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Journal of Clinical Microbiology, October 2004, p. 4726-4734, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4726-4734.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Differential Expression of Secretory Aspartyl Proteinase Genes (SAP1-10) in Oral Candida albicans Isolates with Distinct Karyotypes

Arianna Tavanti,1,{dagger} Giacomo Pardini,1 Daniele Campa,2 Paola Davini,1 Antonella Lupetti,1 and Sonia Senesi1*

Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Sezione di Microbiologia e Virologia,1 Dipartimento dell'Uomo e dell'Ambiente, Sezione di Genetica, Università di Pisa, Pisa, Italy2

Received 29 March 2004/ Returned for modification 21 April 2004/ Accepted 10 June 2004


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ABSTRACT
 
Two karyotypes of oral Candida albicans isolates, named b and c, constituted >80% of a collection from healthy carriers (22 b and 16 c isolates) and oral candidiasis patients who were either infected (31 b and 16 c isolates) or uninfected (13 b and 38 c isolates) with human immunodeficiency virus (HIV). The prevalence of the b and c karyotypes within HIV-positive and HIV-negative patients, respectively, who were suffering from oral candidiasis (P ≤ 0.0001) suggested that these two types possessed different virulence potentials. Since C. albicans proteinases (Saps) are virulence factors in oral candidiasis, we evaluated whether the b and c karyotypes secreted different levels of Saps and expressed different patterns of Sap-encoding genes (SAP1-10). We found that the mean value of Sap activity was significantly lower (P = 0.003) in the commensal type than in the infectious b karyotype, whereas Sap activity in the commensal c type was as high as that registered for the infectious c strains. Marked differences in SAP mRNA expression were observed in commensal strains under non-Sap-inducing conditions, with all SAP genes being expressed only by strains with the c karyotype; interestingly, none of the commensal b strains expressed SAP2. In addition, while all of the SAP1-10 genes were detectable under Sap-inducing conditions, the timing of their expression during growth differed significantly, with mRNAs of SAP1-10 genes detected at 8 and 24 h postinoculation in c and b commensal strains, respectively. This provides the first evidence that commensal oral C. albicans isolates with distinct karyotypes are characterized by different patterns of SAP1-10 gene expression and different levels of Sap secretion.


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INTRODUCTION
 
Candida albicans is an opportunistic pathogen of humans with an increasing medical relevance, causing superficial as well as systemic infections in susceptible individuals (28). Nevertheless, C. albicans may behave as a harmless commensal that is adapted to colonize virtually all body districts in healthy individuals. The major factor that determines whether C. albicans behaves as a commensal colonizer or as a pathogen is the integrity of the host immune response, with cell-mediated immunity playing a crucial role in generating protection, especially towards oral candidiasis (6). However, C. albicans possesses an array of virulence traits which may contribute to the severity of symptomatic infections in hosts with impaired defense mechanisms (3, 9) and may account for the development of symptomatic episodes in healthy individuals (24, 30). Virulence factors expressed by C. albicans include phenotypic switching, adhesins, dimorphism, and the secretion of hydrolytic enzymes, such as aspartyl proteinases and phospholipases (3). Among these, secretory aspartyl proteinases (Saps) have been recognized as virulence factors since their discovery (36). Saps are encoded by a multigene family encompassing at least 10 different highly regulated genes (SAP1-10) (15, 26). The differential expression of SAP genes has been detected under a variety of laboratory conditions (13, 42), in experimental models of reconstituted human cutaneous, oral, and vaginal C. albicans infection (32, 33, 34), and in vivo, both in animal models of infection and in humans suffering from oral candidiasis (25, 27). The presence of 10 SAP genes, their temporal activation during distinct stages of infection, and their coordinate regulation with other virulence C. albicans traits constitute a robust body of evidence showing that individual members of this gene family have a major role in the adaptive response of C. albicans to its environment, including its host.

Although the regulation of Sap secretion and SAP gene expression have been extensively explored in different C. albicans strains (14, 16, 17, 31, 37, 38), little is known on whether distinct genotypic groups of clinical C. albicans isolates have the propensity to express distinctive patterns of SAP1-10 genes and secrete different amounts of Saps. Only one report showed that C. albicans isolates harboring intronless 26S rRNA genes secreted higher levels of Saps than intron-containing strains (39). In an early report, two C. albicans karyotypes, named b and c, were found to constitute roughly 70% of an isolate collection (19). Of these two types, the b karyotype was recognized as the prevalent type harbored in the oral cavities of both healthy carriers and human immunodeficiency virus (HIV)-infected patients suffering from oral candidiasis, suggesting that such a karyotype is particularly prone to colonizing oral cavities and causing symptomatic infections in immunocompromised hosts. In this investigation, C. albicans strains with the c karyotype were predominantly isolated from non-HIV-infected individuals suffering from oral candidiasis who were otherwise healthy. This observation suggested that C. albicans strains with the c karyotype may be more likely to behave as pathogens than the b type.

