Center for Medical Mycology, University Hospitals of Cleveland and Department of Dermatology, Case Western Reserve University, Cleveland, OH 44106-5028, USA1
Department of Microbiology and Immunology, Medical College of Ohio, Toledo, OH 43614-5806, USA2
Author for correspondence: Mahmoud A. Ghannoum. Tel: +1 216 844 8580. Fax: +1 216 844 1076. e-mail: mag3{at}po.cwru.edu
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ABSTRACT |
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Keywords: phospholipase B, virulence factor, candidal transmigration, in vivo localization of Plb1
Abbreviations: GI, gastrointestinal; PAS, periodic acidSchiff reagent
a These authors contributed equally to this work.
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INTRODUCTION |
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To develop such new therapies and treatments for candidiasis, it is essential to dissect the infectious process of C. albicans. Several virulence factors have been proposed for C. albicans, which may represent novel molecular targets for antifungal development. These factors include extracellular phospholipases and proteinases (Hube et al., 1998 ; Ghannoum, 2000
; Mukherjee & Ghannoum, 2001
). Several bacterial and protozoan phospholipases have also been shown to contribute to the infectious processes of these pathogens (Ravdin et al., 1985
; Saffer & Schwartzman, 1991
; Silverman et al., 1992
; Schmiel & Miller, 1999
; Zhou et al., 2001
). Like their bacterial and protozoan counterparts, the phospholipases of C. albicans also are considered important virulence determinants (Ibrahim et al., 1995
), and could potentially facilitate increased penetration of fungal hyphal elements by directly damaging host cell membranes. To determine if phospholipases have a role in candidal virulence, we have previously cloned and disrupted PLB1, the gene encoding candidal phospholipase B (Plb1), and showed that virulence of the plb1 null mutant was significantly attenuated compared to that of its isogenic parental counterpart when tested in a murine model of haematogenously disseminated candidiasis (Leidich et al., 1998
).
C. albicans is a member of the gastrointestinal (GI) microflora in normal individuals. In immunocompromised hosts, migration of this fungus across the GI tract represents one of the mechanisms by which disseminated candidiasis is established (Cole et al., 1989 ). An oralintragastric infant mouse model of GI candidiasis has previously been established which is designed to mimic the process by which C. albicans traverses the human GI tract and haematogenously disseminates to other body organs (Cole et al., 1990
, 1996
). This model allows precise control of challenge dose and maintains the natural host barriers, e.g. gastric and intestinal secretions, peristalsis and mucin (Cole et al., 1996
).
In our earlier investigations, we showed that the plb1 null mutant is attenuated in its virulence (Leidich et al., 1998 ). However, these studies did not involve a revertant strain containing the reintroduced PLB1 gene, due to the unavailability of such a strain at that time. To unequivocally prove the association of PLB1 with candidal pathogenesis, it was necessary to reintroduce the functional PLB1 gene back into the plb1 null mutant, and determine if the revertant has similar virulence to the parental strain. In this study, we report the successful construction of the PLB1 revertant strain and show that it has similar virulence to the wild-type strain in an intravenous murine model of haematogenously disseminated candidiasis. Additionally, using an oralintragastric infant mouse model of candidiasis, we provide evidence that Plb1p is secreted during C. albicans transmigration of the GI tract. Furthermore, deletion of the PLB1 gene results in significant reduction in the ability of the pathogen to traverse the stomach mucosa and disseminate haematogenously to the liver.
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METHODS |
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Reagents.
Restriction endonucleases, T4 DNA ligase, DNA polymerase (Klenow fragment), calf intestinal alkaline phosphatase and Taq DNA polymerase were purchased from Boehringer Mannheim. Oligonucleotide DNA primers were synthesized by Bio-Synthesis. Glass beads (25600 µm), used for total chromosomal DNA extraction, and all other chemicals were obtained from Sigma. Guinea pig anti-Plb1 polyclonal antiserum was produced by Pocono Rabbit Farm & Laboratories.
