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Coccidioides posadasii is a fungal respiratory pathogen of humans that can cause disease in immunocompetent individuals. Coccidioidomycosis ranges from a mild to a severe infection. It is frequently characterized either as a persistent disease that requires months to resolve or as an essentially asymptomatic infection that can reactivate several years after the original insult. In this report we describe a mechanism by which the pathogen evades host detection during the pivotal reproductive (endosporulation) phase of the parasitic cycle. A metalloproteinase (Mep1) secreted during endospore differentiation digests an immunodominant cell surface antigen (SOWgp) and prevents host recognition of endospores during the phase of development when these fungal cells are most vulnerable to phagocytic cell defenses. C57BL/6 mice were immunized with recombinant SOWgp and then challenged with a mutant strain of C. posadasii in which the MEP1 gene was disrupted. The animals showed a significant increase in percent survival compared to SOWgp-immune mice challenged with the parental strain. To explain these results, we proposed that retention of SOWgp on the surfaces of endospores of the mutant strain in the presence of high titers of antibody to the immunodominant antigen contributes to opsonization, increased phagocytosis, and killing of the fungal cells. In vitro studies of the interaction between a murine alveolar macrophage cell line and parasitic cells coated with SOWgp showed that the addition of anti-SOWgp antibody could enhance phagocytosis and killing of Coccidioides. We suggest that Mep1 plays a pivotal role as a pathogenicity determinant during coccidioidal infections and contributes to the ability of the pathogen to persist within the mammalian host.
Coccidioides is a fungal pathogen of humans which can cause a mild to severe respiratory disease (coccidioidomycosis; San Joaquin Valley fever) in immunocompetent individuals (10). The fungus inhabits desert and semiarid regions of the Southwestern United States, as well as parts of Mexico and South and Central America, where it grows as a filamentous saprobe in soil. Two species of Coccidioides have been reported on the basis of molecular and biogeographical differences; Coccidioides immitis is found primarily in the San Joaquin Valley of California, while Coccidioides posadasii is widespread throughout regions of endemicity in the Americas (18). Although the growth rate of the saprobic phase of C. posadasii on high-salt media is significantly lower than that of C. immitis (18), no differences in the in vitro/in vivo morphogenesis or experimental infectivity of these two species have been identified. Inhalation of the airborne spores (arthroconidia) by a mammalian host is followed by the initiation of an elaborate parasitic cycle which is unique among the medically important fungi (6). Only about half of the immunocompetent people infected with Coccidioides develop atypical pneumonia-like symptoms, and the majority of these recover during the subsequent few weeks to several months (10, 38). The majority of other Coccidioides-infected individuals who are otherwise healthy may experience only mild discomfort and usually do not seek medical intervention. Since there is no person-to-person transmission of Coccidioides, with the rare exception of maternal-fetal transmission (39), it would appear that infection of the mammalian host is an evolutionary dead end for the pathogen. On the other hand, a large body of clinical evidence suggests that reactivation of coccidioidomycosis may occur later in life if infected individuals contract an immunocompromising disease, undergo elective immunosuppression for organ transplantation, or are otherwise subjected to long-term drug therapy that compromises their cellular immunity (51, 52). Certain microbial agents of disease have evolved pathogenicity determinants which allow them to coexist with their host rather than exacerbate the infection process (33). In this report we describe a mechanism used by C. posadasii to escape host detection during the pivotal reproductive stage of the parasitic cycle. We suggest that this evasive mechanism contributes significantly to the survival of the pathogen within lung tissue and potentially to the establishment of a persistent coccidioidal infection in the mammalian host.
The saprobic and parasitic phases of C. posadasii were grown in vitro under conditions described previously (25). Parasitic-phase cells were harvested at various times (36 to 132 h) after inoculation of the culture medium with arthroconidia as reported elsewhere (21).
The native spherule outer wall (SOW) fraction was isolated from parasitic-phase cultures as described elsewhere (9). Extraction of the major, water-soluble SOW glycoprotein (SOWgp) component of the SOW fraction, which was obtained from parasitic-phase cultures at 96 h after inoculation, was conducted as previously reported (25). The SOWgp, which consists of two polypeptides (60 and 82 kDa), was purified as previously described (25). The same extraction procedure was used with the SOW fraction obtained from 132-h parasitic-phase cultures for isolation of the metalloproteinase (Mep1) reported in this paper. The 30-kDa and 34-kDa bands observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separations of this fraction were electrotransferred to an Immobilon-P membrane (Millipore) and subjected to N-terminal amino acid sequence analysis as reported elsewhere (25).
The 30-kDa and 34-kDa Coomassie blue-stained protein bands described above were excised, destained, and subjected to in-gel digestion with sequencing-grade trypsin (Promega, Madison, Wis.) at 37°C as reported elsewhere (46). Peptides were applied to a reverse-phase high-performance liquid chromatography column (Aquasil C18 Picofrit column; New Objectives Inc., Woburn, Mass.) and introduced into an ion-trap mass spectrometer equipped with a nanospray source (LCQ DecaXP plus; Finnigan Corp., San Jose, Calif.). The tandem mass spectrometer was operated in the double-play mode, in which the instrument was set to acquire a full MS scan (400 to 2,000 m/z) and an MS/MS spectrum of the most intense ion. Collision-induced dissociation spectra were obtained that yielded amino acid sequences of the peptides. A search for matching sequences in the translated C. posadasii genome database (13) (www.tigr.org) was conducted using the TurboSEQUEST software package, version 3.0 (Finnigan). The genome database was derived from nucleotide sequence analysis of C. posadasii strain C735. A sequence match was identified within a single contig, which was then used to direct the synthesis of gene-specific oligonucleotide primers and subsequent cloning of the full-length MEP1 gene as described below.
The putative full-length genomic sequence of the MEP1 gene obtained from the C. posadasii genome database was translated and used to conduct a BLAST (basic local alignment search tool) search (1) of matching amino acid sequences in the nonredundant NCBI protein database as previously reported (13). A BLASTX match (1) revealed sequence homology (expect [E] value, 9 × 10−34) with a reported fungal metalloproteinase (Mep) of Metarhizium anisopliae (GenBank accession no. CAB63909). Oligonucleotide primers selected from the contig sequence were synthesized and used to amplify a 3.4-kb genomic DNA fragment by PCR employing high-fidelity Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, Calif.). The 3.4-kb fragment was cloned into the pCR2.1-TOPO plasmid vector according to the manufacturer's instructions (Invitrogen). The sense and antisense primers derived from the contig sequence were located 495 bp upstream of the putative N terminus and 1,793 bp downstream of the predicted stop codon, respectively. The primer sequences were 5′-GAATCGATTCGTCCACGA-3′ and 5′-CCGATCGATTGTCGAGGA-3′, respectively (ClaI restriction sites are underlined). The rapid amplification of cDNA ends (RACE) procedure (26) was used to obtain the full-length cDNA sequence of the C. posadasii MEP1 gene. Comparison of the genomic and cDNA sequences confirmed the locations of the exons and introns. The PROSITE algorithm was used to identify conserved motifs in the translated polypeptide with homology to reported proteins (24), and the PSORT II algorithm was used for prediction of the signal peptide (35). A BLAST search of the translated genome database of C. posadasii for homologs of Mep1 resulted in retrieval of nine additional putative members of a family of metalloproteinases. Structural comparison of the translated Mep1 to Mep10 proteins was conducted by alignment of the nine predicted metalloproteinase sequences with Mep1 using the CLUSTALX algorithm (13), followed by construction of a hypothetical phylogenetic tree using the TreeView program (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).