To explore such a possibility, the present study aimed to evaluate whether oral C. albicans isolates with b and c karyotypes could be characterized by their propensities (i) to secrete different levels of aspartyl proteinases and (ii) to express different patterns of SAP1-10 genes during in vitro propagation.


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MATERIALS AND METHODS
 
Collection of C. albicans oral isolates. Oral C. albicans isolates were collected by oral swabs or oral saline rinses from the following groups of patients. (i) Sixty-one AIDS patients who exhibited evident oral lesions (76% males and 24% females, 25 to 35 years old) constituted the first group. These patients (referred to as HIV-positive patients) were being treated as outpatients at the Divisione Malattie Infettive, Ospedale di Cisanello, Pisa, Italy, were not receiving any antimycotic therapy at the time of sampling, and were not receiving current (or recent) protease inhibitor therapy. (ii) Forty-three healthy subjects (42% males and 58% females, 18 to 21 years old) who presented no signs of oral thrush at the time of sampling, referred to as healthy carriers, constituted the second group. (iii) Sixty-five non-HIV-infected patients suffering from symptomatic oral candidiasis but otherwise regarded as healthy individuals (52% males and 48% females, 55 to 83 years old) constituted the third group. These subjects are referred to as HIV-negative patients. Samples from the healthy carriers and HIV-negative patients were collected at the Unità Operativa di Micologia, Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Università di Pisa. Samples from HIV-negative patients were collected mainly from individuals bearing dental prostheses. The following major risk factors, potentially enhancing the susceptibility of HIV-negative subjects to develop symptomatic candidiasis, were considered exclusion criteria: a state of immunodeficiency, including iatrogenic immunosuppression, chronic disease (diabetes or heart, respiratory, or kidney failure), long-term medical treatments, hospitalization within 3 months prior to sampling, and oral disease aside from oral candidiasis. HIV-negative patients were tested for HIV infection at the start and at the end of the study. All of the subjects studied were residents of Pisa. Cultures of the samples were grown on Sabouraud plates at 37°C for 24 h. The isolated strains were analyzed for both the production of germ tubes after 2 h of incubation in fetal calf serum at 37°C (Biological Industries, Kibbutz Beth Haemek, Israel) and the typical C. albicans pattern of sugar utilization (API ID32C; bioMérieux, Marcy l'Etoile, France). The isolates were stored at –80°C in 30% glycerol.

Karyotype determination. Determinations of electrophoretic karyotypes were performed as previously described (19). Briefly, yeast cells were grown for 12 to 14 h (early stationary phase) at 37°C on a rotary wheel in 100 ml of malt extract broth (Difco, Detroit, Mich.). The cells were harvested, embedded (109 cells/ml) into plugs of 1% low-melting-point agarose (Nalgene, Rochester, N.Y.) to preserve the DNA integrity, and then digested with lyticase (720 U/mg; Sigma Chemical Co., St. Louis, Mo.) and proteinase K (>20 U/mg; Gibco, Life Technologies, Milan, Italy) in the presence of ß-mercaptoethanol (Sigma). Karyotypes were determined by pulsed-field gel electrophoresis in a CHEF-DR II system (Bio-Rad, Richmond, Calif.). Chromosomal DNA bands were resolved by two sets of electrophoretic parameters (19) in order to achieve maximum resolution of the smaller and larger chromosomes. C. albicans isolate SC5314 (10) was used as a reference strain for the C. albicans standard karyotype (20). Chromosomes of Saccharomyces cerevisiae strain YNN295 (Bio-Rad) were used as reference markers for the determination of the molecular sizes of the C. albicans DNA bands obtained by pulsed-field gel electrophoresis. The stability of the karyotypic patterns obtained for each strain was confirmed by running three separate preparations of agarose plugs containing yeast DNA from the same strain of C. albicans.

