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1 Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, Building 10, Room 11C304, National Institutes of Health, Bethesda, MD 20892, USA
2 Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
3 Zhangzhen Hospital, Second Military Medical University 200003 Shanghai, China
4 Zhenyi Medical College, Zhenyi, China
Correspondence
Kyung J. Kwon-Chung
june_kwon-chung{at}nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Two supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 1998, 1999; Chang et al., 1996) and melanin (Kwon-Chung et al., 1982; Liu et al., 1999; Perfect et al., 1998; Williamson, 1997) have long been recognized as the major virulence factors. Urease activity has also been reported to play an important role in cryptococcal pathogenesis in studies in which three separate models of infection were used (Cox et al., 2000). These studies, which included the murine intravenous and inhalation models as well as the rabbit meningitis model, revealed that virulence of the urease-negative strains was comparable to the wild-type strains in one model while being attenuated in others. Such contrasts in virulence further emphasize the importance of virulence genes with regard to the stages/sites of infection as well as the strains used. Although there have been case reports of urease-negative isolates of C. neoformans causing infection in humans (Bava et al., 1993; Ruane et al., 1988) as well as those isolated from the environment (Li & Wu, 1992; Li et al., 1993), these are extremely rare. Urease activity is still considered one of the hallmarks of the genus Cryptococcus (Kwon-Chung et al., 1987) and related genera among the Heterobasidiomycetes (Kwon-Chung & Bennett, 1992).
Urease is a metalloenzyme that facilitates the hydrolysis of urea to ammonia and carbamate, and under physiological conditions results in an increase in pH (Curtis et al., 1994). The latter property has been regularly used as a diagnostic tool for the identification of C. neoformans in clinical laboratories (Canteros et al., 1996; Zimmer & Roberts, 1979). Such pH increases caused by urease, in other organisms, have been associated with survival in animal tissues and the cause of tissue damage induced by the production of ammonium hydroxide (Mobley, 1996). Furthermore, urease has also been postulated to play a pivotal role in allowing C. neoformans to convert urea to a more readily usable form of nitrogen in its ecological niche. The exact role of urease in cryptococcal pathogenesis, however, is unclear. In bacterial pathogenesis, the enzyme appears to alter the host's immune function by increasing the microenvironmental pH at the infection site (Curtis et al., 1994). While urease in bacteria that infect the stomach mucosa and urinary tract (Chen et al., 1997; Mobley et al., 1991) allows the microbes to survive at low pH, it appears to act differently in C. neoformans pathogenesis. It has been suggested that urease activity promotes sequestration of the organism in microcapillaries prior to dissemination into the brain and its inhibition correlates with a significant decrease in eliciting immune defence cells in the lungs of mice (Olszewski et al., 2004).
In this study, we investigated the genetic basis of a urease-negative C. neoformans strain that was isolated from the spinal fluid of an immunocompetent patient. A novel urease gene, URE2, was identified in this strain by complementation cloning and its roles in urea utilization and virulence were examined.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. A clinical urease-negative strain, B-4587, was isolated from the spinal fluid of an immunocompetent patient in China who had suffered from severe headaches for 2 months. The strain was determined to be of serotype A according to the test results with the Iatron Crypto Check kit (Iatron Laboratories). Strain B-4587FOA is a spontaneous ura5 mutant obtained by plating strain B-4587 on 5-fluoroorotic acid (5-FOA) agar and subsequent plating on media containing orotidine or orotic acid (Kwon-Chung et al., 1992). B3501A is a serotype D reference strain that was sequenced at Stanford University (http://www-sequence.stanford.edu/group/C.neoformans/). The TAV strains are genetically manipulated strains either of B-4587 or H99 as stated in Table 1. All strains were maintained on slants of YPD agar (per litre: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g Bacto-agar) and stored at 4 °C until use. For each experiment, 24-h-old yeast cells grown on YPD at 30 °C were used. Min agar (YNB) contained 6.7 g yeast nitrogen base without amino acids, 20 g glucose and 20 g Bacto-agar per litre. Urea agar was composed of 1.7 g l–1 yeast nitrogen base without amino acids and ammonium sulfate (Difco), 20 g l–1 glucose and supplemented with 3 g l–1 urea (Sigma) as the sole nitrogen source. This medium was used to isolate urease-positive transformants. Christiansen's urea agar was also used to confirm their urease activity (Kwon-Chung & Bennett, 1992). Transformants resistant to geneticin (G418, Gibco) were screened on YPD plates supplemented with 100 µg G418 ml–1.
