5 cells as described previously (8). As shown in Fig. 1B, recombinant GFP-laccase was enzymatically active, producing melanin (as determined by a plate assay) similar to the melanin produced by the wild type in vitro. Laccase expression was also fully glucose repressible, as described previously for the native enzyme (Fig. 1C, upper panel) (37). In addition, expression and localization could be monitored by epifluorescence, which demonstrated that there was cellular transport of the recombinant protein within secretory vesicles and time-dependent delivery of the enzyme to the cell wall, as described previously for the native enzyme (39). Calcofluor white (Fig. 1C) was used to colocalize cell wall GFP-laccase, which stains cell wall chitin (30). In addition, extensive washing failed to remove cell wall fluorescence, suggesting that there was strong cell wall attachment by the GFP-laccase, similar to the wild-type enzyme (39). Interestingly, induction of GFP-laccase expression at lower pHs, such as pH 4.5 to 5.5 (Fig. 2), resulted in partial inhibition of secretory transport, and the enzyme was trapped within cytoplasmic vesicles at 24 h. Interestingly, these pHs are similar to the pH of the macrophage phagolysosome after cryptococcal uptake, which has been estimated to be approximately pH 5 by analysis of pH-dependent chromophoric molecules correlated to bulk solvent pH values (18). Analysis of 10 cells induced in acidic conditions revealed that there were 3.9 ± 0.2 vesicles per largest-diameter field (average ± SEM), compared to only one small vesicle in 1 of 10 cells induced at physiological pH (3.9 versus 0.1 vesicle/largest-diameter field; P < 0.001). This suggests that the acidic compartment of the phagolysosome may be an effective tool for partial mistargeting of virulence factors, such as laccase, in an effort to limit host cell damage.

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FIG. 1. Expression of a GFP-laccase recombinant protein and localization to the cell wall. (A) Schematic diagram of GFP-laccase expression construct obtained using PCR-amplified C. neoformans DNA and primers shown in Table 1. 3'Term, 3' terminus. (B) Laccase production by constructs after incubation on norepinephrine agar overnight. wt, wild type. (C) Microscopic localization of GFP-laccase after incubation in the presence of glucose (+Glu) or after transfer to starvation conditions (–Glu) at different times and colocalization with either DAPI or calcofluor white. Bright field (BF) magnification, x580. Bars = 5 µm.

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FIG. 2. Localization of GFP-laccase at neutral and acidic pHs. C. neoformans cells expressing GFP-laccase were induced for laccase in asparagine media without glucose at different pHs for 24 h at 30°C, and the epifluorescence was observed. Bright field (BF) magnification, x720. Bars = 5 µm.

Laccase from C. neoformans contains a unique C-terminal sequence involved in cell wall targeting. Since laccase from C. neoformans is the only cell wall-targeted laccase that has been described, we analyzed the structural protein to determine if there were unique attributes of the cryptococcal protein that may help to explain cell wall targeting. As shown in Fig. 3A, comparison of the laccase from C. neoformans to laccases from fungi, plants, and insects showed that the cryptococcal protein has the largest polypeptide backbone and contains a unique C terminus not present in other proteins in its class. Figure 3B shows a expanded view of the C terminus and that there is a high level of conservation between the C termini of serotype A (H99) and serotype D (JEC21) strains but poor conservation in laccases having the longest polypeptide backbones (Rhus [lacquer tree] and Drosophila melanogaster) due to a lack of homologous C termini in the other laccases. Interestingly, in our hands, direct comparisons of C-terminal laccase-GFP and N-terminal laccase-GFP fusion proteins revealed that there was more homogeneous cell wall localization for an N-terminal GFP fusion, suggesting that there is some interference with a C-terminal motif by the GFP protein (data not shown). In addition, in previous work Missall et al. (23) observed vesicular localization of the cryptococcal Lac2 protein, which is missing 30 amino acids at the C terminus, lending support to the hypothesis that the C terminus has a role in cell wall localization. To investigate further the role of the protein sequence in cell wall targeting of cryptococcal laccase, sequential C-terminal deletions of the laccase protein were obtained using PCR-amplified truncated fragments as described in Materials and Methods. As shown in Fig. 4A, expression of truncated GFP-laccases resulted in the absence of melanin production from whole cells and localization within cytoplasmic vesicles, in contrast to the cell wall targeting of the full-length construct. Enzyme activity was also not detectable in lysates from cells expressing the 571-624 truncated protein, compared to the robust activity (5.1 x 10⁴ U/mg) in lysates from cells expressing the full-length construct, as determined using a previously described colorimetric assay (38). Not surprisingly, the 571-624 truncated GFP-laccase also did not properly localize to the cell wall in fungal cells recovered by bronchoalveolar lavage 48 h after intranasal injection of cells into CBA/J mice (Fig. 4B). These data show that the unique cryptococcal C terminus (positions 571 to 624) is required for effective cell wall targeting and enzyme activity. Interestingly, since the C-terminal sequence does not contain consensus sequences such as the copper-binding histidine regions previously implicated in laccase activity (37), the data suggest that the C terminus may confer a required conformation or that secretion per se is required for enzyme activity.