Determination of Sap activities. C. albicans isolates and C. albicans ATCC 48867 (American Type Culture Collection, Rockville, Md.), which was used as a highly proteolytic reference strain (2), were routinely propagated on Sabouraud agar at 37°C. For the determination of Sap activities, yeast cells were transferred into 50 ml of broth containing 1% yeast extract (Difco), 2% mycological peptone (Oxoid, Basingstoke, Hampshire, England), and 2% dextrose and were grown at 37°C overnight. The yeast cells were washed twice in phosphate-buffered saline (pH 7.5) and used to inoculate, at a final concentration of 105 cells/ml, yeast nitrogen base-bovine serum albumin (YNB-BSA) medium, which contained 0.17% yeast nitrogen base without amino acids and ammonium sulfate (Difco), 0.2% glucose, and 0.1% BSA (Sigma) at pH 5.5. Cultures (200 ml), performed in triplicate on separate days, were incubated at 37°C in a shaking rotary incubator unless stated otherwise. All C. albicans isolates were screened for Sap secretion after 48 h of growth, since the maximum amount of Saps released into the culture medium by C. albicans ATCC 48867 was detected at 48 h postinoculation (see below). Culture aliquots were taken, and the optical density (OD) values at 540 nm (Agilent Technologies Deutschland GmbH, Waldbronnen, Germany) did not differ among strains at 48 h postinoculation. The culture aliquots were centrifuged at 2,000 x g for 10 min, and the supernatants were collected separately. The reaction mixtures for the determination of proteinase activities, performed in triplicate for each culture supernatant, contained different amounts of the supernatant (1.0, 2.0, and 3.0 ml) as the source of proteinases in a total volume of 3.0 ml in sterile YNB medium, with 3.0 ml of 1% BSA in 0.1 M citrate buffer (pH 3.2) as the substrate. As controls, reaction mixtures were run without either the supernatant, which was replaced with sterile YNB-BSA medium, or the substrate, which was replaced with citrate buffer. The mixtures were incubated at 37°C, aliquots (0.5 ml) were taken at 0, 15, 30, 60, 90, 120, 240, and 300 min and poured into separate test tubes, and the enzymatic reactions were stopped by adding 0.5 ml of 0.44 M trichloroacetic acid (20%; Sigma). After 40 min at 4°C, each mixture was centrifuged at 2,700 x g for 10 min, and the absorption of clear supernatants was measured at 280 nm (Agilent Technologies), with a {Delta}E280 of 0.66 for 100 µg of BSA hydrolyzed ml–1. The enzyme's specific activity was expressed in micrograms of BSA hydrolyzed per milliliter per minute or in micrograms of BSA hydrolyzed per milliliter when it was measured at the completion of the catalyzed reaction (5 h). The kinetics of Sap secretion during growth were monitored for six commensal isolates as well as for C. albicans ATCC 48867. Culture aliquots were taken at different time intervals during growth and treated as previously described. Although all of the supernatants were usually used without being concentrated to measure proteinase activities, the supernatants from 4- to 12-h cultures were concentrated 10- to 20-fold (Vacuum System Amicon, YM3 membrane; Millipore Corporation, Bedford, Mass.) prior to assays of Sap activities. When the enzyme activity was expressed per a constant number of cells (109 cells), the number of viable yeast cells was counted on Sabouraud agar plates.

RNA isolation. Total RNAs were extracted from C. albicans blastoconidia by use of an RNeasy mini kit (Qiagen GmbH, Hilden, Germany). Briefly, yeast cells (107) were centrifuged and suspended in 600 µl of highly denaturing guanidine isothiocyanate buffer containing 0.14 M ß-mercaptoethanol and 600 µl of glass beads (400 to 600 µm in diameter; Sigma). The cell suspensions were vigorously vortexed for 30 min. The cell lysates were centrifuged to remove cell debris, and the supernatants (350 µl) were mixed with an equal volume of 70% ethanol. Samples were applied to RNeasy mini spin columns and centrifuged for 15 s at 8,000 x g to bind the RNAs to a silica gel membrane. After a washing step with RW1 buffer (Qiagen), the total RNAs were treated twice with DNase (2.7 U/µl). Samples of total RNAs were stored at –80°C or were used immediately.

cDNA synthesis by RT-PCR. SAP1-10 expression during growth was measured for the six commensal C. albicans isolates chosen for monitoring the kinetics of Sap secretion during growth in a Sap-inducing medium (YNB-BSA medium). Aliquots of cultures, taken at the same time intervals as for Sap determinations, were centrifuged at 2,000 x g, and 107 blastoconidia were used to extract total RNAs and for reverse transcriptase PCRs (RT-PCRs). For each sample of C. albicans RNA, 10 RT-PCRs were performed. These included reactions to detect SAP1-10 genes (SAP1, SAP2, SAP3, SAP4/6, SAP7, SAP8, SAP9, and SAP10), the C. albicans ACT1 gene, which was used as an internal mRNA control for evaluation of the sensitivity and efficacy of the RT-PCR analysis, and RNase-free water, which was used as a negative control. For RT-PCRs and PCRs, one pair of primers each for the SAP1, SAP2, SAP3, SAP4-6, SAP7, SAP8, SAP9, SAP10, and C. albicans ACT1 genes was used (Table 1). Total RNA (100 ng) was added to the RT-PCR mix (50 µl), which contained 1x Tfl-avian myeloblastosis virus (AMV) buffer, 1 mM MgSO4, a 0.1 mM concentration of each deoxynucleoside triphosphate, a 15 mM concentration of each primer, 3.75 U of AMV RT, and 2.5 U of Tfl DNA polymerase (Access RT-PCR system; Promega, Madison, Wis.). The annealing temperatures used for touchdown cycling were described by Naglik et al. (25) and were as follows: 66°C for 2 cycles, 65°C for 2 cycles, 64°C for 2 cycles, 63°C for 2 cycles, and 62°C for 35 cycles. Cycling times were as follows: denaturation at 94°C, annealing at 62°C, and extension at 72°C, for 30 s each, followed by a final extension at 72°C for 10 min. The purified RNA from each sample was confirmed to be DNA-free by the absence of an amplified product after a PCR performed with specific primers that were complementary to the C. albicans actin gene. The PCR conditions were optimized by the protocol described above for RT-PCR by replacing the AMV RT with an equal volume of RNase-free water. The amplified DNA products were separated by electrophoresis in 1.8% agarose gels containing 0.5 µg of ethidium bromide/ml. Tris-borate-EDTA was used as the running buffer, and the pBR322 plasmid digested with MspI was used as a molecular weight marker. DNA bands were visualized with a UV transilluminator (ImageMaster VDS apparatus; Pharmacia Biotech, Uppsala, Sweden). The expression of SAP1-10 mRNAs in all C. albicans isolates was also measured in yeast cells grown for up to 24 h in Sabouraud broth.


