1 Laboratorio de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Avda. Italia 3318, CP11600, Montevideo, Uruguay
2 Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK
Correspondence
Pablo Zunino
pablo{at}iibce.edu.uy
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ABSTRACT |
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INTRODUCTION |
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Virulence of P. mirabilis has been related to several potential factors including swarming motility mediated by flagella, urease production, cell invasiveness, haemolysin production, cleavage of IgA and IgG by proteolytic enzymes, lipopolysaccharide, capsular polysaccharide, resistance to normal serum, outer-membrane proteins, binding of environmental iron and adherence to the uroepithelium due to fimbriae (Rozalski et al., 1997). This last factor is thought to be one of the most important virulence properties of uropathogenic bacteria. P. mirabilis represents a particular case in which various types of fimbriae can be expressed simultaneously by the same cell (Mobley & Belas, 1995
). The different fimbriae include mannose-resistant Proteus-like haemagglutinin (MR/P), P. mirabilis fimbriae (PMF), uroepithelial cell adhesin (UCA) and ambient-temperature fimbriae (ATF).
Massad et al. (1994) suggested that PMF could have a role in the colonization of the bladder but not in the colonization of kidney tissue. Massad & Mobley (1994)
also described the pmf gene cluster, encoded by 5655 bp that predicted five polypeptides: PmfA, PmfC, PmfD, PmfE and PmfF. They determined that the pmf operon shares features with fimbrial gene clusters from enteric bacteria but that it also displays features that are unique.
The purpose of the present study was to assess the role of PMF in UTIs using different experimental approaches. An isogenic pmfA mutant was generated and characterized, and used in a co-infection ascending UTI model and a haematogenous UTI model in the mouse. It was also used in different tests to evaluate in vitro bacterial adherence to uroepithelial cells. Expression of PmfA was determined in numerous P. mirabilis strains isolated from different origins.
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METHODS |
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PCR and DNA sequencing.
PCR using chromosomal DNA from Pr2921 as template was performed to amplify the DNA flanking pmfA for allelic replacement mutagenesis. Primers used for the PCR were derived from the published pmf fimbrial operon sequence from strain HI4320, GenBank accession number Z35428 (Massad & Mobley, 1994). Primers 5'pm1 (5'-CAAATTAATCTAGAACCACTC-3') and 3'pm1 (5'-ATTATAGAGGATCCCTTGAAGGTA-3'), spanning positions 22122232 and 28062829 of the published sequence, respectively, were used to amplify a 5' pmfA DNA fragment (pm1). The cycle used was 94 °C for 2 min, followed by 30 cycles of 94 °C for 1 min, 54 °C for 1 min and 72 °C for 1 min, with a final cycle at 72 °C for 5 min.
Primers 5'pm2 (5'-TCAAGCTGGATCCGCTGACGCTAAAG-3') and 3'pm2 (5'-AGGGTCGATGCATGCCACGGAATGAT-3'), spanning positions 33043329 and 38223847 of the published sequence, respectively, were used to amplify a DNA fragment (pm2) that corresponds to the 5' end of pmfC, the putative molecular usher of PMF, which is the first gene downstream of pmfA in the PMF operon. The cycle used was 94 °C for 2 min, followed by 30 cycles of 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min, with a final cycle at 72 °C for 5 min.
PCR was also performed to amplify pmfA for cloning into pET-21a(+), using Pr2921 chromosomal DNA as template. In order to facilitate protein expression, the amplified DNA fragment did not include the leader peptide sequence. The 3'-end oligonucleotide carried a C-terminal fusion of six histidine (His6) residues to enable one-step purification of the expressed protein using Nickelagarose columns. The 5'-end and 3'-end primers were 5'-TCTGTTGCTCATATGGAAAATGAAACGCCT-3' and 5'-TCAATAAGCTTAATGGTGATGGTGATGGTGCTGATAATCAACTTG-3', respectively. NdeI and HindIII restriction sites were incorporated into the 5' and 3' primers, respectively, for cloning procedures. The cycle used was 94 °C for 3 min, followed by 30 cycles of 94 °C for 1 min, 40 °C for 1 min and 72 °C for 1 min, with a final cycle of 72 °C for 5 min.