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FIG. 3. Comparison of laccases from C. neoformans and other organisms. (A) Comparison of the sequence of C. neoformans LAC1 (CnLAC1) (GenBank accession no. DQ897640) and the sequences of the laccases of Manduca sexta (MsLac1 [GenBank accession no. AY135185], MsLac2 [GenBank accession no. AY135186], and MsLac1[GenBank accession no. AY135186]), Thanatephorus cucumeris (TcLac3 [Swiss-Prot accession no. Q02079]), D. melanogaster (DmLac4 [GenBank accession no. NM165431], DmLac1 [accession no. NM133021], and DmLac2 [GenBank accession no. NM135443]), Rhus (lacquer tree) (RhusLac [GenBank accession no. AB06262449]), Anopheles gambiae (AgCP153 [GenBank accession no. EAA10258]), Pinus taeda (PtLac1 [GenBank accession no. AF132119]), Nicotiana tabacum (NtLac1 [GenBank accession no. U43542]), Lolium perenne (LpLac2 [GenBank accession no. AF465469]), Liriodendron tulipifera (LtLac2 [GenBank accession no. AAB17191]), Phlebia radiate (PrLac [Swiss-Prot accession no. Q01679]), Trametes versicolor (TveLac4 [Swiss-Prot accession no. Q12719]), and Tramates hirsute (Thlac [Swiss-Prot accession no. Q02497]). Relationships were established by performing Clustal W analysis (3). (B) Comparison of the C termini of C. neoformans LAC1 from serotype A strain H99 (CnLac1-A) and serotype D strain JEC21 (CnLac1-D) and their closest relatives.

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FIG. 4. C-terminal deletion results in mislocalized laccase. (A) C. neoformans lac1 cells expressing recombinant GFP-LAC1 having different C-terminal deletions were incubated on asparagine media (pH 7.4) without glucose for 24 h, and epifluorescence was observed after staining with 2.5 mg/ml of DAPI for nuclear localization. Bright field (BF) magnification, x820. Bars = 5 µm. Melanin production was determined by incubation of the cells on norepinephrine agar for 24 h. (B) Expression of truncated GFP-laccase in fungal cells recovered by bronchoalveolar lavage from CBA/J mice 48 h after intranasal infection.

Expression of GFP-laccase in alveolar macrophages shows that formation of intracellular cytoplasmic vesicles and cell wall targeting occur. Since alveolar macrophages are some of the first intracellular host cells to phagocytose C. neoformans during infection (1, 9), we inoculated CBA/J mice intranasally with fungal cells expressing GFP-laccase or a control episomal plasmid, sacrificed the animals, and performed bronchoalveolar lavage to recover macrophage-associated fungal cells 24 and 48 h after inoculation. At 24 h, the fungal cells that were recovered showed no cellular epifluorescence above the background fluorescence level (data not shown). However, as shown in Fig. 5, recovery and examination of fungal cells 48 h after infection showed that there was expression of GFP-laccase that was localized to the cell wall and was also localized to cytoplasmic vesicles, similar to what was observed for identical cells induced in vitro at an acidic pH. This was observed for a majority of the macrophage-associated C. neoformans cells, and 3.6 ± 1.2 vesicles (average ± 95% confidence interval) were observed in a single plane at the largest diameter (n = 10). Fungal epifluorescence was distinguished from autofluorescence of adherent macrophages by the specificity of the GFP signal using GFP filter sets, whereas autofluorescence was detectable for all filter sets examined. Extensive attempts to induce GFP-laccase expression in macrophage cultures, using cell line J774.1, failed to produce significant results, so cell wall targeting could not be studied under these conditions.