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  		TABLE 1. Primer sequences used for detection of C. albicans secretory aspartyl proteinase genes SAP1-10 and ACT1

Statistical analysis. Results are presented as means ± standard deviations (SD) of at least three independent experiments. Differences between the values were analyzed by Student's t test, for which P values of <0.05 were considered significant. Differences in karyotype distribution and Sap secretion among C. albicans isolates collected from the three populations studied were analyzed by the {chi}2 test. Differences in SAP1-10 gene expression were compared by a two-way analysis of variance with the Bonferroni posttest for the significance of individual comparisons.


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RESULTS
 
Distribution of C. albicans strains with b and c karyotypes within healthy carriers and oral candidiasis patients. Two prevalent karyotypic profiles were obtained for the C. albicans strains under study (Fig. 1). C. albicans karyotypic patterns were analyzed on the basis of the disposition and number of chromosome-sized DNA bands within the four mobility groups obtained by pulsed-field gel electrophoresis, as previously described (19). According to such criteria, the two prevalent C. albicans karyotypes were identified as b and c types. C. albicans strains with b and c karyotypes constituted >80% of the isolate collection (136 of 169), being the prevalent karyotypes in healthy carriers (38 of 43) as well as in HIV-positive (47 of 61) and HIV-negative (51 of 65) oral candidiasis patients. However, the b and c karyotypes were differently distributed within the three populations studied: the isolation frequencies of the b karyotype were 65.8, 57.9, and 25.5% for HIV-positive patients, healthy carriers, and HIV-negative patients, respectively, while those of the c karyotype were 74.5, 42.1, and 34.1% for HIV-negative patients, healthy carriers, and HIV-positive patients, respectively (Table 2). While no significantly different distributions between the b and c karyotypes from healthy carriers and those from HIV-positive patients were observed, a significant difference was found for the distribution of b and c karyotypes from HIV-negative oral candidiasis patients compared to both healthy carriers (P = 0.004) and HIV-positive oral candidiasis patients (P ≤ 0.0001) (Table 2). Therefore, for the oral candidiasis patients, strains with b and c karyotypes were mainly collected from HIV-positive and HIV-negative patients, respectively.



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  		FIG. 1. Electrophoretic karyotypes of representative C. albicans oral isolates. Lane 1, reference C. albicans strain SC5314 showing the standard karyotype; lane 2, C. albicans isolate with b karyotype; lane 3, C. albicans isolate with c karyotype; lane M, molecular weight marker.


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  		TABLE 2. Isolation frequencies of oral C. albicans strains with b and c karyotypes from healthy carriers and oral candidiasis patients

Sap secretion by C. albicans strains with b and c karyotypes isolated from healthy carriers and oral candidiasis patients. The screening of all C. albicans isolates for Sap secretion was performed with culture supernatants taken at 48 h postinoculation, with BSA as the substrate. To evaluate differences in Sap secretion within the strain collection, we only considered the rate of proteinase activity, expressed in micrograms of BSA hydrolyzed per milliliter per minute. Indeed, while activity values measured at the completion of catalyzed reactions (4 to 5 h), in micrograms of BSA hydrolyzed per milliliter, led to an artificial homogenization of the amounts of Saps secreted by the strain collection, the rate of product formation (micrograms of BSA hydrolyzed per milliliter per minute) remained constant for at least 60 to 90 min during the time course of the catalyzed reaction (Table 3), thus allowing us to better quantify differences in the amounts of BSA hydrolyzed by individual C. albicans isolates.