Levels of expression of MEP1 during development of spherule initials, segmented spherules, and endosporulation-stage spherules of C. posadasii (cultured parasitic cells isolated after 36 h, 96 h, and 132 h of incubation, respectively) were determined by quantitative real-time PCR (QRT-PCR) analysis using a LightCycler PCR system (Roche Diagnostics, Indianapolis, Ind.). The generation of cDNA from parasitic-cell-derived total-RNA preparations was conducted as reported elsewhere (13). MEP1-specific primers were designed using LightCycler Probe Design software (version 1.0; Roche). The sequences of the sense and antisense primers were 5′-ACCTTCATCCGTGATGGATGC-3′ and 5′-TGTGGAAGAGACCCATCCAGTG-3′, respectively. This primer pair amplified a 121-bp PCR product using single-stranded template cDNA generated from total RNA that was isolated from the 36-h, 96-h, or 132-h parasitic-cell preparations cited above. A 191-bp amplicon used for normalization of the assay was derived from the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of C. posadasii (GenBank accession no. AF288134) as previously reported (13). The CT value (number of cycles at the fluorescence threshold) versus the known amount of the GAPDH or MEP1 cDNA template was determined to generate standard curves as reported elsewhere (34). The data derived from the standard curves were imported into the LightCycler relative quantification software program (Roche) to calculate the amount of specific cDNA transcript in each of the parasitic-cell-derived samples. The data are presented as the ratio of the amount of the MEP1 transcript to the amount of the GAPDH transcript in each sample. The QRT-PCR experiments were performed in triplicate.
Estimates of relative amounts of the metalloproteinase (Mep1) in total homogenates of parasitic cells grown in vitro for 36, 96, or 132 h (26), in extracts of the SOW fraction (25), and in concentrated parasitic-phase culture filtrates (43) were performed by SDS-PAGE followed by immunoblot analysis. The SOW extracts were not concentrated by centrifugal filtration in this case. Immunoblotting was conducted using a murine polyclonal antibody raised against the recombinant Mep1 protein expressed by Escherichia coli strain BLR(DE3). In brief, the full-length cDNA of MEP1 (0.83 kb) was inserted into the NdeI/XhoI site of pET28b (Novagen, Madison, Wis.), and the pET28b-MEP1 plasmid was used to transform the E. coli strain. Induction of expression, protein purification, and amino acid sequence analysis of recombinant Mep1 (rMep1) were performed as reported elsewhere (21). The purified protein was used to immunize BALB/c mice for production of a specific antiserum as previously described (21). Immunoblot analysis was conducted as reported elsewhere (7).
The bacterially expressed rMep1 was not enzymatically active. Therefore, the native Mep1 protein purified from the SOW extract (25) derived from 132-h parasitic-phase cultures was used to evaluate enzymatic activity. The SOW extract was first passed through a centrifugal filter with a 50-kDa cutoff (Millipore). The filtrate was subsequently applied to a second centrifugal filter with a 10-kDa cutoff. The retentate was examined by SDS-PAGE and subjected to substrate gel electrophoresis using essentially the same procedure as previously reported (12). The native Mep1 fraction (0.04 μg) was incubated with purified SOWgp (2.0 μg) in the presence or absence of EDTA (5.0 mM) in 10 mM Tris buffer, pH 7.5, containing 0.125 M NaC1 plus 3.0 μM ZnSO4. The incubation reaction was conducted at 37°C for 2 h. Incubation of the purified SOWgp with or without EDTA in the absence of native Mep1 served as control reactions. The reaction products were examined by SDS-PAGE.
A pΔmep1 plasmid was designed as a gene replacement vector (14) and constructed using a 4.1-kb fragment of pAN7.1 that contained the hygromycin resistance gene (HPH) (41). The 3.4-kb genomic clone of MEP1 was digested with HapI followed by BstBI, which resulted in deletion of most of the coding region of the gene (i.e., nucleotides [nt] 1292 to 2679). The 4.1-kb pAN7.1 fragment was then inserted into the deletion site of the C. posadasii genomic clone to yield a 6.1-kb pΔmep1 plasmid construct. The latter was linearized by digestion with ClaI. The subsequent steps of transformation of C. posadasii and isolation of the mutant strain were performed as reported elsewhere (27, 43). Confirmation of homologous recombination was based on results of Southern hybridization using nucleotide probes derived from the HPH gene and targeted MEP1 gene. The HPH gene probe has been described previously (27). The MEP1 probe consisted of a 566-bp PCR product generated by use of a sense primer (5′-GTCGAACTTGTGGCTCTGTA-3′) and an antisense primer (5′-GCATCCATCACGGATGAAGGT-3′) in the presence of a genomic DNA template. Genomic DNAs isolated from the parental and mutant strains were separately digested with either HindIII or SalI and then subjected to Southern hybridization as reported elsewhere (27).
The revertant strain containing the restored full-length parental MEP1 gene was generated by the strategy previously reported (27). A 4.6-kb fragment of the pAN8.1 plasmid, which contains the BLE gene conferring resistance to phleomycin (44), was inserted into the PCR2.1-TOPO plasmid containing the wild-type MEP1 gene. The resulting plasmid construct, pMEP1R, was used for transformation of the Δmep1 mutant strain and generation of the parental phenotype. The transformation and screening procedures have been reported elsewhere (27). Southern hybridization and immunoblot analyses were conducted to confirm that the functional parental MEP1 gene was restored to the revertant strain. The HPH and MEP1 probes used for Southern hybridization were the same as those described above. The BLE gene probe consisted of a 346-bp PCR product generated by use of a sense primer (5′-AGTGCCGTTCCGGTGCTCACC-3′) and an antisense primer (5′-CGGCCACGAAGTGCACGCAGT-3′) in the presence of a pAN8.1 plasmid DNA template.
Immunofluorescence microscopy was conducted to examine the localization of SOWgp in parasitic cells. Intact or sectioned parasitic cells from 24-, 96-, and 132-h cultures were examined as previously described (8, 25). Parasitic cells of the parental, Δmep1 mutant, and Mep1R revertant strains were separately reacted with an anti-SOWgp antiserum followed by goat anti-mouse secondary antibody conjugated with fluorescein isothiocyanate (FITC; Sigma, St. Louis, Mo.) as reported elsewhere (27). Infected lungs from C57BL/6 mice challenged intranasally with a lethal inoculum of C. posadasii arthroconidia of the parental or Δmep1 mutant strain, as reported elsewhere (13), were excised from animals sacrificed at 15 days postchallenge. The lung tissue was chemically fixed, embedded in plastic resin or paraffin, sectioned, and stained with FITC-conjugated anti-SOWgp or hematoxylin-eosin in preparation for immunofluorescence or conventional light microscopy as reported elsewhere (8, 47).