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FIG. 5. Localization of laccase during infection of bronchoalveolar macrophages: expression of GFP-laccase during infection of bronchial alveolar cells 48 h after inoculation. The control consisted of infection with equivalent cells expressing an empty episomal vector. Bright field (BF) magnification, x520. Bars = 5 µm. Mac, macrophage; Cn, C. neoformans cell; Cap, fungal capsule.

Expression of GFP-laccase in the lung and brain. To visualize the location of the laccase enzyme during pathogenesis at a later time in infection, GFP-LAC1-expressing cells were inoculated intranasally into CBA/J and Swiss Albino mice and also intravenously into Swiss Albino mice as described previously (26), and cells were recovered after tissue homogenization from lungs (after 9 days and 3 weeks) and brains (after 9 days), respectively. As shown in Fig. 6A, laccase was found to reside principally within the cell wall during lung infection after 3 weeks in CBA/J mice. In addition, numerous cytoplasmic epifluorescent vesicles were also observed; 2.7 ± 0.5 large vesicles of GFP-laccase (average ± SEM) were present. In the Swiss Albino mouse strain (Fig. 6B, C, and E), intranasal infection resulted in recovery of fungal cells from lungs that also showed cytoplasmic localization of GFP-laccase (number of vesicles at 9 days, 6 ± 0.5; number of vesicles at 3 weeks, 3 ± 0.5 [averages ± SEM]), but in this case laccase was predominantly intracellular and only a few cells (3 of 20 cells) showed detectable cell wall localization as determined by colocalization with calcofluor white stain. An insufficient number of cells disseminating to the brain prevented analysis of fungus infection by this route. Thus, we also inoculated Swiss Albino mice with fungal cells intravenously and found that at 9 days the cells recovered from the brain showed cell wall localization almost exclusively, with only an occasional, barely visible, small cytoplasmic vesicle (0.7 ± 0.3 vesicle [average ± SEM]) (Fig. 6D, inset). Statistical analysis of 10 cells from each group showed a significantly greater number of vesicles in all lung groups than those from the brain. (P < 0.05).

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FIG. 6. Expression of GFP-laccase during infection of lungs and brains. C. neoformans cells expressing full-length GFP-LAC1 or C. neoformans cells with a control plasmid not containing GFP-LAC1 (control) were inoculated intranasally (105 cells) into CBA/J mice (A) or Swiss Albino mice (B, C, and E), fungal cells were recovered from homogenized lungs, and epifluorescence was observed; colorization was made with calcofluor white (E). Similar cells were inoculated intravenously (106 cells) into Swiss Albino mice and recovered from homogenized brains, and epifluorescence was observed (D). The inset is an enlargement of the region indicated; the arrow indicates a cytoplasmic vesicle. Bright field (BF) magnification, x820. Bars = 5 µm.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The implications of the cellular localization of laccase for pathogenesis are important, because the ability of this enzyme to act as an immune modulator at the host-parasite interface is most likely dependent on its proximity to the extracellular space. For example, oxidation of low levels of exogenous brain catecholamines (20) and transient host cell Fe(II) species in the phagolysosome (19) would be most effective for an enzyme exposed directly to the reagents at a relatively superficial location and would obviate the need for additional dopamine and iron transport mechanisms, respectively. This could be accomplished by the fungus using a cell wall-localized enzyme, because the capsule is relatively permeable to small molecules (12).

In the present study, we found that there was robust localization of an N-terminal GFP-tagged laccase to the cell wall in vitro, similar to the localization of the native enzyme observed by use of a monoclonal antibody using immunoelectron microscopy and biochemical methods (39). In addition, expression under the native promoter allowed analysis of cellular localization under conditions that normally induce the wild-type enzyme (for example, exhibition of suppression by glucose as described previously [37]). Interestingly, under acidic conditions, cell wall targeting of GFP-laccase was partially inhibited at 24 h and was found to persist in cytoplasmic vesicles. This observation may explain a longstanding question concerning C. neoformans laccase: the apparent insolubility of enzyme preparations from cells incubated under neutral conditions and the generation of soluble, enzymatically active preparations from cells grown under acidic conditions (13), the latter of which was key for the original purification of the enzyme (37).