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  		TABLE 3. Time course of BSA hydrolysis by commensal oral isolates of C. albicans

The amounts of BSA hydrolyzed by C. albicans isolates ranged from 0.09 up to 0.38 µg ml–1 min–1. Depending on the amount of BSA hydrolyzed, the isolates were arbitrarily subdivided into three major groups, groups I, II, and III, encompassing, respectively, strains exhibiting low (<0.15 µg of BSA hydrolyzed ml–1 min–1), medium (0.15 to 0.30 µg of BSA hydrolyzed ml–1 min–1), and high (>0.30 µg of BSA hydrolyzed ml–1 min–1) proteinase activities. Within group I, 61.5% of the strains were from healthy carriers (Fig. 2), with a significant difference (P < 0.01) from the numbers of both HIV-positive (27.2%) and HIV-negative (28.1%) patients. Within group II, there was a significant difference (P < 0.01) between the percentages of strains isolated from healthy carriers (23.1%) and HIV-positive (51.9%) as well as HIV-negative (58.5%) patients. Strains secreting high proteinase activities (group III) were less frequently collected (Fig. 2). When the b and c karyotypes were distributed into groups I, II, and III (Fig. 3), a significantly different distribution of the b and c karyotypes was observed within group I (P = 0.017) as well as within group II (P = 0.00057) when we compared the three populations. A significantly (P = 0.0009) different distribution into groups I, II, and III was also found when symptomatic oral candidiasis patients were compared with healthy carriers. It was apparent that symptomatic oral candidiasis was mainly caused by medium-proteinase-activity producers (group II), which had the b karyotype in HIV-positive patients and the c karyotype in HIV-negative patients. This observation was further strengthened by the findings (Fig. 4) that (i) within healthy carriers, the mean value of secreted Saps was significantly (P = 0.044) lower for the b karyotype than for the c karyotype, and (ii) within oral candidiasis patients, the mean values of secreted Saps did not differ between strains with b and c karyotypes. Consequently, only for the b karyotype was the mean value of secreted Saps significantly (P = 0.002) lower in strains from healthy carriers than in those from symptomatic patients, since the mean value of Sap secreted by commensal c strains was as high as that secreted by infectious c karyotypes. These data clearly indicate that commensal strains with the c karyotype share the propensity to secrete higher levels of proteinases than strains with the b karyotype.



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  		FIG. 2. Isolation frequency of oral C. albicans isolates secreting low (group I: <0.15 µg of BSA hydrolyzed ml–1 min–1), medium (group II: 0.15 to 0.30 µg of BSA hydrolyzed ml–1 min–1), and high (group III: >0.30 µg of BSA hydrolyzed ml–1 min–1) proteinase activities from healthy carriers (closed bars) and from oral candidiasis patients who were either infected (open bars) or uninfected (diagonally hatched bars) with HIV. The enzyme activity was assayed in 48-h culture supernatants. *, significant (P = 0.01) difference within group I between healthy carriers and oral candidiasis patients, either infected or uninfected with HIV. **, significant (P = 0.01) difference within group II between healthy carriers and oral candidiasis patients, either infected or uninfected with HIV.



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  		FIG. 3. Isolation frequency of oral C. albicans strains with b (right) and c (left) karyotypes secreting low (group I: <0.15 µg of BSA hydrolyzed ml–1 min–1), medium (group II: 0.15 to 0.30 µg of BSA hydrolyzed ml–1 min–1), and high (group III: >0.30 µg of BSA hydrolyzed ml–1 min–1) proteinase activities from healthy carriers (closed bars) and oral candidiasis patients who were either infected (open bars) or uninfected (diagonally hatched bars) with HIV. Significant differences within group I (P = 0.017) and within group II (P = 0.00057) were found in the isolation frequency of the b karyotype compared to the c karyotype in the populations under study.



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  		FIG. 4. Secreted aspartyl proteinases in oral C. albicans isolates with b (closed bars) and c (open bars) karyotypes from healthy carriers and oral candidiasis patients who were either infected or uninfected with HIV. Enzyme activities were assayed in 48-h culture supernatants. Results are means plus SD of at least three independent experiments. *, significantly (P = 0.044) different levels of secreted Saps in b compared to c karyotypes isolated from healthy carriers. **, significantly (P = 0.002) different levels of secreted Saps within b strains isolated from healthy carriers compared to HIV-positive symptomatic patients.