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) with either plasmid pPM8 (vector only), the plasmids harbouring the H99 genomic DNA library (Mondon et al., 2000), the plasmid containing the truncated/intact URE2 gene sequence (this study), or the plasmid containing the intact URE1 gene sequence (Cox et al., 2000), a gift from Gary Cox, Department of Medicine, Duke University Medical Center, Durham, NC, USA. Plasmids containing the gene conferring resistance to G418, plasmids harbouring the URE2 : : G418 disruption constructs and plasmids containing the URE2 : : ADE2 disruption constructs were used in biolistic transformations (Toffaletti et al., 1993) to delete the native URE2 gene from either the LP1 or the H99 strain.
Cloning and sequencing of the URE2 gene.
A genomic DNA library of the serotype A strain H99 of C. neoformans, constructed in the URA5-containing plasmid pPM8 (Mondon et al., 2000), was linearized and used to complement the urease-negative ura5 strain B-4587FOA by electroporative transformation. Genomic DNA was extracted from eight independent urease-positive transformants grown on urea agar (Varma & Kwon-Chung, 2000). Genomic DNAs from these urease-positive clones were separately used to transform the urease-negative clinical isolate B-4587. The genomic DNA from these transformants was electrophoresed undigested on 10 g l–1 agarose gels and analysed via Southern blot hybridizations to detect the presence of episomes in each. The episomal plasmids were recovered from four of the urease-positive transformants (TAV-756, TAV-758, TAV-759, TAV-760). Genomic DNA from each of the urease-positive transformants was digested with NotI to remove telomeric sequences. The DNA was then ligated with DNA polymerase to recircularize the plasmids prior to amplification in Escherichia coli. The episomal plasmids (pAV-756, pAV-758, pAV-759 and pAV-760) recovered from each of the urease-positive transformants were also used to electrophoretically transform strain B-4587, and the DNA inserts harboured by the plasmids were subsequently sequenced using the standard dideoxy method.
Functional analysis of the URE2 gene.
To determine whether expression of an intact and functional URE2 gene was required for urease activity in the B-4587 background, two approaches were taken. In the first approach, a truncated version of the URE2 gene was generated by digestion with SnaBI that resulted in a deletion of 1395 bp across the conserved coding region of the gene. In parallel experiments, plasmids containing either the deletion construct or the intact URE2 gene were linearized with NotI prior to being used for transformations of the strain B-4587FOA by electroporation. The transformants were then tested for urease activity by growth on urea agar. In the second approach, a disruption construct of the URE2 gene was designed in which a significant portion of the internal coding sequence of the gene was replaced by either the ADE2 or the G418 gene. Genomic DNA sequences extending 1 kb proximal to the 5' and 3' regions of the URE2 coding region were amplified by PCR from the H99 or the LP1 strain. Primer sequences were used from the respective flanking regions as well as those that created overlaps with the 5' and 3' ends of the G418 gene sequence or restriction enzyme matches with the ADE2 gene for restriction digestion and ligation. A DNA fragment containing the gene conferring resistance to G418 was amplified by PCR using specific primers with homology to the overlapping template DNA sequences between the URE2 and G418 genes. This enabled the generation of an overlapping DNA sequence by PCR that contained the G418 gene sequence flanked by DNA sequences specific to the 5' and 3' ends of the URE2 gene from H99. The biolistic method was used to transform the PCR fragment containing the ure2 : : G418 disruption construct into the G418-sensitive strain H99. The putative ure2-disruption strains were grown on YPD agar supplemented with 100 µg G418 ml–1. Disruption of the native URE2 gene was confirmed by PCR and by Southern blot analysis. Specific oligonucleotides were designed to generate a PCR product that initiated from within the inserted G418 gene sequence and extended to the flanking DNA sequences derived from the genomic sequence of the URE2 gene. A similar strategy was employed to obtain a disruption of the URE2 gene in the serotype D strain LP1. In this case, however, the URE2 gene was disrupted by insertion of the ADE2 gene in its coding region that was then used as a selection marker for transformation. Oligonucleotide primers used to generate overlapping PCR fragments for insertion were as follows. For the ADE2 gene: 5'-GTACTAGTCATCTTCAGACCCCAATCTCACAC-3' and 5'-CAGGATCCCGCACAGTTTATCACGACCACG-3'; 5'-CAGAATCCGTCAAACAGCAACTTCTTCTGGG-3' and 5'-GTATCGATCAATGCGAAAAGCGAAATGGAG-3'; for the G418 gene: 5'- GCTCTCCAGCTCACATCCTCCCAACTCCCTCAACTCTTCATCCG-3' and 5'-GCATGATGAACAAAGTAAGG-3'; 5'-TCGTCCTTCCTGCAGATAGGAGGG-3' and 5'-GCTAGTTTCTACATCTCTTCCGTGCGATAAAGGTGATTGAAAAGG-3'. For Southern analysis, genomic DNA from each of the transformants was digested with different restriction enzymes and screened with the URE2/G418 gene sequences to identify hybridization patterns consistent with disruption of the native URE2 gene. This allowed discrimination of the ure2 disruption strain from the untransformed strains. The resulting ure2 disruption strains were then tested for urease activity by culturing them on urea agar medium.