SOWgp is an immunodominant parasitic cell surface antigen that stimulates humoral and cellular immune responses of the infected host (25). We designed experiments to identify the major epitope(s) of SOWgp responsible for host seroreactivity. The cDNAs which encode the mature protein (MP; amino acids [aa] 69 to 328, representing the full-length protein less the signal peptide, propeptide, and 6 residues of the adjacent nonrepeat region) (27), the N-terminal fragment (N-t; aa 1 to 83), the repeat domain (Rpt.; aa 80 to 278), and the C-terminal region (C-t; aa 273 to 328) of SOWgp produced by the Silveira strain of C. posadasii were cloned into pET28b (Novagen) and expressed by E. coli BL21(DE3). The SOWgp of the Silveira strain has four repeats of 47 residues each, while the SOWgp produced by the C735 strain has six repeats (27). For unknown reasons, the N-terminal, repeat, and C-terminal fragments encoded by the SOWgp gene of the Silveira strain were more easily expressed in E. coli than the respective fragments of the C735 strain, and therefore the recombinant proteins of the former were used for this part of the study. The primer sequences used for PCR amplification of the respective cDNAs are reported in Table Table1.1. The recombinant proteins were purified as described previously (21). Sera from patients with confirmed coccidioidal infection were tested for reactivity with each purified recombinant protein by immunoblot analysis and for reactivity with the purified recombinant Rpt. fragment by an indirect enzyme-linked immunosorbent assay (ELISA) as described previously (25). Human control sera were obtained from hospital admissions with no systemic or pulmonary mycosis as reported previously (7).
The loss of Mep1 activity in the Δmep1 strain results in persistence of SOWgp at the endospore surface. We designed experiments to test whether the loss of Mep1 activity leads to reduced virulence of C. posadasii in mice. Specifically, we investigated whether high titers of anti-SOWgp antibody provide protection against coccidioidal infection in the absence of Mep1. Two groups of C57BL/6 mice (6 to 8 weeks old, 30 animals per group; supplied by the National Cancer Institute, Bethesda, Md.) were separately immunized with either phosphate-buffered saline (PBS) or the seroreactive recombinant SOWgp Rpt. protein described above (1 μg/dose) plus adjuvant (10 μg of CpG prepared in 50 μl of PBS plus 50 μl of complete Freund's adjuvant [CFA; Sigma]). A previously reported immunization protocol was used (13). The immunized mice were challenged by the intranasal route (80 arthroconidia suspended in PBS) (13) 4 weeks after the second immunization (boost) with either the parental strain (C735), the Δmep1 mutant strain, or the revertant strain (10 mice per group). Sera were collected from 4 mice per group (approximately 50 μl each) by puncture of the saphenous vein (23) just prior to challenge and at 10 and 20 days postchallenge. Titers of immunoglobulins (immunoglobulin G1 [IgG1] and IgG2c isotypes determined separately) that were reactive with the purified recombinant repeat protein of SOWgp were measured by the indirect ELISA as described elsewhere (30). C57BL/6 mice have the Igh1-b gene, which encodes the IgG2c isotype rather than IgG2a (31). An IgG2c-specific antiserum was obtained from Southern Biotechnology Associates, Inc. (Birmingham, Ala.). The percent survival of each group of mice was evaluated over 50 days postchallenge. This experiment was repeated twice. Survival differences between groups of mice were calculated by the Kaplan-Meier method as reported elsewhere (27, 43).
An in vitro immunoassay was designed to test the hypothesis that retention of SOWgp at the surface of parasitic cells of the Δmep1 mutant in the presence of high titers of anti-SOWgp antibody enhances phagocyte engulfment and killing of the parasitic cells compared to the interaction between phagocytes and the wild-type strain of C. posadasii. An essential control in this assay is the preincubation of SOWgp-coated parasitic cells with enzymatically active Mep1, followed by incubation of the viable, SOWgp-stripped cells with phagocytes in the presence or absence of anti-SOWgp. The large amounts of Mep1 necessary for this experiment required development of an expression system for isolation of active recombinant enzyme.
Our novel expression system involved transformation of Uncinocarpus reesii (American Type Culture Collection, Manassas, Va.; catalog no. 34534) with a plasmid construct containing the C. posadasii MEP1 gene. Transformants of this nonpathogenic ascomycetous fungus, which is closely related to C. posadasii (37), secreted large amounts of the enzymatically active recombinant metalloproteinase. The plasmid construct used for transformation contained the promoter and terminator of the C. posadasii heat shock protein gene (CpHSP60) (49), which flanked a 1.1-kb genomic fragment of the MEP1 gene (ATG to nt 1621). A 21-nt sequence, which encoded six histidine residues (His tag) upstream of a stop codon (TAA), was inserted between the 3′ terminus of the MEP1 gene and the 5′ end of the CpHSP60 terminator. This MEP1 gene complex was first cloned into the pZErO 2.1 plasmid vector (Invitrogen), followed by insertion of a 3.9-kb fragment of the pAN7.1 plasmid which included the hygromycin resistance gene cassette (41). Transformation of U. reesii was performed using the same method reported elsewhere for C. posadasii (27, 43). Transformants were selected by their ability to grow on hygromycin-containing agar. The culture filtrates of eight putative transformants were screened by immunoblot analysis using an anti-His tag monoclonal antibody (Novagen, San Diego, Calif.) for detection of secreted rMep1 (rMep1ur). The transformant with the largest amount of secreted recombinant protein was selected, and rMep1ur purification was performed by a two-step process. Total secreted protein was first precipitated with ammonium sulfate (90% saturation at 4°C), followed by nickel affinity chromatography as reported elsewhere (27, 43). The amino acid sequence of the purified rMep1ur was examined by LC-MS/MS, and enzyme activity was confirmed by substrate gel electrophoresis as described above.
To evaluate whether the in vitro digestion of SOWgp by Mep1 influenced antibody-dependent phagocytosis and killing of parasitic cells, it was necessary to isolate fungal cells which were nearly synchronized in development. It was not possible to obtain adequate numbers of endospores from cultures of the Δmep1 mutant to conduct these studies. This was due both to the asynchronous release of endospores from mature spherules and to the tendency for endospores to form dense clusters as they emerge from ruptured spherules in vitro. As an alternative, spherule initials isolated from 24-h parasitic-phase cultures were used for these studies. The parasitic cells proved to be developmentally synchronized in vitro and were coated with SOWgp. To simulate the condition whereby SOWgp of endospores produced by the wild-type strain is stripped from the cell surface, spherule initials were incubated with rMep1ur (5 × 106 cells suspended in 5 ml of defined glucose-salts medium plus 30 μg of the recombinant metalloproteinase). The reaction mixture was incubated at 39°C for 3 h in a shaking incubator. Digestion of SOWgp at the fungal cell surface was confirmed by immunofluorescence as described previously.