Standard procedures were followed for the propagation and selection of plasmids, the growth of their bacterial hosts, and for the subcloning of DNA fragments (Sambrook et al., 1989 ).
Construction of plasmids.
To reintroduce the PLB1 gene, the integration plasmid pPMB3 was constructed as follows. Plasmid pMB-7, which contains the C. albicans URA3 gene flanked by 1·1 kb direct repeats of the Salmonella typhimurium hisG DNA, was kindly provided by Dr W. Fonzi (Georgetown University, Washington, DC, USA). A 2·9 kb fragment containing the URA3 gene was isolated by digesting pMB-7 with BamHI and cloned into the BamHI site of the plasmid pPCR-Script Amp (Stratagene) to generate plasmid pPMB1. A SwaI/ApaI genomic fragment containing the intact PLB1 gene was ligated into the EcoRV site of plasmid pcDNA3.1 (Invitrogen) to form plasmid pGN. Digestion of pGN with EcoRI and XhoI led to the release of a 2·2 kb PLB1 fragment, which was subcloned into the EcoRI/SalI sites of plasmid pPCR-Script Amp (Stratagene) to generate plasmid pPMB2. Next, plasmid pPMB2 was digested with PstI/XhoI to release the 2·2 kb PLB1 fragment, which was then cloned into the PstI/XhoI sites of plasmid pPMB1 to generate the integration plasmid pPMB3. The presence of URA3 and PLB1 genes in pPMB3 was confirmed by restriction enzyme analysis using URA3- or PLB1-specific restriction enzymes (data not shown). Integration plasmid pPMB3 was used in transformation of the plb1-disrupted strain of C. albicans.
Reintroduction of the C. albicans PLB1 gene.
The integration plasmid (pPMB3, constructed as described above), containing the functional PLB1 and URA3 genes, was used to transform the null mutant strain in order to reintroduce these two genes. Plasmid pPMB3 was digested with BglII, which has a single recognition site in PLB1 but not in the URA3 or vector regions (Fig. 1a). This linearized plasmid was then transformed into the PLB1
2-derived Ura- strain (PMY53, generated by selection on 5-fluoroorotic acid as described above) using a lithium acetate-based transformation protocol (Rose et al., 1990
). Ura+ prototrophs were selected on minimal medium lacking uridine. Total chromosomal DNA was isolated (Rose et al., 1990
) from cultures produced by the growth of individual colonies (transformants). Insertion of the functional PLB1 gene into the null mutant as a result of spontaneous recombination was confirmed by Southern blot analyses.
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Southern blot analyses.
Total chromosomal DNA was isolated from the respective C. albicans strains as described previously (Rose et al., 1990 ). The isolated DNA was digested with (i) KpnI and SacI or (ii) KpnI only [6 U (µg DNA)-1], electrophoresed through 1·2% agarose and transferred to nylon membranes (Boehringer Mannheim) following standard protocols (Sambrook et al., 1989
). The transferred DNA was cross-linked to the membrane using a UV Stratalinker 228 (Stratagene) delivering 120000 µJ. The probes used were (a) a 3 kb HindIII fragment containing a portion of the 5'-terminus of the intact PLB1 gene, (b) a 736 bp BamHI fragment corresponding to the portion of PLB1 deleted during the disruption process (BamHI internal) (Fig. 1a
) or (c) a 1·3 kb XbaIPstI fragment of the pPMB1 plasmid which contains the S. typhimurium hisG region. Probes were labelled with digoxigenin (DIG)dUTP using the DIG High Prime DNA Labelling and Detection Kit (Boehringer Mannheim) according to the manufacturers instructions. Cross-linked membranes were prehybridized for 1 h at 65 °C, then hybridized overnight with DIG-labelled probe (30 ng ml-1) at 65 °C. After stringency washes, hybridizing DNA fragments were detected using the kit mentioned above, according to the manufacturers instructions.