To determine if the urease-negative phenotype of strain B-4587 was the result of anomalies within its URE2 gene sequence, the latter was sequenced and compared with that of the wild-type strain H99. Since the URE2 gene is quite large, specific DNA primers were designed to generate overlapping DNA fragments from the URE2 gene of strain B-4587. The use of short overlapping PCR fragments for sequencing not only minimized potential polymerase-generated sequence errors but also allowed for multiple sequencing reactions of each fragment to confirm the gene sequence. The sequences of these PCR fragments were then compiled to generate the intact URE2 gene sequence from strain B-4587. This sequence was then compared with the URE2 genomic sequence in the C. neoformans database of strain H99 (http://cneo.genetics.duke.edu/).
Analysis of URE1 and URE2 transcriptional levels by RT-PCR.
Cells of the urease-positive strain H99, the urease-negative ure2 disruption strain, the urease-negative clinical strain B-4587 and the urease-positive URE2-complemented transformant B-4587C were grown overnight in YPD broth at 30 °C. The cells were harvested by centrifugation and washed twice with sterile water. Total RNA was extracted from each strain in the exponential growth phase by using the FastRNA kit-Red (Qbiogene). The total RNAs extracted from each strain were treated with DNase I, and RT-PCR was performed for each sample using the Qiagen One-Step RT-PCR kit (B-D Sciences). Oligonucleotide primers used for RT-PCR were specifically designed to generate DNA fragments from RNA products of the URE2 gene (5'-ACCTTGATGGCGTCTTTGGAC-3'; 5'-TCCTGTTGATGAGGGTCTGTCG-3'), the URE1 gene (5'-CGTTCAAGTTGGCTCCCACTAC-3'; 5'-TGTCCTGTAAAACATCTTCGGCTG-3') and the constitutively expressed product of the GPD gene (5'-TGCTATTGGTCGAATTGTTC-3'; 5'-TGGTGCAAGAAGCGTTGGACA-3').
Virulence studies in mice.
Virulence in mice was determined for the urease-negative strains B-4587, TAV-4587V (ectopically integrated, stable, vector-only transformant of B-4587) and TAV-H99SD (ure2 : : G418 disruption strain of H99) as well as the urease-positive wild-type strain H99 and B-4587C (ectopically integrated, stable, urease-positive clone of B-4587 transformed with a wild-type copy of the URE2 gene). Animal studies were performed using 7-week-old female BALB/c mice and their mortality was monitored following two different routes of infection – 1x106 cells per mouse for intravenous injection via the tail vein and 2x107 cells per mouse for intranasal inhalation – using sets of 10 mice for each strain. Overnight cultures of each strain in the exponential growth phase were harvested by centrifugation, washed and suspended in 9 g l–1 saline solution. Appropriate dilutions of each cell suspension were made in saline to yield the desired number of cells in the required volumes (200 µl for tail vein intravenous and 20 µl for intranasal inhalation) needed to administer per mouse. The inoculum sizes were confirmed by quantifications of expected colony-forming units on agar media.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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strain of C. neoformans that was urease-negative. The patient was treated with ketoconazole plus 5-fluorocytosine (5-FC) and his CSF became culture-negative after 5 months of treatment. Strain B-4587 is highly encapsulated, strongly melanin positive on birdseed agar and grows well at 37 °C. However, it failed to grow on agar with urea as the sole source of nitrogen and was also negative on Christiansen's urease testing medium, which is routinely employed by clinical microbiology laboratories for the diagnosis of urease-positive organisms.