With the above recombinant Mep1 and isolated spherule initials of C. posadasii, we were able to evaluate whether opsonization of the parasitic cells by anti-SOWgp antibody influenced the ability of a murine alveolar macrophage cell line (MH-S; ATCC catalog no. CRL-2019) to phagocytose and kill the pathogen. A purified IgG fraction of the antiserum raised against the recombinant Rpt. of SOWgp (anti-Rpt.) was obtained by affinity chromatography using protein A-agarose beads (Bio-Rad Laboratories, Hercules, Calif.). The MH-S cell line was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (ATCC catalog no. 30-2001 and 30-2020) as reported elsewhere (32). The fungal cells (5 × 106) were first suspended in Hanks ' buffer (ATCC; catalog no. 30-2213) and stained with a fluorochrome (Blankophor-P fluessig; Bayer AG, Leverkusen, Germany) at a concentration of 0.2 μg/ml. This fluorochrome has been shown to bind with high avidity to cell wall glucan and chitin, and it has no effect on cell viability (45). The stained spherule initials were then incubated with anti-Rpt. IgG or nonspecific murine IgG (100 μg/ml) for 30 min at room temperature before they were added to the nonactivated MH-S macrophages which had been cultured as above in 24-well flat-bottom tissue culture plates (Falcon 3047; Fisher, Pittsburgh, Pa.). The antibody-labeled fungal cells were coincubated with macrophages at a ratio of 1:5 for 3 or 6 h at 37°C under an atmosphere of 5% CO2. A total of 3.5 × 104 spherule initials were added to 1.8 × 105 macrophages per well. After two washes with Hanks' buffer, the macrophages were incubated with trypan blue (1 mg/ml in the same buffer) for 15 min at 25°C to quench the fluorescence of bound (uningested) organisms. The macrophages were washed again and then fixed with 1% paraformaldehyde at 4°C for 1 h. Phagocytosis was quantified by phase-contrast and fluorescence microscopy as reported elsewhere (36). At least eight fields were counted per well, and three wells were examined for each measurement. Ingestion of intact spherule initials in the presence of anti-Rpt. IgG was compared to the phagocytosis of parasitic cells pretreated with recombinant Mep1 in the presence or absence of antibody. The phagocytic index was calculated on the basis of the total number of ingested organisms per 100 macrophages.
Quantitation of macrophage fungicidal activity was conducted essentially as reported elsewhere (2, 19). The unbound fungal cells collected in the supernatant of each well, plus the ingested and the bound, uningested spherule initials collected after osmotic lysis of the macrophages, were dilution plated on 1% glucose plus 0.5% yeast extract agar, and the CFU were determined. All experiments were conducted twice, and the results are presented as means ± standard deviations.
The C. posadasii MEP1 nucleotide sequence reported in this paper has been submitted to GenBank under accession no. AF500214 (genomic).
SOW material was isolated from 36-h and 96-h parasitic-phase cultures and extracted to obtain the water-soluble components of this lipid-rich parasitic cell surface fraction (9, 25). SDS-PAGE gel separations of these concentrated SOW extracts derived from C. posadasii isolate C735 typically showed two prominent polypeptide bands with estimated molecular sizes of 60 kDa and 82 kDa (Fig. (Fig.1A).1A). On the other hand, gel separations of the concentrated water-soluble components of SOW isolated from 132-h endosporulating cultures revealed two bands with molecular sizes of 30 kDa and 34 kDa and the absence of the SOWgp polypeptide bands. When the extract of this latter gel separation was subjected to immunoblot analysis with a murine anti-SOWgp antiserum (Fig. (Fig.1A),1A), diffuse regions of antibody reactivity were observed in the low-molecular-weight range (<45 kDa), suggesting the presence of multiple seroreactive peptide fragments.
Separate N-terminal amino acid sequence analyses of the Immobilon membrane-transferred 30-kDa and 34-kDa polypeptides revealed identical sequences of residues (Fig. (Fig.1B).1B). LC-MS/MS sequence analysis of trypsin digests of the same two gel-isolated protein bands also revealed identical peptides (underlined sequences in Fig. Fig.1B).1B). On this basis, we concluded that the two prominent bands revealed by SDS-PAGE separation of the SOW extract obtained from the 132-h parasitic-phase culture in Fig. Fig.1A1A are derived from the same protein. A search of the translated C. posadasii genome database was conducted using the peptide sequences listed in Fig. Fig.1B.1B. Sequence matches were identified with a contig containing a hypothetical 1.1-kb gene. The 1.1-kb genomic fragment was used to perform a BLASTX search of the nonredundant NCBI protein database. A fungal metalloproteinase of Metarhizium anisopliae showed high homology (E, <10−34) with a 435-bp open reading frame (ORF) of the putative C. posadasii gene. Upon restriction analysis of the entire contig containing the 435-bp ORF, two ClaI sites were identified which flanked a 3.4-kb genomic fragment. The latter was cloned and subjected to nucleotide sequence analysis. The sequence of the putative 1.1-kb gene was shown to be identical to a nucleotide sequence reported in the genomic database of C. posadasii. RACE was used to confirm the cDNA sequence of an 828-bp ORF and the location of four introns within the 1.1-kb full-length C. posadasii gene (Fig. (Fig.1C).1C). Alignment of the predicted C. posadasii protein sequence was conducted with reported homologs in the GenBank database by CLUSTAL X analysis (50). The translated gene showed 40 to 55% sequence identity and 54 to 69% sequence similarity to six putative fungal metalloproteinases reported in GenBank (Aspergillus nidulans [EAA62282], Fusarium graminearum [EAA69367], Magnaporthe grisea [EAA57072], Neurospora crassa [CAD71082], Ustilago maydis [EAK86013], and Metarhizium anisopliae [CAB63909]). We also conducted TblastN searches (1) of all the available fungal genomic databases on the websites of The Institute for Genomic Research (http://www.tigr.org/tdb/fungal/) and the Broad Institute (http://www.broad.mit.edu/annotation/fungi/fgi/). A single match was found with a contig of Aspergillus fumigatus. Alignment of the C. posadasii Mep1 sequence with that of the translated A. fumigatus homolog showed 38.1% amino acid sequence identity and 58.3% similarity. On the basis of these analyses, homologs of Mep1 have so far been identified only in filamentous fungi.
We propose that the C. posadasii gene (MEP1) encodes a metalloproteinase containing 276 residues with a predicted molecular mass of 29.6 kDa and an isoelectric point (pI) of 5.96. No N-glycosylation sites were predicted. However, some of the serine and threonine residues (at positions 29 and 14, respectively), could serve as O-glycosylation sites and may account for the higher molecular mass of the native protein. Sequence analysis of the putative metalloproteinase performed by PSORT revealed a signal peptide with a predicted cleavage site between A18 and A19. Two conserved motifs were predicted on the basis of the PROSITE algorithm: a zinc-binding active site (H191EVGHWMGLFH201X202-245M246) (20) and an ATP/GTP binding site (P-loop) with a consensus sequence of A181PYDLGKT188 (29) (Fig. (Fig.1C).1C). The glutamate (E192) active site within the zinc-binding motif and the conserved methionine (M246) that is believed to be important for the integrity of the active site characterize the metalloproteinase subfamily, M43B, to which C. posadasii Mep1 belongs (42). Although a single N-terminal amino acid sequence of Mep1 was identified, two polypeptide bands of unequal intensity were consistently observed in SDS-PAGE gels (the 30-kDa band typically stained more intensely than the 34-kDa band). These observations suggest that either cleavage of a C-terminal peptide or modification of a glycosylation site occurs during protein maturation.