Western blot analysis and assays for Plb1 enzyme activities were performed with culture supernatants from the respective strains, as described previously (Leidich et al., 1998 ).
Haematogenous model of disseminated candidiasis.
The protocol described earlier by our group was followed (Ghannoum et al., 1995 ). Briefly, strains of C. albicans were grown in SD medium at 30 °C until mid-exponential phase. Cells were harvested, washed, counted and resuspended at a density of 1·25x107 cells ml-1 in PBS. Female BALB/c mice (78 weeks old) were injected intravenously via the lateral vein with 200 µl (5x105 blastospores) of this fungal suspension. Cages were checked twice daily for dead or moribund mice. Mice were categorized as moribund if they displayed the following symptoms: lethargy, wasting and vertigo. Such mice were killed by CO2 asphyxiation. To determine tissue fungal burden, mice were injected with the respective C. albicans strain as described above. Mice in each group were killed 48 h post-infection and their kidneys were removed. The organs were weighed, homogenized separately in 5 ml sterile PBS, and serial dilutions were plated on SDA plates supplemented with chloramphenicol (50 µg ml-1; Sigma). The plates were incubated at 30 °C for 2448 h, after which the number of c.f.u. was determined.
GI infection of mice (oralintragastric model).
Outbred mice [crl:CFW (SW) BR] obtained from Charles River Farms (Wilmington, MA, USA) were used to establish a breeding colony and the offspring of these animals were used in all experiments. Infant mice, 6 d old (34 g), were separated from their mother 4 h prior to challenge. Candidal cells grown on SDA slants at 30 °C were harvested, washed with saline, and the challenge dose was standardized by haemocytometer counts. The inoculum size was confirmed by dilution plating. Infant mice were inoculated with 2x108 blastospores in a total volume of 0·05 ml by the oralintragastric route using a 24-gauge animal feeding needle (Popper & Sons) as described previously (Cole et al., 1990 ). Following inoculation, the pups were kept at 35 °C for 1 h before being returned to their mother. We determined that C. albicans delivered by this route was not recovered from the lungs or blood of the pleural cavity when these tissues were cultured within 10 min post-challenge, thus making it unlikely that spread to body organs resulted from faulty inoculation technique or aspiration.
Faecal pellet assay and separation of test groups.
Mice infected by oralintragastric inoculation with C. albicans were selected for further study on the basis of the presence of yeast in their faecal pellets at 9 d post-challenge (Cole et al., 1989 ). Animals were marked for identification using picric acid at the time of the assay. Fresh faecal pellets were immediately homogenized in 1·0 ml chilled, sterile saline and 0·1 ml of the homogenate was plated on SDA (Difco) supplemented with 50 µg chloramphenicol ml-1. The plates were incubated at 37 °C for 48 h. Mice identified as culture positive for C. albicans were immunocompromised as described below and used to compare the systemic spread of the parental (SC5314), plb1 null mutant (PLB1
2) and revertant (PMY106) strains from the site of colonization in the GI tract.
Immunosuppression.
An immunocompromising treatment was initiated on day 11 after oralintragastric challenge as previously reported (Cole et al., 1989 ). Mice selected on the basis of positive faecal pellets received cyclophosphamide (Adria Laboratories) by the intraperitoneal route at doses of 0·2 mg (g body wt)-1 and 0·1 mg (g body t)-1, on days 11 and 14 post-challenge, respectively. In addition, 1·25 mg cortisone acetate (Merck, Sharp & Dohme) was administered by the intraperitoneal route on days 11 and 14 post-challenge. This combined drug treatment was chosen on the basis of its severe immunocompromising effects, which lead to systemic spread of C. albicans from sites of colonization in the GI tract (Guentzel & Herrera, 1982
; Cole et al., 1991a
). Mice were killed at day 20 post-challenge by CO2 asphyxiation. Stomachs and livers were aseptically removed and prepared for microscopic analyses or quantification of fungal c.f.u. as described previously (Pope et al., 1979
). For enumeration of candidal c.f.u., the stomach and liver of each infected animal were removed under aseptic conditions, visually inspected for gross candidal foci, and homogenized separately in 5·0 ml sterile saline (Travenol Laboratories) using a tissue grinder equipped with a Teflon pestle (Cole et al., 1989
). The c.f.u. of C. albicans in each organ were determined by plating serial dilutions of the respective homogenate on SDA supplemented with chloramphenicol (50 µg ml-1) as above. Plates were incubated at 37 °C for 48 h and then colonies were counted.