Cloning of the URE2 gene
In order to functionally complement the urease-negative phenotype, B-4587FOA was used as the host strain for cloning the URE2 gene from an H99 genomic library (Mondon et al., 2000). Strain B-4587FOA was electroporatively transformed using the library and the transformed cells were plated on YNB medium that contained urea as the sole source of nitrogen. Several transformants grew on urea medium within 3 days, indicating either complementation or reversion of the urease-negative phenotype. When these transformation plates were incubated for several more days, satellite colonies appeared around the original transformants that suggested the feeding from the urease-positive clones by secretion of urease (Fig. 1). Genomic DNA from each of these urease-positive transformants successfully complemented the urease-negative phenotype of the clinical strain B-4587 (Fig. 2). Southern blot analysis revealed the presence of an episomal plasmid in each of the transformants tested (data not shown). The recovered episomal plasmid from each of the urease-positive transformants was able to successfully complement the urease-negative phenotype of strain B-4587. This indicated that the urease-positive phenotype of the transformants was the result of gene complementation and not reversion. Besides, the urease-negative phenotype of the strain B-4587 was so stable that revertants had never been observed. Four of the episome-harbouring urease-positive transformants were analysed further. Sequence analysis of the 6 kb genomic insert in the plasmid harboured by each of the transformants revealed the presence of a 4.5 kb gene that exhibited no sequence similarity to the previously reported C. neoformans urease gene URE1. This gene was named URE2 since it functionally complemented the urease-negative phenotype, although it showed extensive homology, especially at the N- and C-termini, with the protein encoded by the sudA gene of Aspergillus nidulans (see Fig. S1, available as supplementary data with the online version of this paper). Transformation with a plasmid containing the urease-encoding gene URE1 failed to complement the urease-negative phenotype of the strain B-4587 (data not shown). An analysis of the genomic DNA sequence of the URE2 gene with its predicted cDNA sequence (TIGR, http://www.tigr.org) revealed the presence of fourteen introns within the coding region of the gene. A C-T-rich stretch was observed in the putative promoter region upstream of the 5' end of the gene which also contained a TAATA box upstream from the initiating ATG. No other possible ORF was detected proximal to the 5' and 3' ends of the gene. The URE2 gene of C. neoformans encodes a putative protein composed of 1243 amino acids. A search of the C. neoformans cDNA sequence database revealed homology of the URE2 genomic DNA sequence to an ORF annotated to encode a hypothetical protein.
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) and turned Christiansen's urease test medium colour to magenta within 24 h. This indicated that the URE2 gene product was involved in restoring the ability of B-4587 to utilize urea as the sole nitrogen source. To determine if this was indeed the case, two approaches were taken. First, 1395 bp of the URE2 gene's internal coding region were deleted. The resulting truncated gene construct was used in parallel with a plasmid containing an intact URE2 gene to transform the urease-negative strain B-4587. The transformants with the truncated version of the URE2 gene were not able to grow on medium with urea as the sole nitrogen source while those with the intact version did (data not shown). Second, the coding region of the native URE2 gene in the wild-type strain H99 was disrupted by insertion with the DNA sequence of the gene conferring resistance to G418. Disruption of the native URE2 gene was confirmed by Southern blot analysis as well as by PCR analysis (Fig. 3). The ure2 : : G418 disruption transformant of strain H99 (H99SD) failed to grow on agar medium supplemented with urea as the sole nitrogen source (Fig. 2). Retransformation of strain H99SD with a plasmid containing the URE2 gene restored the strain's ability to grow on urea agar medium. A similar disruption of the URE2 gene in the serotype D strain LP1, by insertion of the ADE2 gene in the coding region, also led to failure of growth on urea agar (data not shown).
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The DNA sequence of the URE2 gene in the urease-negative strain B-4587 was compared with the URE2 gene of strain H99. The sequence of the URE2 gene was remarkably well conserved between H99 and B-4587. The comparison revealed only three single-nucleotide substitutions in the DNA sequence of the URE2 gene between strains B-4587 and H99. Two of these (C to T, and A to G) were observed within the 2nd and 11th exons, respectively. The third (C to T), however, was observed 15 bp downstream of the coding sequence of the gene.