Expression of SAP1-10 genes by C. albicans strains with b and c karyotypes isolated from healthy carriers and oral candidiasis patients. To evaluate whether the differences in the amounts of Saps secreted by C. albicans strains with b and c karyotypes reflected differences in the patterns of SAP1-10 gene expression, we screened commensal strains with the b and c karyotypes for SAP gene expression after 48 h of growth under Sap-inducing conditions. All SAP1-10 genes were expressed by all of the commensal b and c strains analyzed (see below), including the SAP7 gene, whose expression has so far been detected only in vivo (13, 25, 26, 27). Therefore, to evaluate whether the b and c karyotypes had the propensity to differently express SAP1-10 genes, we analyzed the patterns of SAP1-10 expression after 48 h of growth under non-Sap-inducing conditions (Sabouraud broth) (Table 4). The results revealed that, within healthy carriers, SAP2 (P = 0.0009) and SAP7 (P = 0.0002) were expressed more frequently by c than by b karyotypes, and for the b karyotype, SAP2 was expressed by a larger number of strains that were isolated either from HIV-positive (P = 0.0004) or HIV-negative patients (P = 0.002) than by those from healthy carriers (Table 4). In addition, for healthy carriers, only 25% of C. albicans isolates with the b karyotype were found to simultaneously express the SAP1,3-10 genes, and none of the commensal b strains expressed SAP2. A lack of the SAP2 coding region was excluded, since the SAP2 gene could be detected by genomic DNA amplification in all b-type commensal strains (data not shown). In contrast, C. albicans isolates with the c karyotype (50%) expressed SAP2, and up to 80% of the strains expressed the SAP1,3-10 genes. For oral candidiasis patients who were either infected or uninfected with HIV, the b and c karyotypes expressed a larger number of SAP genes than b and c strains collected from healthy carriers, with >70% of c strains versus nearly 45% of b strains from oral candidiasis patients expressing all SAP1-10 genes (Table 4).


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  		TABLE 4. SAP1-10 gene expression under non-Sap-inducing conditions in C. albicans isolates with b and c karyotypes from healthy carriers and oral candidiasis patients

Kinetics of Sap secretion and SAP1-10 gene expression in commensal C. albicans strains with b and c karyotypes. Since commensal b and c karyotypes were found to express all SAP1-10 genes under Sap-inducing conditions, at least after 48 h of growth, we evaluated whether the different levels of Saps secreted by b and c commensal isolates could be related to differences in the timing of SAP1-10 gene expression during growth under Sap-inducing conditions (Fig. 5). We found that strains with the c karyotype (three strains from group II of Sap secretion) expressed the SAP1,3-10 genes from 4 up to 48 h of growth, with only SAP2 being detectable starting from 8 h postinoculation. In contrast, strains with the b karyotype (three strains from group I of Sap secretion) expressed only mRNAs of SAP1, SAP4/6, SAP9, and SAP10 at 4 h postinoculation, and the completion of SAP gene expression required as long as 24 h of growth (Fig. 5). The notable differences observed in the expression of individual SAP genes did not change when yeast cells were grown at 30°C (data not shown). The finding that SAP2 was the last gene expressed by the c (after 8 h of growth) and b (at 24 h postinoculation) types was of some interest since SAP2 has been described as the most frequently expressed SAP gene during the log phase of growth in proteinase-inducing media by both laboratory and clinical C. albicans strains (13, 42). Marked differences in the timing of gene expression were observed for SAP7 and SAP3: their mRNAs were detected after 4 h of growth for the c karyotype and after 16 (SAP7) and 24 (SAP3) h of growth for the b karyotype. Moreover, the expression of SAP9, which was reported to occur preferentially in late growth phases (23), was detected from early to late phases of growth in c and b karyotypes. However, for the C. albicans reference strain ATCC 48867, the expression of SAP2 and SAP9 was detected starting from early (4 h) and late (12 h) growth phases, respectively (Fig. 5).



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  		FIG. 5. Kinetics of Sap secretion (left) and timing of SAP1-10 gene expression (right) in C. albicans ATCC 48867 and in representative C. albicans commensal isolates with b and c karyotypes grown in Sap-inducing medium. At the growth time points indicated, culture aliquots were assayed for absorption (diamonds) and were centrifuged: supernatants and cells were used to measure the amounts of secreted Saps (open bars) and to detect SAP1-10 gene expression, respectively. M, molecular weight standard; A, positive control (actin gene); nc, negative control. OD, optical density. The results are means plus SD of at least three independent experiments.

The secretion of Saps during growth (Fig. 5) appeared to closely account for the marked differences in the in vitro timing of SAP1-10 gene expression by b and c commensal C. albicans isolates. Indeed, Saps were undetectable in culture supernatants, even when concentrated 20-fold, of both b and c strains during the first 4 h postinoculation. Thereafter, the enzyme activities were first detected in supernatants of c strains (8-h cultures) and later in those of the b karyotype (12-h cultures). The amounts of Saps released into the growth medium increased up to 48 h postinoculation, with higher activities detected for the c karyotype at any growth time examined (Fig. 5). Higher levels of Saps in c than in b types were also registered when the kinetics of Sap secretion were expressed per a constant number of cells (109), although the highest levels of Saps were detected around 24 instead of 48 h of growth for both the b and c karyotypes (data not shown).


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DISCUSSION
 
This report analyzes the expression of SAP1-10 mRNA genes and the secretion of Saps in a collection of C. albicans isolates exhibiting two distinct karyotypes, designated as b and c types. The major finding of this study was the demonstration that C. albicans strains with b and c karyotypes are characterized by marked differences in the expression of SAP1-10 genes and by the ability to secrete significantly different levels of Saps during in vitro propagation.