Nine ORFs with homology to Mep1 have been identified in the translated C. posadasii genome database (GenBank accession no. AY987806 to AY987814) and represent a family of putative secreted metalloproteinases produced by this fungal pathogen. Mep2 to Mep7 show structural homology with both the deuterolysin of Aspergillus flavus (48) and the penicillolysin of Penicillium citrinum (28). The latter are members of the M35 subfamily of metalloproteinases, which are characterized by the presence of two zinc-binding histidines and a catalytic glutamate in an HEXXH motif (42). In addition, members of the M35 subfamily contain an aspartate residue which serves as a third zinc ligand and is present in a GTXDXXYG motif located adjacent and C-terminal to the histidines. Members of subfamily M35 do not contain the conserved methionine which is thought to be important for the integrity of the active site of metalloproteinases in subfamily M43B (e.g., Mep1). The preferred substrates of the deuterolysins and penicillolysins include basic proteins such as histones and protamines (15). The structures of Mep8 to Mep10 show homology to those of members of the M36 subfamily, exemplified by the fungalysin of A. fumigatus, which is an elastinolytic metalloproteinase. All members of subfamilies M35 and M36 are probably not able to digest the coccidioidal SOWgp antigen, which is an acidic proline- and aspartate-rich polypeptide (pI 4.2) (27). Mep1 and the other putative metalloproteinases were classified according to the MEROPS database (42). The nine predicted metalloproteinase sequences of C. posadasii were aligned with Mep1 by using the CLUSTAL X software, and a phylogenetic tree was then constructed by analysis of the output of this algorithm using the TreeView program (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) (not shown). The structural features of Mep1 that differentiate it from Mep2 to Mep10 in both the phylogenetic tree and the classification system suggested that this secreted metalloproteinase is distinct from its C. posadasii homologs. The sum of the data presented in this study indicates that Mep1 is uniquely accountable for digestion of SOWgp.
We have previously shown that the first generation of the parasitic cycle of C. posadasii isolate C735 is characterized by a near-synchronous sequence of developmental events in vitro (13). Total RNA was isolated either from spherules prior to endospore release at incubation times of 36 h and 96 h or after endospore differentiation and during the early stage of endospore release from the maternal spherule at 132 h. QRT-PCR was used to evaluate levels of expression of the C. posadasii MEP1 transcript during these three developmental stages of the first generation of the parasitic cycle (Fig. (Fig.1D).1D). The data are presented as ratios of the amount of the MEP1 transcript to that of the constitutive GAPDH gene transcript. A sharp rise in MEP1 expression (approximately ninefold) occurred during endospore differentiation and release compared to that in the preendosporulation stages of development. These data correlate with the SDS-PAGE results presented in Fig. Fig.1A,1A, which show that the Mep1 protein was detected only in 132-h parasitic-phase cultures.
Further analysis of the production of Mep1 by in vitro-grown parasitic cells was conducted by immunoblotting. Equal amounts of total protein from spherule homogenates, nonconcentrated SOW extracts, and concentrated culture filtrates of parasitic cells grown in vitro for 36 h, 96 h, or 132 h were examined by SDS-PAGE (Fig. (Fig.2A).2A). Mep1 was detected in these samples by use of a polyclonal murine anti-rMep1 antiserum (Fig. (Fig.2B).2B). Faint single 34-kDa protein bands were visible in spherule homogenates from 36-h and 96-h cultures (Fig. (Fig.2B).2B). Faint single bands were also visible in the SOW extracts of 36-h and 96-h cultures. A single prominent 34-kDa protein band was visible in the immunoblots of both the total spherule homogenate and the culture filtrate obtained from 132-h parasitic-phase cultures. Only the immunoblot of the SOW extract isolated from 132-h endosporulating cultures revealed two prominent bands of 30 kDa and 34 kDa.
The native metalloproteinase (Fig. (Fig.2C)2C) was isolated by size exclusion filtration from the SOW extract of 132-h parasitic-phase cultures as described above. When subjected to substrate gel electrophoresis, the native enzyme showed a well-defined zone of clearance of gelatin, which appeared to be composed of two bands, in the Coomassie-stained gel (Fig. (Fig.2D).2D). We also tested the ability of native Mep1 to digest purified SOWgp (Fig. (Fig.2E).2E). The latter was isolated by electroelution from an SDS-PAGE gel separation of a 96-h parasitic-culture-derived extract of SOW. When EDTA was added to the reaction mixture, the 60-kDa and 82-kDa components of SOWgp remained visible after 2 h of incubation with the metalloproteinase. In the absence of EDTA, however, the SOWgp was completely digested. The purified SOWgp incubated with or without EDTA in the absence of Mep1 showed no digestion of the glycoprotein.
A linearized, 6.1-kb transformation plasmid construct, pΔmep1 (not shown), was designed to integrate into C. posadasii chromosomal DNA by homologous recombination. Transformation of approximately 107 germ tube-derived protoplasts with 3 μg of the ClaI-linearized plasmid DNA construct yielded 18 hygromycin-resistant colonies. PCR screening of these candidate transformants revealed that five had undergone homologous recombination. One of these transformants, referred to as the Δmep1 strain, was selected for further analysis by Southern hybridization. A restriction map was constructed of the sequenced 3.4-kb genomic fragment that included the MEP1 gene. A restriction map was also generated based on the hypothetical structure of the chromosomal fragment (Δmep1) that contained the integrated pΔmep1 plasmid (Fig. (Fig.3A).3A). A 566-bp MEP1 amplicon and a 600-bp HPH amplicon were used as probes to conduct Southern hybridization analyses of the hygromycin-resistant transformants, whose chromosomal DNA contained the integrated pΔmep1 plasmid. The results of hybridization of the HindIII and SalI digests of genomic DNA from the parental strain and the selected transformant provided evidence that homologous recombination occurred at a single locus in the Δmep1 mutant strain (Fig. (Fig.3B).3B). Single bands of the predicted size were detected by Southern hybridization with the restricted genomic DNA of the transformant, which indicated that the MEP1 gene fragment had been deleted and the Δmep1 strain was homokaryotic.
A pAN8.1 plasmid containing the BLE gene, which encodes a phleomycin resistance protein, was ligated to the parental MEP1 gene and used for transformation of the Δmep1 mutant and generation of the revertant strain. In this case, transformation was conducted using the circular (undigested) plasmid. Transformation of approximately 107 protoplasts with 5 μg of the 4.6-kb plasmid DNA yielded 17 colonies which were resistant to both hygromycin and phleomycin. Each of the putative transformants was screened by immunoblot analysis of endosporulating spherule homogenates for production of Mep1. Four were positive, and two of these transformants were further screened by Southern hybridization. A hypothetical restriction map of the integrated plasmid in chromosomal DNA of the revertant strain is shown in Fig. Fig.3C.3C. Results of Southern hybridization revealed that the pMEP1R construct in one of the two selected transformants had successfully integrated into the chromosomal DNA of the Δmep1 mutant host strain by homologous recombination at a single crossover site. Genomic DNA of this transformant was restricted with HindIII and SalI and was then hybridized with MEP1-, HPH-, and BLE-derived nucleotide probes. Bands of the predicted size were detected, providing evidence that the revertant strain was homokaryotic and contained a single copy of the wild-type MEP1 gene. This transformant, referred to as the Mep1R strain, was chosen for further study. Results of Southern hybridization of the restricted genomic DNA of the second transformant revealed that the plasmid construct had integrated into the host chromosomal DNA either at multiple sites or as a tandem repeat (not shown).