Role of URE2 in murine cryptococcosis
Virulence in mice was studied in sets of 7-week-old female BALB/c mice that were infected by two separate modes. Each mouse was infected either by intravenous tail vein injections with 1x106 organisms or via inhalation with 2x107 organisms. No apparent difference in virulence was observed between the strains when administered via the intravenous route except that mortality in strain H99SD appeared to be slightly delayed (P=0.03, log-rank test) (Fig. 4a). All mice infected via this route died of cryptococcal infection within 7–13 days regardless of the urease phenotype of the infecting organism (Fig. 4a). Although mortality among mice infected via the inhalation route was generally delayed by 20–30 days compared to the intravenous route, mice infected with H99 and the urease-negative B-4587 strain succumbed to cryptococcosis at the same rate (Fig. 4b). Furthermore, the urease-negative vector transformant (B-4587V) and the urease-positive URE2 transformant of B-4587 (B-4587C) were equally virulent (Fig. 4c). Virulence with the ure2 : : G418 disruption isolate of H99 (H99SD), however, was significantly reduced compared to both B-4587 (P=0.02, log-rank test) and its URE2 complemented transformant (B-4587C), where it was found to be comparable (Fig. 4c). This indicated that the importance of the urease phenotype in pathogenicity of C. neoformans is strain dependent, and the route of infection emphasizes this importance.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ruane et al., 1988) and two from pigeon droppings (Li & Wu, 1992; Li et al., 1993). Unlike our urease-negative strain, however, the two urease-negative clinical isolates reported had caused disseminated infection in AIDS patients. Our case, therefore, is believed to be the first report of a urease-negative C. neoformans infection in an immunocompetent patient who was apparently cured with a regimen of 5-FC and ketoconazole. The combination of ketoconazole and 5-FC is rarely used as the drug treatment of choice for cryptococcosis of the central nervous system (Kwon-Chung & Bennett, 1992), and its therapeutic success in this case is likely due to the patient's normal immune status. The circumstances under which the patient might have been exposed to C. neoformans have not been determined. This encapsulated, melanin-positive and urease-negative strain became urease-positive, as evidenced by growth on urea agar, when transformed with a genomic plasmid library from the serotype A reference strain H99. The appearance of satellite colonies proximal to the transformants suggested secretion of urease by the transformants that degraded the urea in the agar into ammonia and carbamate, thereby allowing growth of urease-negative untransformed clones. Involvement of the URE2 gene product in the utilization of urea for growth in C. neoformans was further evidenced by the observation that strains containing the truncated versions of the URE2 gene failed to grow on medium with urea as the sole nitrogen source. A similar failure of the ure2 disruption stains of H99 or LP1 to grow on urea agar, coupled with restoration of this ability upon retransformation with the URE2 gene, indicated a prominent role of the URE2 gene product in the organism's ability to utilize urea as the sole nitrogen source. A search of the C. neoformans genomic database, with the DNA sequence of the gene contained within the insert, found the putative protein encoded by the ORF to be annotated as a hypothetical protein. It is not uncommon to find genomic databases littered with ORFs annotated to encode hypothetical proteins. We find this especially to be true when screening the genomic database of C. neoformans. The use of complementation cloning, as we have done to identify the gene with an apparent association with urease metabolism, can be a useful tool to assign function to such genes. Although a homology-based search of the NCBI database using the sequence of our gene revealed the ORF to have significant similarity to the sudA gene of A. nidulans, the gene was named URE2 due to its role in urease activity.
In A. nidulans, SudAp is involved in chromosome scaffolding that facilitates segregation during mitosis (Holt & May, 1996). The product of this gene, which appears to be well conserved, belongs to a family of extragenic suppressor proteins known as the DA-box protein family (Peterson, 1994). These DA-proteins, by genetic and biochemical analysis, have been shown to contribute directly to the composition of chromosomes in order to facilitate organization and segregation fidelity of chromosomes during mitosis (Peterson, 1994).The interaction of scaffold proteins with histones in the nucleus is known to affect several functional aspects of cell physiology, from structural aspects involving chromosomal condensation during cell division to gene regulation via transcriptional control (Peterson, 1994; Saitoh et al., 1994; Saka et al., 1994). It is possible that Sud1p of C. neoformans is a structural component of the mechanism integral to urease activity and may function in the secretion of urease, as suggested by the appearance of satellite colonies around the transformants.