Light microscopy.
The stomachs of five animals from each group (parental, plb1 null mutant and revertant strain infected) were used for light microscopic histological and immunofluorescence labelling. In addition, stomachs of five more animals from each group were used for electron microscopic and immunogold labelling studies. Following removal, stomachs were immediately placed in chilled saline and dissected open to expose the cardial-atrial fold (Cole et al., 1990 ). Tissues were fixed in 4% paraformaldehyde (v/v in PBS; 0·1 M, pH 7·4) for 12 h at 4 °C. Tissues were then washed with buffer, dehydrated in ethanol, and embedded in paraffin wax. Sections were stained with periodic acidSchiff reagent (PAS) for demonstration of fungal elements (Luna, 1968
).
Transmission electron microscopy.
C. albicans-infected stomachs were chemically fixed for 12 h at 4 °C in glutaraldehyde (3%, v/v) and paraformaldehyde (2%, v/v), each prepared separately in cacodylate buffer (0·1 M, pH 7·4) and mixed just before use. The tissues were rinsed five times in buffer, post-fixed in 2% osmium tetroxide (2 h) prepared in the same buffer, dehydrated and embedded in Spurrs low viscosity resin as described previously (Seshan & Cole, 1994 ). Thick sections (approx. 1 µm) were stained with azure 11-methylene blue for light microscopy as reported by Cole et al. (1989)
. Thin sections were mounted on copper grids, stained with uranyl acetate and lead citrate and examined with a Phillips CM-10 transmission electron microscope. Osmium tetroxide post-fixation was omitted for tissues intended for immunofluorescence and immunogold labelling described below.
Production of antibodies.
Guinea pig anti-Plb1 polyclonal antibodies were produced commercially (Pocono Rabbit Farm & Laboratory). A standard protocol was followed for the antibody production. Briefly, on day 0 a preimmune (base-line) bleed was performed. This preimmune serum was stored at -80 °C until assayed. On the same day, 50 µg purified Plb1 was injected intramuscularly in Freunds complete adjuvant. On day 14, 1020 µg Plb1 was injected intramuscularly in Freunds incomplete adjuvant. A test bleed was performed on day 42 post-immunization and anti-Plb1 antibody levels were titred. Guinea pigs were boosted every 4 weeks with 10 µg Plb1 antigen until peak antibody titres were achieved. Antiserum samples were stored frozen (-80 °C) until assay.
Immunofluorescence microscopy.
Immunofluorescence detection of Plb1 was performed on chemically fixed, resin-embedded stomach tissue harvested from mice infected with the parent, plb1 null mutant or revertant strains. Ten thick (1·0 µm) sections of stomach tissues of each strain cut with an ultramicrotome (MT-5000; Dupont) were mounted on a specially prepared gelatin-coated glass slide for immunolabelling. Sections were blocked with 1% bovine serum albumin in PBS (0·15 M, pH 7·4) for 10 min, reacted with primary antibody (guinea pig anti-Plb1) diluted 1:500 in PBS for 1 h and incubated with goat anti-guinea pig IgGFITC secondary antibody (Sigma; diluted 1:30 in PBS) for 1 h. The sections were washed and examined with a Zeiss microscope equipped with an FITC filter set. Control sections were reacted either with FITC alone, or with normal guinea pig preimmune serum followed by secondary antibodyFITC conjugate. These experiments were repeated five times.