We conducted morphogenetic studies of parasitic-phase cultures of the three strains. Each strain was grown in defined glucose-salts medium under identical incubation conditions (25). The cultures were each inoculated with an equal number of viable arthroconidia (1 × 105/ml) obtained from GYE plates of each strain. Aliquots of cells were examined by light microscopy at 36 h, 96 h, and 132 h after inoculation of the cultures. The stage of development of the parasitic cells produced by each strain appeared to be identical for the three strains. After incubation for 132 h, the percentage of spherules that had endosporulated (ruptured spherules plus mature spherules just prior to endospore release) was calculated for each strain. At least 500 spherules from each culture were examined. Our results indicated that there was no significant difference in the percentage of endosporulating spherules between the parental (57% ± 12%), Δmep1 (63% ± 8%), and revertant (58% ± 7%) strains. Approximately equal amounts of total protein extracted from separate isolates of SOW were applied to an SDS-PAGE gel as shown in Fig. Fig.4A.4A. As predicted, the characteristic 60-kDa and 82-kDa bands of SOWgp were visible in extracts of SOW isolated from 96-h parasitic-phase cultures of the parental, mutant, and revertant strains and were absent in extracts of SOW obtained from 132-h cultures of the parental and revertant strains. However, two prominent Coomassie blue-stained SOWgp bands (60 kDa and 82 kDa) were present in the extract of the 132-h parasitic-phase culture of the Δmep1 mutant strain. Immunoblot analysis of the SDS-PAGE gel was conducted using an anti-recombinant Mep1 antiserum and confirmed that the doublet (30 kDa, 34 kDa) of Mep1 was visible only in SOW extracts isolated from the 132-h parental and revertant cultures (Fig. (Fig.4B).4B). No bands were visible in immunoblots of the Δmep1 extracts obtained from either 96-h or 132-h cultures. In correlation with our previous observation (compare Fig. Fig.2B),2B), faint anti-Mep1-reactive bands were visible in the 96-h culture-derived SOW extracts of the parental and revertant strains. Substrate gel electrophoresis was performed using SOW extracts of the parental and Δmep1 mutant strains; it revealed that only the extract from the 132-h parental culture showed distinct enzymatic activity (Fig. (Fig.4C).4C). A faint digestion band was visible in the lane containing the extracted SOW fraction from the 96-h parental culture, which correlated with the light band in the corresponding immunoblot (compare Fig. Fig.4B).4B). Similar results were obtained using SOW extracts derived from the revertant strain (not shown).
Parasitic cells isolated from 36-h and 96-h cultures (Fig. 5A and B, respectively) were plastic embedded, sectioned, and stained with FITC-conjugated anti-SOWgp as previously reported (8). The results show that the antigen is localized both at the surface of spherule initials and in the cytoplasm, probably within vesicles (Fig. (Fig.5A).5A). During early stages of endospore differentiation (Fig. (Fig.5B),5B), SOWgp is associated with the newly formed wall material as well as the outer spherule wall. Intact spherules isolated at the endosporulation stage (132-h culture) were examined by both phase-contrast and immunofluorescence microscopy (Fig. 5C and D, respectively). Note the absence of fluorescent label on the surface of endospores released from the maternal spherule in Fig. Fig.5D.5D. The same observation was made when fluorescence-labeled endosporulating spherules of the revertant strain were examined (not shown). Paraffin sections of murine lung tissue infected with the parental strain (Fig. (Fig.5E)5E) or the Δmep1 mutant strain (Fig. (Fig.5F)5F) show a striking difference in the amount of SOWgp detected at the endospore surface. Only the endospores of the Δmep1 mutant were stained with the anti-SOWgp-specific fluorescent label. Examinations of in vitro-grown parasitic cells of the two strains also showed that only the endospores of the Δmep1 mutant strain were labeled. The persistence of SOWgp at the endospore surface in the mutant strain may influence the outcome of coccidioidal infection with this genetic variant. Macrophages present in infected murine lung tissue are frequently observed within ruptured spherules (Fig. (Fig.5G).5G). Opsonization of endospores by antibody which binds to cell surface-expressed SOWgp in the Δmep1 mutant strain may enhance phagocytosis and clearance of the pathogen. We tested this possible outcome of host infection with the Δmep1 mutant by using our murine model of coccidioidomycosis.
The titers of SOWgp-specific antibody in C. posadasii-infected C57BL/6 mice are typically low because the animals become moribund or die within 2 to 3 weeks after intranasal challenge. High titers of anti-SOWgp antibody, therefore, were first raised in mice by immunization with the recombinant protein prior to infection. To optimize antibody production, we mapped the B-cell epitopes of SOWgp by comparing murine and patient seroreactivity with the recombinant MP versus the recombinant N-t, Rpt., and C-t fragments (Fig. (Fig.6A).6A). SDS-PAGE separations of these E. coli-expressed recombinant proteins are shown in Fig. Fig.6B.6B. The 9-kDa recombinant C-t protein preparation included a 30-kDa contaminant derived from E. coli. The disparity between the predicted molecular sizes and the SDS-PAGE estimates of the molecular sizes of the recombinant MP and Rpt. protein is due to their high proline content (Table (Table1)1) (27, 40). The immunoblot in Fig. Fig.6B6B is representative of the reactivity between sera from patients with confirmed coccidioidal infection and the recombinant fragments of SOWgp. The repeat domain contains the B-cell epitopes. Additional evidence of patient seroreactivity with this domain is shown in Fig. Fig.6C.6C. Ten patients with confirmed coccidioidomycosis revealed antibody titers of 1:1,600 to 1:12,800 by ELISA. Control patients with no evidence of fungal infections showed essentially no seroreactivity with the recombinant Rpt. protein.
C57BL/6 mice were either immunized with the recombinant Rpt. fragment of SOWgp (1 μg/dose) plus adjuvant (CpG plus CFA) and then challenged by the intranasal route with one of three strains of C. posadasii or immunized with adjuvant alone and then challenged. The titers of IgG1 and IgG2c antibody to the recombinant Rpt. were measured by ELISA just prior to challenge and at 10 and 20 days postchallenge (Fig. 7A and B). High titers of antigen-specific IgG1 were detected (1:50,000 to >1:100,000), while significantly lower titers of IgG2c were found, irrespective of the fungal strain that was used to infect the mice. Control mice immunized with adjuvant alone showed consistently lower titers of IgG1 and IgG2c to the purified recombinant repeat peptide of SOWgp (approximately 1:100).
The same groups of immunized and control mice as above were evaluated for their ability to survive a lethal intranasal challenge of arthroconidia over a period of 50 days postchallenge (Fig. (Fig.7C).7C). The majority of immunized and nonimmunized mice challenged with the parental or revertant strain died within 3 weeks postchallenge. Only the mice immunized with the recombinant Rpt. protein plus adjuvant and challenged with the Δmep1 mutant strain showed a significant increase in the number of survivors compared to the other groups of mice tested in this experiment.