While the NCBI database identified significant overall homology to the sudA gene in A. nidulans, a 288 bp region of the gene exhibited homology to DNA sequences present in genes encoding urease accessory proteins in other organisms. Urease accessory proteins, such as UreD, UreE and UreF, have been reported in Lycopersicon esculentum (tomato), Glycine max (soybean), Arabidopsis thaliana (thale cress) and the fission yeast Schizosaccharomyces pombe (Bacanamwo et al., 2002). Although the DNA sequences of the UreD and UreF genes appear to be well conserved among plants and some species of divergent bacteria, the homology of these genes appears to be intermediate when compared to the fungus S. pombe (Bacanamwo et al., 2002). The accessory proteins UreD and UreF are apparently essential for urease activity and reportedly form a complex to which the chaperone protein UreE delivers nickel, thus activating the urease enzyme (Lee et al., 1992; Soriano et al., 2000). Synthesis of metal-containing enzymes, such as urease, which requires the binding of nickel for function (Bacanamwo et al., 2002; Eitinger et al., 2000), often elicits the participation of several accessory proteins. It is postulated that the UreD gene is in some way involved in the production or incorporation of a nickel co-factor essential for urease activity. In previous reports, supplementing the urea growth media with nickel salts with or without bicarbonates was shown in other organisms to augment or restore the urease activity in some urease mutant strains which were otherwise unable to bind nickel (Soriano & Hausinger, 1999). However, the URE2 mutant strains of C. neoformans, B-4587 and the ure2 : : G418 strain of H99, were not able to grow on urea agar media supplemented with different concentrations and/or combinations of nickel and bicarbonate salts (data not shown).
The inability of strain B-4587 to grow on urea agar is apparently not due to a lack of URE1 transcription, as evidenced by the RT-PCR result and the failure of the URE1 gene to complement the urease-negative phenotype. This is not surprising since there have been reports of urease-negative strains in which the URE1 gene is expressed (Tanaka et al., 2003). Since the clinical isolate B-4587 was urease-negative, we tried to determine if the basis of this malfunction was the result of any mutation in the DNA sequence of the SUD1 gene. Although three nucleotide substitutions were detected, no discernible characteristics of the regions housing these substitutions were apparent. While these substitutions may play a role, their influence on the function of the protein is as yet unknown.
Virulence was recorded as mortality of the mice after infection. Two models of infection were used: the intravenous and the intranasal routes. Although URE2 disruption strains were constructed in serotype A as well as serotype D backgrounds, the disruptant in the serotype A background was chosen for virulence comparisons since both the reference wild-type strain H99 and the clinical isolate B-4587 were of serotype A. For mice infected intravenously, virulence was comparable between the urease-negative strain B-4587, its vector-only transformant (B-4587V) and the urease-positive strains H99 and the URE2-complemented transformant B-4587C. The urease phenotype apparently had no effect on the virulence of strains in the B-4587 background when a systemic model of infection was used. This is in contrast to the report of Cox et al. (2000), where a significant attenuation in virulence was observed for mice infected intravenously with the urease-negative H99 mutant ure1. However, virulence in the ure2 : : G418 disruption isolate of H99 (H99SD) was slightly attenuated compared to the wild-type strain H99 (P=0.03, log-rank test). This suggested that the urease-negative phenotype of H99 derived from a disruption of the URE2 gene did affect its virulence in mice. In the inhalation model, mortality in mice was generally delayed compared to mice infected intravenously. Intranasal infection of mice is probably not the ideal method of organism delivery due to the inherent lack of inoculum control, which may result in variations in reproducible mortality times. As in the intravenous model, mortality of mice infected intranasally was also similar between the urease-negative strain B-4587, the urease-positive transformants, and strain H99. This showed that in both infection models, urease activity in strain B-4587 and its derivatives was not important for virulence in mice. However, in mice infected with the ure2 disruption strain of H99 (H99SD), virulence via the inhalation model was found to be significantly more attenuated (P=0.02, log-rank test) than in strain B-4587 and its derivatives compared to the intravenous model. While all the mice infected intranasally with strains H99, B-4587 and B-4587C succumbed to