Cryofixation and freeze-substitution.
To better preserve the association between the soluble Plb1p antigen and the fungal cells, the infected tissue was subjected to cryofixation and freeze-substitution (Cole et al., 1991b ). In brief, blocks of dissected stomach tissue (approx. 3 mm2) from the region of the cardial-atrium fold were dropped onto a polished copper block prechilled with liquid nitrogen. The frozen tissue was then immediately transferred to a vial containing the substitution fluid (anhydrous acetone plus 0·05% uranyl acetate), which was maintained at -80 °C. The tissue was then freeze-substituted at -80 °C for 48 h, -40 °C for 24 h, -20 °C for 24 h, 4 °C for 4 h, and then gradually brought to room temperature. After two rinses with absolute acetone, the tissues were embedded in Spurrs resin (Spurr, 1969
).
Immunogold labelling for localization of Plb1.
Stomach tissue was prepared for immunoelectron microscopy as previously described (Kruse & Cole, 1992 ). Briefly, three to four thin sections of cryofixed, infected stomach tissue were mounted on a Formvar-coated nickel grid. A total of 15 grids containing about 4560 thin sections from each group were used for immunological labelling. The grids were floated on 10% ovalbumin in 20 mM Tris/HCl (pH 7·4) for 10 min. The grids were then transferred to droplets of the guinea pig anti-Plb1 primary antibody (diluted 1:500) prepared in 1% ovalbumin in Tris/HCl buffer. Control sections were incubated in preimmune guinea pig serum diluted as above. Sections were incubated for 48 h at 4 °C. The grids were rinsed with buffer and incubated on droplets of goat anti-guinea pig secondary antibody conjugated with colloidal gold (20 nm diameter). The secondary antibody was prepared in Tris/HCl buffer (pH 8·2) and diluted 1:20 in 1% ovalbumin. The grids were finally washed in buffer followed by distilled water, and then stained with uranyl acetate and lead citrate. The specimens were examined with a transmission electron microscope as described above.
Statistical analysis.
The numbers of c.f.u. per organ were expressed on a log scale. Because these values did not fall into normal distribution, the MannWhitney U test was used to compare medians. Statistical comparisons were performed using the StatView version 4.5 software package for Windows 95, or SPSS version 9.0 software package for Windows.
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RESULTS |
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Reintroduction of the functional PLB1 gene
Strain PMY53 (the Ura- derivative of the plb1 null mutant) was selected as the recipient for transformation with the BglII-digested integration plasmid pPMB3 containing a functional PLB1 gene. Growth and morphology of the resulting transformants were compared and found to be similar (data not shown); one of these transformants, PMY106, was selected as the representative revertant strain and further analysed. PCR analysis was performed to determine the correct insertion of the functional PLB1 gene into PMY53. Total chromosomal DNA was isolated from PMY106 and subjected to PCR using a PLB1-specific 751 bp region as template. Fig. 1(b) shows that the revertant (PMY106) and parental (SC5314 and CAI-4) strains contained the functional PLB1 allele amplified as a 751 bp fragment, while no signal corresponding to the amplified functional PLB1 could be detected in the null mutant.
To confirm the reintroduction of the PLB1 gene into the mutant strain, total chromosomal DNA isolated from the transformant PMY106 was analysed by Southern blotting using a C. albicans PLB1-specific, as well as a bacterial hisG-specific probe. Fig. 1(c) shows that the 3·6 kb band corresponding to the functional PLB1 gene present in the parent SC5314 and CAI-4 strains is also detected in PMY106, thus proving the introduction of functional PLB1 in the transformant. To make sure that PMY106 was not a contaminant strain, total chromosomal DNA from the revertant strain was digested with KpnI, and hybridized with a hisG-specific probe. An 8 kb band representing the URA3hisG
plb1::hisG fragment and a 4·7 kb band representing the
plb1::hisG fragment were detected (Fig. 1d
). The parent strains (SC5314 and CAI-4) did not show any detectable fragment, which was expected since the probe used (hisG) represents a bacterial sequence. These results clearly show that the PLB1 gene was successfully reintroduced into the mutant strain.