As stated above in Materials and Methods, it was necessary to develop an expression system for production of enzymatically active Mep1 which could be used in our in vitro studies. The purpose of these investigations was to test whether parasitic cells (spherule initials; diameter, 10 to 15 μm) coated with SOWgp are readily engulfed and killed by alveolar macrophages in the presence of antibody raised against the repeat fragment of SOWgp (anti-Rpt. IgG). Conversely, we tested whether preincubation of the spherule initials with active Mep1 abrogated opsonophagocytosis in the presence of the same antibody. We successfully transformed Uncinocarpus reesii with the plasmid construct containing the C. posadasii MEP1 gene shown in Fig. Fig.8A.8A. Putative transformants, initially selected for their ability to grow on hygromycin-containing agar (100 μg/ml), were cultured in GYE liquid medium at 30°C for 4 days and then at 37°C for 1 day. A single transformant was selected (Fig. (Fig.8B)8B) based on positive results of immunoblot analysis of the culture filtrate using an anti-His tag monoclonal antibody. The ammonium sulfate-precipitated total-protein fraction of the culture filtrate was subjected to nickel affinity chromatography for purification of rMep1ur. The purified protein was enzymatically active, as demonstrated by the results of substrate gel electrophoresis (Fig. (Fig.8B).8B). The ability of rMep1ur to digest SOWgp at the surface of intact spherule initials is demonstrated in Fig. 8C and D. Immunoblot analysis of the homogenate of these enzyme-treated cells showed a marked reduction in the amount of SOWgp detected compared to that in the homogenate of the untreated spherule initials (Fig. (Fig.8E).8E). The persistence of SOWgp in homogenates of the treated cells is accounted for by the pool of antigen in the cytoplasm of the parasitic cells (compare Fig. Fig.5A5A).
The results of the in vitro opsonophagocytosis and killing experiments are summarized in Fig. 9A and B, respectively. Typically, those macrophages which had engulfed the pathogen contained a single spherule initial. After 3 h of incubation, the MH-S alveolar macrophages engulfed spherule initials in significantly higher numbers when the cells were not pretreated with rMep1ur but were opsonized with anti-Rpt. IgG than when the cells were enzyme treated and opsonized (P = 0.001) or enzyme treated or untreated and not opsonized (P = 0.001 and 0.004, respectively). Similar differences in the phagocytic index were observed after 6 h of incubation. As expected, spherule initials that were not pretreated with recombinant enzyme but were opsonized with anti-Rpt. IgG were also more efficiently killed after 3 h of incubation than untreated control cells (P = 0.002). Fungal cells that were stripped of their SOWgp by preincubation with rMep1ur and then exposed to anti-Rpt. IgG were killed less efficiently by the alveolar macrophages than nondigested, opsonized parasitic cells (P = 0.03).
A lipid-rich, membranous SOW layer is produced during in vitro growth of Coccidioides (7, 9). Thin-section electron microscopic studies of the isolated SOW fraction revealed tripartite membranes reminiscent of phospholipid bilayers (9). Antibody from patients with confirmed coccidioidal infection showed high affinity for the SOW associated with the parasitic cell surface, as well as the membranous material released into the culture media (7, 9). To identify the component(s) responsible for this immunoreactivity, we first extracted the isolated SOW with a nonionic detergent, N-octyl-β-d-glucopyranoside (OG), and examined the protein composition of the OG-soluble fraction by SDS-PAGE (7, 9). Two major Coomassie blue-stained bands (60 kDa and 82 kDa) were revealed in the detergent extract of SOW obtained from C. posadasii strain C735. The two polypeptides were subsequently shown to be components of a single glycoprotein, designated SOWgp, which is responsible for patient seroreactivity with the isolated membranous SOW fraction (25). The purified glycoprotein was also demonstrated to be a potent stimulant of both murine immune T-cell and human peripheral blood monocytic cell (PBMC) proliferation in vitro (7, 25). The PBMCs were obtained from individuals who showed a skin test-positive reaction to spherulin, which is a parasitic-phase antigen of Coccidioides used to measure immunological reactivity (7). SOWgp, therefore, has been determined to be a major cell surface antigen of Coccidioides that elicits both antibody-mediated and cellular immune responses in patients with coccidioidal infections (25). We have shown that a mutant strain of C. posadasii with a targeted disruption of the SOWgp gene is significantly less virulent in mice during pulmonary infection than the parental strain (27). Preliminary studies have revealed distinct differences in the major cytokines produced by C57BL/6 mice in response to pulmonary infection with the Δsowgp mutant versus the parental strain (11). We propose that the SOW components, which include immunodominant SOWgp antigen, influence the host to react to respiratory infections with the wild-type strain of C. posadasii by activation of a T-helper 2 (Th2) pathway of immune response. A substantial body of evidence derived from studies of coccidioidal infections in mice, on the other hand, suggests that Th2 immunodominance compromises host protection against coccidioidomycosis and exacerbates the course of disease (10, 13, 30).
Endospores that emerge from ruptured spherules of wild-type strains of Coccidioides are not recognized by sera of patients with coccidioidal infection or by antisera raised against the purified SOWgp. The surface of mature spherules of the same strains, on the other hand, shows high affinity for anti-SOWgp antibody (7, 27). We have also noted that wild-type strains of the pathogen grown in vitro show a sharp reduction in both the transcript level of the SOWgp gene and the amount of SOWgp recovered from cultures of endosporulating spherules compared to preendosporulating spherules (27). These results suggested that a developmental-stage-specific mechanism(s) of downregulation exists which controls the level of production of this immunodominant cell surface antigen. A possible function of such a regulatory system is to permit the endospores to evade cell surface antibody binding and host detection during early development, when the parasitic cells are most vulnerable to killing by host phagocytes. In this study, we discovered that the disappearance of SOWgp at the endospore surface is at least partly under the control of a specific metalloproteinase expressed during the endosporulation phase of the parasitic cycle.
The absence of the SOWgp of C. posadasii strain C735 in detergent extracts of SOW obtained from endosporulation-phase cultures correlated with the appearance of a new protein not previously detected in this cell wall fraction. The protein was determined to be a metalloproteinase that belongs to the metzincin superfamily (4). Its full-length amino acid sequence was deduced from results of MS/MS analyses of peptides derived from trypsin digests of the purified protein, combined with BLAST searches of the translated genomic database of C. posadasii for matches with the peptide sequences. The amino acid sequence of the translated ORF of the newly cloned gene (MEP1) of C. posadasii predicted the existence of an elongated zinc-binding motif, HEXXHXXGXXH, and a conserved Met residue spatially located in close proximity to three His residues that have been reported to comprise the active site of other reported metalloproteinases (22). These features are characteristic of the M43B subfamily of metzincins (3). A BLASTX search of the nonredundant NCBI protein database was conducted using the translated MEP1 gene as a probe. Sequence similarities with six reported fungal metalloproteinases were found. TblastN searches of the genomic databases of medically important fungi revealed a possible sequence match with a putative protein of Aspergillus fumigatus. The full-length MEP1 gene was also used to conduct a BLAST search of the translated C. posadasii genomic database. Nine additional secreted metalloproteinase homologs classified in two separate subfamilies were identified. However, Mep1 proved to be a structurally unique member of the Coccidioides metalloproteinases, which suggests that it may also have a unique function.