infection in 40 days, 60 % of the mice infected with the ure2 disruption strain H99SD were still alive after 50 days. Our results suggest that virulence of C. neoformans in mice is not necessarily dependent on urease activity by itself but it is strongly influenced by other factors such as products of other related genes like URE2 and/or the route of infection. It is also possible that the differences in virulence observed between our URE2-derived urease-negative strains and the URE1-derived urease-negative strains used by Cox and colleagues are in fact the result of the specific genes involved, the fungal strains used or the strains of mice (BALB/c or A/Jcr) rather than just urease activity. While the mechanism involving urease in pathogenicity is not known, genes affecting virulence may be important during certain stages or at specific sites of infection, and this may account for the difference in the two models. Urease is reported to be a major site-specific virulence factor in bacteria infecting the tissue lining of the stomach and urinary tract (Mobley et al., 1991). It has also been reported that sequestration of the fungus within microcapillaries after inhalation is important prior to dissemination of C. neoformans to the brain (Olszewski et al., 2004). The exact mechanism by which urease facilitates such sequestration is not known but the hydrolysis of urea proximal to endothelial cells may promote adhesion, or the release of ammonia could result in cell toxicity affecting astrocytes by weakening the cellular junctions of the blood–brain barrier. Since the ure1 and ure2 strains are indistinguishable in their urease activity, urease itself may not be important for the virulence in certain strains of C. neoformans. The relationship between urease activity and virulence of C. neoformans therefore warrants further study.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Bava, A. J., Negroni, R. & Bianchi, M. (1993). Cryptococcosis produced by a urease negative strain of Cryptococcus neoformans. J Med Vet Mycol 31, 87–89.[Medline]
Canteros, C. E., Rodero, L., Rivas, M. C. & Davel, G. (1996). A rapid urease test for presumptive identification of Cryptococcus neoformans. Mycopathologia 136, 21–23.[CrossRef][Medline]
Chang, Y. C. & Kwon-Chung, K. J. (1994). Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol 14, 4912–4919.
Chang, Y. C. & Kwon-Chung, K. J. (1998). Isolation of the third capsule-associated gene, CAP60, required for virulence in Cryptococcus neoformans. Infect Immun 66, 2230–2236.
Chang, Y. C. & Kwon-Chung, K. J. (1999). Isolation, characterization, and localization of a capsule-associated gene, CAP10, of Cryptococcus neoformans. J Bacteriol 181, 5636–5643.
Chang, Y. C., Penoyer, L. A. & Kwon-Chung, K. J. (1996). The second capsule gene of Cryptococcus neoformans, CAP64, is essential for virulence. Infect Immun 64, 1977–1983.[Abstract]
Chen, G., Fournier, R. L., Varanasi, S. & Mahama-Relue, P. A. (1997). Helicobacter pylori survival in gastric mucosa by generation of a pH gradient. Biophys J 73, 1081–1088.
Cox, G. M., Mukherjee, J., Cole, G. T., Casadevall, A. & Perfect, J. R. (2000). Urease as a virulence factor in experimental cryptococcosis. Infect Immun 68, 443–448.
Curtis, J. L., Huffnagle, G. B., Chen, G. H., Warnock, M. L., Gyetko, M. R., McDonald, R. A., Scott, P. J. & Toews, G. B. (1994). Experimental murine pulmonary cryptococcosis. Differences in pulmonary inflammation and lymphocyte recruitment induced by two encapsulated strains of Cryptococcus neoformans. Lab Invest 71, 113–126.
Edman, J. C. & Kwon-Chung, K. J. (1990). Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation. Mol Cell Biol 10, 4538–4544.
Eitinger, T., Degen, O., Bohnke, U. & Muller, M. (2000). Nic1p, a relative of bacterial transition metal permeases in Schizosaccharomyces pombe, provides nickel ion for urease biosynthesis. J Biol Chem 275, 33184.
Holt, C. L. & May, G. S. (1996). An extragenic suppressor of the mitosis-defective bimD6 mutation of Aspergillus nidulans codes for a chromosome scaffold protein. Genetics 142, 777–787.[Abstract]
Kwon-Chung, K. J. & Bennett, J. E. (1992). Medical Mycology. Philadelphia: Lea & Febiger.
Kwon-Chung, K. J., Polacheck, I. & Popkin, T. J. (1982). Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol 150, 1414–1421.
Kwon-Chung, K. J., Wickes, B. L., Booth, J. L., Vishniac, H. S. & Bennett, J. E. (1987). Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans. Infect Immun 55, 1751–1754.
Kwon-Chung, K. J., Varma, A., Edman, J. C. & Bennett, J. E. (1992). Selection of ura5 and ura3 mutants from the two varieties of Cryptococcus neoformans on 5-fluoroorotic acid medium. J Med Vet Mycol 30, 61–69.[Medline]
Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y. & Hausinger, R. P. (1992). Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. J Bacteriol 174, 4324–4330.