Cell wall localization of laccase is a unique attribute of the cryptococcal enzyme; most laccases are completely secreted into the environment for activity in degrading lignin or protection against parasites (24). The present data suggest that the unique cryptococcal C terminus plays a role in cell wall targeting. In addition, a comparison with previous X-ray structural analyses of laccases from Coprinus cinereus (7) and Trametes versicolor (28) showed that the C termini of laccases are highly mobile superficial polypeptides. The superficial position should allow participation in secretory targeting of the cryptococcal C-terminal extension identified in the present study. The cryptococcal targeting region contains no previously described targeting motifs, although several serines which could represent O-glycosylation sites are present. However, previous analysis of the laccase protein did not demonstrate that there is O glycosylation (37), although it is not known whether the C terminus was cleaved during processing. Further analysis of the cryptococcal targeting motif is under way, which may help shed light on these questions.

Since alveolar macrophages are essential to the primary, initial defense against C. neoformans (1, 9), we investigated the cellular localization of laccase showing macrophage adherence after bronchoalveolar lavage. The macrophage-adherent fungal cells selected for observation in this study had most likely been phagocytosed by macrophages, although it was difficult to assess fungal internalization directly by dye exclusion methods (17) because of the physical shearing forces that the cells were subjected to during lavage. These macrophage-adherent cells had a predominant cell wall localization, which may explain the effectiveness of protection by the laccase enzyme against alveolar macrophages described previously (19). However, a subpopulation of the laccase protein appeared to be mislocalized based on the observation of numerous epifluorescent GFP-laccase cytoplasmic vesicles, and this could represent a novel antifungal property of macrophages. The secretory inhibition may be due to specialized conditions within macrophages, such as a pH which is lower within the phagolysosome (18), and is suggested by the pH-dependent mislocalization observed in vitro. These results appear to confirm previous results obtained by immunoelectron microscopy which showed that there was intracellular localization of laccase during lung infection (11). Since the laccase C terminus was also found to be important for cell wall targeting of the protein during early lung infection, it is interesting to speculate whether this unique structural motif could have evolved in a similar cryptococcal environment, such as amoebae (33), to produce an optimized virulence factor that is also useful against macrophages.

Intracellular GFP-laccase vesicles appeared to persist during infection of the lung and were observed up to 3 weeks after inoculation. Interestingly, there appeared to be differences in the pattern of mislocalization between the two mouse strains during infection of the lung, with CBA/J mice having large vesicles and retaining cell wall localization and Swiss Albino mice producing more punctuate vesicles with almost no visible cell wall-localized GFP-laccase. Such differences in patterns of laccase localization in fungal cells may be due to differences in the host response, as many attributes, such as the cellular response to vaccination, have been shown to be highly variable in different mouse strains (6). Although macrophage residence of these cells could not be confirmed in our study later in infection due to the method used for cell preparation, macrophage residence of C. neoformans has been shown to predominate during infection of the lung for up to 2 weeks (9). In contrast, recovery of organisms from the brain showed that there was almost no cytoplasmic vesicularization of laccase, and almost all epifluorescence was present within the cell wall. While mislocalization in cells from the lungs of Swiss Albino mice appeared to have reduced the level of cell wall laccase, we could not directly compare the absolute levels of cell wall laccase in cells recovered from brains and lungs because we could not control for possible tissue-dependent quenching and autofluorescence in cells from different tissues. However, mislocalization of a fraction of the expressed enzyme during infection of the lung, especially in the Swiss Albino strain, would be expected to reduce the potential total amount of laccase available for host cell damage. Interestingly, infection of the brain in most mouse strains by C. neoformans results in less cellular response than infection of the lung results in, and the fungal cells are predominantly extracellular (26). Thus, differences in GFP-laccase localization between tissues could represent differences in the amounts of the fungus that are intracellular and extracellular, or there may be other unidentified factors similar to low pH which result in this mislocalization. Interestingly, laccase also plays a greater role in dissemination to the brain and a lesser role in pulmonary persistence (26), suggesting that tissue-specific protein secretion by the fungus, as observed in the present study, could play a role in these tissue-specific contributions of virulence factors to the overall virulence composite.

 
This work was supported in part by United States Public Health Service grants NIH-AI38258 and A14599. We also acknowledge use of the C. neoformans Genome Project, Stanford Genome Technology Center (http://www-sequence.stanford.edu), funded by the NIAID/NIH under cooperative agreement AI47087.

 
* Corresponding author. Mailing address: Division of Infectious Diseases, Rm. 888, m/c 735, University of Illinois at Chicago College of Medicine, 808 S. Wood St., Chicago, IL 60612. Phone: (312) 996-6070. Fax: (312) 413-1657. E-mail: prw{at}uic.edu.

Published ahead of print on 13 November 2006.

Editor: A. Casadevall

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, February 2007, p. 714-722, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.01351-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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