The oral C. albicans isolates with b and c karyotypes constituted >80% of a strain collection (136 of 169) from healthy carriers and oral candidiasis patients who were either infected or uninfected with HIV. All C. albicans isolates exhibiting non-b and non-c karyotypes (33 of 169) were excluded from this investigation, since b and c karyotypes were overrepresented in the three populations studied (Table 2). The high percentage of b and c karyotypes was not due to artificial homogenization of such karyotypes since the patients were not related, yeast sampling was done only once from each subject, and one isolate per patient was collected. The prevalence of a few karyotypic patterns could be due, however, to the poor discriminatory power of karyotyping, as this molecular method may be more useful to delineate genomic background similarities shared by C. albicans populations (20) than to distinguish individual C. albicans strains (35). On the other hand, the high frequency of isolating C. albicans strains exhibiting few different karyotypes in a defined geographical area could reflect the widespread distribution of some strain lineages that are more prone than others to colonize the human oral cavity; this possibility is consistent with the idea that the population structure of C. albicans is primarily clonal, as no definitive genetic evidence for meiosis has yet been presented (11, 40). In such a view, the high prevalence of b and c karyotypes was thought to reflect the in vivo adaptation of these karyotypes to survive and replicate over oral mucosal surfaces, thus becoming the prevalent karyotypes isolated during carriage and infection. However, the significantly (P ≤ 0.0001) different distribution of b and c karyotypes found in HIV-negative oral candidiasis patients compared to that for HIV-positive patients, with b strains mainly collected from HIV-positive patients and c strains mainly collected from HIV-negative subjects, addressed the question of whether these two patterns were selected depending on both the HIV status of the host and/or a different ability of such karyotypes to express a different pathogenic potential. To explore such a possibility, we screened all of the C. albicans isolates for Sap secretion, since a close association between the amount of Saps secreted in vitro and the virulence exerted in vivo by oral C. albicans has been convincingly demonstrated (27).

The overall results revealed that strains from healthy carriers predominantly secreted lower Sap levels than strains collected from oral candidiasis patients, either infected or uninfected with HIV. It should be emphasized that the use of BSA alone as the substrate for Sap determination has some limitations. Indeed, substrate specificity towards BSA has not yet been defined for all Sap proteins, and it cannot be excluded that non-Sap proteins that hydrolyze BSA may contribute to Sap activity levels. Nevertheless, to the best of our knowledge, Sap1 to Sap10 account for all extracellular proteinases that are secreted in vitro by C. albicans grown under Sap-inducing conditions (26). The high Sap activities detected in strains from oral candidiasis patients were in agreement with several reports documenting that C. albicans strains from HIV-infected patients suffering from oral candidiasis secreted higher levels of Saps in vitro than strains from healthy carriers (4, 44). Only one published report (29) has claimed that strains collected from HIV-negative oral candidiasis subjects secreted lower Sap levels than strains collected from HIV-positive patients suffering from oral candidiasis. In that study, however, C. albicans strains isolated from HIV-negative oral candidiasis patients (50 strains) were mixed together with those from asymptomatic individuals (72 strains), and the strains isolated from symptomatic HIV-positive subjects (85 strains) were mixed together with those from HIV-positive asymptomatic C. albicans carriers (15 strains). Thus, the levels of Saps registered in the study by Ollert et al. (29) for all HIV-negative patients most likely led to an underestimation of the proteinase activity produced by strains from HIV-negative oral candidiasis subjects (29).

In this investigation, the finding of major interest was the demonstration that the mean value of Saps secreted by commensal c strains was significantly higher than that recorded for commensal b karyotypes (P = 0.044). Thus, while the Sap activity registered for b-type C. albicans strains was significantly (P = 0.003) lower in commensal than in infectious strains, the mean values of Sap activity for the c karyotype were very similar in strains from both healthy carriers and oral candidiasis patients. These results highlight the observation that the c-type strains of C. albicans share the propensity to produce higher levels of secretory proteinases in vitro, independent of the host HIV status and of their isolation as commensal or infectious agents. It should be noted that these data could have been lost if the isolates had not been subgrouped on the basis of the karyotype they exhibited and separately considered in the three populations studied.