Lee, H., Chang, Y. C. & Kwon-Chung, K. J. (2005). TUP1 disruption reveals biological differences between MATa and MAT strains of Cryptococcus neoformans. Mol Microbiol 55, 1222–1232.[CrossRef][Medline]
Li, A. & Wu, S. (1992). An urease negative Cryptococcus neoformans. Wei Sheng Wu Xue Bao 32, 68–71.[Medline]
Li, A., Guo, N. & Wu, S. (1993). A strain of urease negative Cryptococcus neoformans isolated from the environment in China. Chin Med Sci J 8, 52–54.[Medline]
Liu, L., Wakamatsu, K., Ito, S. & Williamson, P. R. (1999). Catecholamine oxidative products, but not melanin, are produced by Cryptococcus neoformans during neuropathogenesis in mice. Infect Immun 67, 108–112.
Mobley, H. L. (1996). The role of Helicobacter pylori urease in the pathogenesis of gastritis and peptic ulceration. Aliment Pharmacol Ther 10, 57–64.[Medline]
Mobley, H. L., Hu, L. T. & Foxal, P. A. (1991). Helicobacter pylori urease: properties and role in pathogenesis. Scand J Gastroenterol 187, 39–46.
Mondon, P., Chang, Y. C., Varma, A. & Kwon-Chung, K. J. (2000). A novel episomal shuttle vector for transformation of Cryptococcus neoformans with the ccdB gene as a positive selection marker in bacteria. FEMS Microbiol Lett 187, 41–45.[CrossRef][Medline]
Olszewski, M. A., Noverr, M. C., Chen, G. H., Toews, G. B., Cox, G. M., Perfect, J. R. & Huffnagle, G. B. (2004). Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion. Am J Pathol 164, 1761–1771.
Perfect, J. R., Wong, B., Chang, Y. C., Kwon-Chung, K. J. & Williamson, P. R. (1998). Cryptococcus neoformans: virulence and host defences. Med Mycol 36, 79–86.[Medline]
Peterson, C. L. (1994). The SMC family: novel motor proteins for chromosome condensation? Cell 79, 389–392.[CrossRef][Medline]
Ruane, P. J., Walker, L. J. & George, W. L. (1988). Disseminated infection caused by urease-negative Cryptococcus neoformans. J Clin Microbiol 26, 2224–2225.
Saitoh, N., Goldberg, I. G., Wood, E. R. & Earnshaw, W. C. (1994). ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure. J Cell Biol 127, 303–318.
Saka, Y., Sutani, T., Yamashita, Y., Saitoh, S., Takeuchi, M., Nakaseko, Y. & Yanagida, M. (1994). Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J 13, 4938–4952.[Medline]
Soriano, A. & Hausinger, R. P. (1999). GTP-dependent activation of urease apoprotein in complex with the UreD, UreF, and UreG accessory proteins. Proc Natl Acad Sci U S A 96, 11140–11144.
Soriano, A., Colpas, G. J. & Hausinger, R. P. (2000). UreE stimulation of GTP-dependent urease activation in the UreD-UreF-UreG-urease apoprotein complex. Biochemistry 39, 12435–12440.[CrossRef][Medline]
Tanaka, R., Nishimura, K., Imanishi, Y., Takahashi, I., Hata, Y. & Miyaji, M. (2003). Analysis of serotype AD strains from F1 progenies between urease-positive- and negative-strains of Cryptococcus neoformans. Nippon Ishinkin Gakkai Zasshi 44, 293–297.[Medline]
Toffaletti, D. L., Rude, T. H., Johnston, S. A., Durack, D. T. & Perfect, J. R. (1993). Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol 175, 1405–1411.
Varma, A. & Kwon-Chung, K. J. (2000). Characterization of the L41 gene in Cryptococcus neoformans: its application as a selectable transformation marker for cycloheximide resistance. Yeast 16, 1397–1403.[CrossRef][Medline]
Williamson, P. R. (1997). Laccase and melanin in the pathogenesis of Cryptococcus neoformans. Front Biosci 2, e99–e107.[Medline]
Zimmer, B. L. & Roberts, G. D. (1979). Rapid selective urease test for presumptive identification of Cryptococcus neoformans. J Clin Microbiol 10, 380–381.
Received 14 July 2006;
revised 8 August 2006;
accepted 16 August 2006.
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