Western blot analysis of the culture supernatant from the revertant, mutant and parental strains was performed to detect the Plb1 protein secreted by these strains. Plb1p was detected in both the parental and revertant strains, but not in the mutant strain (data not shown). Additionally, the relative levels of Plb1 enzyme activity in culture supernatant from the parental, revertant and mutant strains were 100%, 98% and 1%, respectively, as determined by the colorimetric free fatty acid assay procedure. This indicated that the enzyme activity in the revertant strain was similar to that of the parental strain.
Reintroduction of PLB1 restores candidal virulence (haematogenously disseminated model)
A murine model of haematogenously disseminated candidiasis was employed to evaluate the effect of PLB1 reintroduction on virulence. All mice injected with either the wild-type strain SC5314 or the revertant PMY106 strain succumbed to candidal infection within 10 d (Fig. 2). In contrast, 80% of the mice injected with PLB1
2 were alive at day 10. The mean survival time (±SD) for mice infected with SC5314 was 5·30±0·790 d, while that for mice infected with the revertant strain was 6·30±0·59 d. In contrast, the mean survival time for mice infected with the plb1-disrupted mutant was 15·80±1·27 d. Statistical comparisons of the survival curves by Logrank (MantelCox) analysis revealed that survival of mice infected with strain SC5314 did not differ significantly from that of mice infected with PMY106 (P=0·48). In contrast, mice infected with the PLB1
2 mutant strain survived significantly longer than mice infected with either SC5314 or PMY106 (P<0·0001 for both comparisons). Candidal c.f.u. were recovered from the kidneys of mice challenged with either the parental (SC5314) or revertant (PMY106) strain. No significant difference was found (P=0·54) between c.f.u. count for mice challenged with the revertant strain [1·3±0·12x106 c.f.u. (g tissue)-1] and those challenged with the parental strain [1·2±0·28x106 c.f.u. (g tissue)-1]. Strain PMY106 recovered from the kidneys of infected mice was incubated in serum for 2 h, and no difference was found in its ability to form germ tubes, as compared to SC5314 (data not shown). Revertant and wild-type strains passed through mice were further analysed by Southern hybridization, which confirmed that passage through animals had no effect on the genetic composition of these strains (data not shown). These results show that the reintroduction of the PLB1 gene restores the virulence properties of C. albicans in the haematogenously disseminated model of candidiasis.
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DISCUSSION |
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The mechanism/s by which Plb1p contributes to virulence is not well understood, but probably involves direct damage or degradation of host cell membranes. Such host cell injury would be expected to facilitate candidal penetration. In this context, Klotz et al. (1983) used an in vitro model to depict the earliest events of metastatic C. albicans infection and showed that the pathogen first adheres to, and then penetrates the endothelium. During transmigration, endothelial cell continuity was found to be disrupted by the yeasts. As destruction of the endothelium progressed, the fungus penetrated deeper into the substance of the vascular tissue. These authors attributed the dissolution of a portion of the endothelial cells to phospholipase activity. In another study, Pugh & Cawson (1977)
reported that phospholipase activity is generally localized at the tips of developing hyphae, i.e. in the direction of candidal penetration. Consistent with these findings, our immunolabelling studies showed that Plb1 localized mostly to the hyphal tips. Although the cell wall of plb1 null mutant cells reacted slightly with the immunolabel, the intensity of the signal was much lower than that of the parental and revertant strains. The low level of reactivity may be due to non-specific binding or the presence of another minor phospholipase with homology to Plb1p. In this regard, we reported the cloning of caPLB2, the second PLB gene in C. albicans (Sugiyama et al., 1999
).