The levels of expression of MEP1 mRNA and production of the enzymatically active Mep1 protein were sharply elevated during the endosporulation phase of C. posadasii compared to other stages of parasitic cell development. Purified Mep1 was shown to be capable of complete digestion of SOWgp in vitro, and proteolysis was inhibited by the presence of the metal chelator EDTA. SDS-PAGE separation of the Mep1 protein revealed two polypeptide components (30 kDa and 34 kDa). Two enzymatically active bands of Mep1 were also visible in substrate electrophoresis gels, but only in detergent extracts of SOW isolated from cultures that contained endosporulating spherules. These extracts revealed significantly higher Mep1 enzyme activity than extracts obtained from preendosporulation stages of development. The N-terminal sequences of the 30-kDa and 34-kDa polypeptide components of Mep1 are identical. The 30-kDa polypeptide could be the product of C-terminal autolytic cleavage, which may occur only in reaction mixtures with large amounts of active Mep1. The functional significance of production of these two polypeptides of Mep1 is unknown.
SOWgp synthesis and cell surface deposition occur during development of first-generation spherule initials, derived from germinated arthroconidia, and continue during subsequent stages of spherule segmentation and early endospore differentiation. It appears that the peak of Mep1 production occurs while the endospores are still contained within the maternal spherule. Endospores are released from mature spherules in large numbers (>200 cells per spherule), undergo rapid isotropic growth, and soon become too large to be engulfed by host phagocytes. To test whether Mep1 plays a role in evasion of host defenses, the MEP1 gene was disrupted by homologous recombination using a strategy which is now well established in our laboratory (27, 43). As expected, the SOW extract from 132-h endosporulation-phase cultures of the Δmep1 mutant showed that SOWgp was present at a concentration which appeared to be comparable to that of the preendosporulation phase. Endospores isolated from mice infected intranasally with the Δmep1 mutant and examined by immunofluorescence microscopy showed abundant anti-SOWgp-FITC label at the cell surface, in contrast to the identically treated but unlabeled endospores of the parental strain which were isolated from separately infected animals. The phenotype of the revertant was the same as that of the parental strain. In vitro growth rates and in vitro/in vivo morphogenesis of the three strains appeared to be identical. With these three strains in hand, we were able to design an animal experiment to test whether the absence of Mep1 activity and retention of SOWgp at the endospore surface influenced the outcome of coccidioidal infection in mice.
The maternal spherules release their contents as the endospores enlarge and cause the outer spherule wall to rupture. The host, in turn, mounts an intense inflammatory response to the released products (10). Neutrophils are the dominant innate cells found associated with endosporulating spherules, although macrophages are also present (5). Many phagocytes are typically found within the ruptured spherule. The fungal products and host factors responsible for this chemotaxis are undefined. In spite of this intense phagocytic response to endosporulation, rarely are the endospores observed within the host cells. Earlier in vitro studies of host phagocyte interaction with Coccidioides have indicated that macrophages and neutrophils are only able to partially inhibit growth of the pathogen; they are unable to kill the organism (2, 16, 19). We speculated that opsonization of SOWgp-coated endospores produced by the Δmep1 mutant strain would result in increased phagocytosis and clearance of the pathogen from infected host tissue. Our strategy was to generate high titers of anti-SOWgp antibody in mice prior to infection by immunization of the animals with the seroreactive fragment of the glycoprotein. Our preliminary epitope-mapping studies indicated that only the 47-aa-repeat domain of SOWgp (27) is recognized by patient sera and elicits production of high titers of antibody in mice. Indirect ELISAs of antibody in immunized C57BL/6 mice prior to challenge with Coccidioides revealed that the titers of SOWgp-specific IgG1 were in the range of 1 × 105. The immune mice inoculated intranasally with 80 viable arthroconidia of the Δmep1 mutant strain showed a significant increase in survival at 50 days postchallenge compared to immunized mice infected with the parental or revertant strain. Since first-generation spherule initials produced by either the wild-type or the Δmep1 mutant strain are coated with SOWgp, it would be expected that these cells would be opsonized in the presence of high titers of anti-SOWgp. However, we argue that since the intranasal challenge dose was high, and a lethal coccidioidal infection of C57BL/6 mice can be established with as few as 10 spores, sufficient numbers of spherule initials developed into endosporulating parasitic cells to establish a pulmonary infection. The results of immunization of mice challenged with the Δmep1 mutant support our hypothesis that persistence of SOWgp on the surfaces of endospores in the presence of high titers of anti-SOWgp antibody enhances phagocytosis and killing of the parasitic cells. Conversely, SOWgp-immunized mice challenged with the parental strain were unable to control progressive disease because of the ability of Mep1 to strip endospores of the immunodominant cell surface antigen and thus evade host detection. Nonimmunized mice that were challenged with the Δmep1 mutant failed to survive. We believe that this was due to the rapid dissemination of the pathogen and early onset of a moribund state, which did not permit the animals to generate a high anti-SOWgp antibody titer.
To further test our hypothesis, we designed an in vitro experiment using a murine alveolar macrophage cell line (MH-S) to evaluate opsonophagocytosis and killing of SOWgp-coated or SOWgp-depleted parasitic cells in the presence and absence of anti-SOWgp antibody. Spherule initials were used for these studies rather than endospores because of their ease of isolation from parasitic-phase cultures and their developmental synchrony in vitro. Production of sufficient quantities of purified, enzymatically active Mep1 was achieved by generation of a novel expression system using Uncinocarpus reesii. The latter is a nonpathogenic, ascomycetous fungus that is phylogenetically related to C. posadasii (37). Incubation of spherule initials with the recombinant Mep1 stripped SOWgp from the cell surface, simulating the condition of endospores released by mature spherules of the wild-type strain of C. posadasii. In our in vitro evaluations of opsonophagocytosis, we prestained the parasitic cells with a fluorochrome and then quenched the noningested cells with trypan blue after their incubation with macrophages. This procedure permitted us to distinguish clearly between engulfed and phagocyte-bound fungal cells and provided more-accurate data on phagocytosis of coccidioidal cells than previously reported (2). The results of our in vitro studies demonstrated that incubation of spherule initials with anti-SOWgp enhanced phagocytosis and killing of C. posadasii in comparison to incubation of the parasitic cells with control murine IgG. We also showed that pretreatment of the spherule initials with recombinant Mep1 abrogated opsonophagocytosis and killing by the alveolar macrophages. Although it has been reported that Coccidioides is resistant to complement-mediated killing (2), we cannot rule out the possibility that complement provided in the macrophage culture medium may contribute to the results observed in our in vitro killing experiments. On the other hand, it has been well documented that Fc receptor-mediated, antibody-dependent phagocytosis plays an important role in host resistance to microbial infection (17, 53).
In summary, the results of this study support our earlier observation that SOWgp presentation during parasitic cell development is cyclic; spherule initials and segmented spherules are coated with the immunodominant antigen, while endospores of the wild-type strain that are released from mature spherules are devoid of SOWgp at the cell surface. The latter is at least partly due to the action of Mep1, which contributes to the ability of the pathogen to evade host detection and establish a persistent infection. It is possible that humans with active disease, who typically have high titers of anti-SOWgp antibody, would benefit from both passive immunization with an anti-Mep1 antibody and chemotherapy with a Mep1 inhibitor. The feasibility of these therapeutic interventions is under investigation in our laboratory.
Support for this study was provided by Public Health Service grants AI19149, AI37232, and UO1-AI50910 (Coccidioides genome sequencing project) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
We are grateful to Nicholas Poitinger for technical assistance.
Editor: T. R. Kozel