In addition, marked differences in the kinetics of Sap secretion were observed for these karyotypes. Saps were detected starting from 8 and 12 h postinoculation in c and b strains, respectively, and higher levels of Saps were secreted by c rather than b karyotypes at any growth time point examined. Since the synthesis and secretion of Saps are tightly coupled and since their regulation occurs predominantly at the mRNA level (42), we explored whether the differences in the kinetics of Sap secretion depended on a different timing of SAP1-10 gene expression. The expression of SAP1-10 genes, detected at identical time points during growth, closely accounted for the notably different amounts of secreted Saps by b and c commensal C. albicans isolates. Indeed, we made the following observations: (i) strains with the c karyotype expressed all SAP genes as early as 4 h postinoculation, with the exception of SAP2, which was detected starting from 8 h of growth; and (ii) the only genes detected at 4 h postinoculation in strains with the b karyotype were SAP1, SAP4/6, SAP9, and SAP10, with the remaining mRNA genes noticed after 8 (SAP8), 16 (SAP7), and 24 (SAP2 and SAP3) h of growth. It should be considered, moreover, that SAP9 and SAP10, which encode proteinases that are incorporated into the cell wall via a glycosylphosphatidylinositol anchor (7, 27), may not contribute to the increased Sap activity secreted by both b and c strains. Remarkably, all SAP genes, once induced, appeared to be expressed up to the last growth time point examined (48 h): this occurrence, together with the different timing of SAP1-10 expression in b and c karyotypes, may account for the different amounts of Saps released into the growth medium by C. albicans isolates that share the corresponding karyotypic patterns. Although these data need further investigation by screening wider collections of oral C. albicans isolates and quantifying the expression of mRNAs of the individual SAP genes, it appears that commensal C. albicans isolates with the c karyotype are more prone than the b strains to rapidly express all SAP genes and to secrete higher Sap levels. Two observations, specifically the detection of SAP2 mRNA after the expression of all other SAP genes had occurred for both the c and b karyotypes and the detection of SAP7 mRNA in all strains of C. albicans analyzed, raised further interest. Indeed, several reports have described that SAP2 is expressed in early growth phases, while the mRNA of SAP7 has been detected only in vivo (25, 27). These observations merit further exploration before it is hypothesized that SAP2 and SAP7 undergo different transcriptional regulation in C. albicans strains with b and c karyotypes. However, the lack of SAP2 expression observed for commensal strains with the b karyotype under non-Sap-inducing conditions, together with the late expression of SAP2 during growth under Sap-inducing conditions, suggests that SAP2 may undergo a different transcriptional regulation.

The patterns of SAP gene expression obtained for all b and c karyotypes under non-Sap-inducing growth conditions (Sabouraud broth; 48 h postinoculation) confirmed the different propensities of these two karyotypes to differently express several SAP genes. Moreover, on the whole, the patterns of SAP1-10 expression clearly demonstrate that strains with the c karyotype, isolated from both healthy carriers and oral candidiasis patients, express a larger number of SAP genes than strains with the b karyotype, although a substantial increase in the number of expressed SAP genes was observed for both the b and c karyotypes from oral candidiasis patients in comparison to healthy carriers.

The demonstration that karyotypic subgroups of C. albicans are characterized in vitro by significant differences in Sap secretion and SAP gene expression does not firmly address the issue of whether they are representative of a different pathogenic potential exerted in vivo. However, clinical isolates and laboratory strains of C. albicans that produce higher levels of Saps in vitro have proved to be more pathogenic in animal models of infection than strains producing lower Sap activities (1, 4, 5, 44). In view of this, the development of symptomatic oral candidiasis may be attributable, apart from the host immune status, to an enhanced virulence of the opportunistic pathogen. The increase in Sap secretion that we observed for C. albicans strains isolated from oral candidiasis patients may be consistent with a preferential selection of strains with higher overall levels of Saps, since isolates that secrete high levels of proteinases are also present among healthy carriers.

However, while b-type C. albicans strains producing high Sap levels are isolated from healthy carriers at a rather low frequency, the majority of commensal c strains secrete high levels of Saps. This observation strongly suggests that C. albicans strains with the c karyotype may be more prone to behave as a virulent strain cluster, thus accounting for their prevalence in causing symptomatic episodes of oral thrush in HIV-negative immunocompetent patients. Nevertheless, since SAP mRNA expression patterns in vivo may significantly differ from those detected in vitro (27), it is imperative that further studies should address the questions of (i) whether C. albicans strains with b and c karyotypes also express diversified patterns of SAP mRNAs in vivo and (ii) whether transcriptional factors influencing SAP gene expression in b and c karyotypes also play a role in the coordinated expression of other C. albicans virulence genes in response to host triggering stimuli in cellular and animal models of infection.


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ACKNOWLEDGMENTS
 
This work was supported by a research grant from the Italian "Ministero dell'Istruzione, dell'Università e della Ricerca," under contract no. 2003062784.

We are grateful to Alessio Bertozzi for excellent technical assistance with karyotyping C. albicans isolates.


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FOOTNOTES
 
* Corresponding author. Mailing address: Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Sezione di Microbiologia e Virologia, Università di Pisa, Via S. Zeno 37-39, 56127 Pisa, Italy. Phone: (39) 050 2213695. Fax: (39) 050 2213711. E-mail: senesi{at}biomed.unipi.it. Back

{dagger} Present address: Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD Aberdeen, United Kingdom. Back


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Journal of Clinical Microbiology, October 2004, p. 4726-4734, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4726-4734.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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