Our results suggest that Plb1 may directly degrade the phospholipid constituents of host cell membranes. Such injury to the protective cell membrane may provide fungal hyphae with rapid access to the cytoplasm. Similar evidence for enzymic activity by C. albicans in the penetration of glossal epithelium in both rats (Howlett & Squier, 1980 ) and humans (Montes & Wilborn, 1968
) has been reported, and studies have been performed that document this process in murine cervical tissue and skin (Scherwitz, 1982
). We previously reported a significant difference in the ability of parental and Plb1-deficient strains to penetrate endothelial and epithelial cells in vitro (Leidich et al., 1998
). The wild-type parental strain penetrated at twice the rate of the Plb1-deficient strain. However, that study did not include the revertant strain, which was necessary in order to unequivocally ascertain the role of Plb1 in candidal virulence, according to Molecular Kochs Postulates (Falkow, 1988
). The successful construction of the revertant and analysis of its virulence in two different animal models enabled us to satisfy Molecular Kochs Postulates and conclusively prove that Plb1p is an important virulence factor in disseminated candidiasis. In the present study, both the parental and the revertant strains penetrated deep into the gastric mucosal and submucosal tissues. In contrast, the Plb1-deficient mutant was not as invasive and was generally sequestered to the stomach lumen. The invasiveness of the parental and revertant strains increased their access to the gastric vasculature, thus allowing the organisms to haematogenously disseminate in the bloodstream more efficiently than the Plb1-deficient strain. Consistent with this interpretation, hyphal elements were observed in blood vessel lumens following challenge with the parental or revertant strains, as compared to the mutant. These differences in penetration and dissemination were reflected by the number of candidal c.f.u. recovered from the liver of mice infected with the parental, revertant or Plb1-deficient strains. c.f.u. counts in livers isolated from mice infected with the revertant strain were similar to those obtained from mice infected with the parental strain. In contrast, c.f.u. counts obtained in livers isolated from mice infected with the revertant or parental strain were significantly higher than those found in liver harvested from mice infected with the Plb1-deficient mutant. Similar observations were reported earlier using genetically unrelated strains, which differed in their ability to secrete phospholipase (Barrett-Bee et al., 1985
; Ibrahim et al., 1995
). The results of our study, using a set of isogenic strains, suggest a possible role for Plb1 in the transmigration of C. albicans across the GI tract, and its ability to cause systemic candidiasis.
Our results show that deleting the PLB1 gene attenuates candidal virulence and reduces the extent of candidal infection in two clinically relevant murine models (intravenous and oralintragastric models), although this targeted disruption does not render the strains completely avirulent. The fact that this attenuation in virulence is the result of deletion of the PLB1 gene is borne out by our results, which show that when the functional PLB1 gene is reintroduced into the null mutant, the strain (revertant) regains virulence properties. The PLB12 strain retains the ability to produce low-grade infections, which is expected because C. albicans virulence is believed to be multifactorial. Other putative virulence factors for C. albicans have been the subject of several gene disruption studies. Targeted deletion of genes encoding such factors does not always result in complete avirulence, suggesting that more than one factor may be required for infection. For example, disruption of INT1 supports this concept since int1 null mutants have a dual phenotype (loss of adherence and germination), both of which have been implicated in candidal virulence (Gale et al., 1998
). The int1 null mutants are essentially avirulent (90% reduction in mouse mortality). Further investigations regarding the mechanism/s of Plb1 action and how this secreted enzyme relates to other virulence factors will provide important clues about the pathobiology of C. albicans. Uncovering the key components and steps involved in the infectious process of C. albicans should provide an impetus for the development of new antifungal agents and improved therapeutic modalities for efficient treatment of disseminated candidiasis.
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ACKNOWLEDGEMENTS |
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Received 29 January 2001;
revised 23 April 2001;
accepted 11 May 2001.