DNA sequencing was performed at the Department of Biochemistry, University of Cambridge, using an Applied Biosystems automated DNA sequencer (model 377). DNA sequences from the cloned genes were compared to published sequences using the BLASTn search tool (Altschul et al., 1990). Oligonucleotides for DNA sequencing (Genosys) in pET-21a(+) were 5'-TAATACGACTCACTATAG-3' (forward primer) and 5'-GCTAGTTATTGCTCAGCG-3' (reverse primer).
Mutagenesis of pmfA in P. mirabilis.
The pmfA isogenic mutant of P. mirabilis, P2, was constructed by conjugating Pr2921 (wild-type) with E. coli SM10pir (pCVD442pmfA : : km) as described previously (Legnani-Fajardo et al., 1996
). After conjugation, bacteria were scraped from the plates, suspended in PBS and plated again at various dilutions on LuriaBertani (LB) agar lacking NaCl and solidified with 2 % (w/v) agar to inhibit any swarming and containing kanamycin (40 µg ml-1), tetracycline (10 µg ml-1) and sucrose (10 %, w/v). Pr2921 is naturally resistant to tetracycline, which was used to counter-select the E. coli donor strain.
Southern blotting.
Southern blots were prepared by standard procedures as described previously (Zunino et al., 2000). The probe DNA was labelled with [
-32P]dCTP (Amersham) using a Prime-it RmT Random Primer Labelling Kit (Stratagene) according to the manufacturer's instructions and added to the pre-hybridization solution; the mixture was then incubated overnight at 65 °C. Next, the membrane was washed four times at 65 °C in the following way: 5 min in 2xSSC/0·1 % (w/v) SDS, 30 min in 1xSSC/0·1 % (w/v) SDS, 30 min in 0·2xSSC/0·1 % (w/v) SDS and 5 min in 2xSSC. Then, the membrane was air-dried and exposed to X-ray film (X-Omat LS; Kodak).
Overexpression and purification of PmfAHis6 fusion protein.
pmfA lacking its N-terminal signal peptide sequence was amplified by PCR for cloning into pET-21a(+). After digestion with NdeI and HindIII, the DNA fragments were ligated into NdeI/HindIII-digested pET-21a(+). The ligation was transformed into E. coli XL-1Blue. Then, the pET-21a(+) construct containing pmfA was moved into the lysogen E. coli BL21(
DE3) by transformation, for expression of recombinant PmfA. Overexpression and purification of PmfA were performed according to instructions provided by the manufacturer (Novagen; pET System Manual, 6th edn, 1985) as described previously (Pellegrino et al., 2003
). The protein concentration was determined by the Lowry method using BSA as a standard.
Antiserum preparation.
The immune serum was prepared by immunization of a New Zealand White rabbit using a four-dose schedule. All doses contained 100 µg of purified PmfA. The first dose was emulsified in Freund's complete adjuvant (Sigma), then the three subsequent doses were given at 2 week intervals in Freund's incomplete adjuvant (Sigma). Serum was collected 1 week after the last immunization and pre-adsorbed with P. mirabilis pmfA mutant to reduce non-specific reactions (Gruber & Zingales, 1995).
SDS-PAGE and Western blot.
Proteins and whole bacteria were suspended in sample buffer and boiled for 5 min before loading onto 15 % (w/v) SDS-polyacrylamide gels. The gels were prepared and run according to Sambrook et al. (1989). For Western blot assays, proteins were transferred from SDS-polyacrylamide gels to nitrocellulose membranes (Bio-Rad), according to Sambrook et al. (1989)
. Western blots were developed using a 1/200 dilution of a rabbit polyclonal immune antiserum raised against purified recombinant PmfA.
Animals and infection models.
Female CD-1 mice weighing 2025 g from the breeding facilities at IIBCE were used and provided with food pellets and tap water as necessary. All animal experiments were conducted in accordance with procedures authorized by IIBCE, Montevideo, Uruguay. To assess the virulence of the wild-type and the pmfA mutant strains, two mouse models of UTI were used.
Initially, a co-challenge experiment was performed in the ascending UTI model described previously (Zunino et al., 2000). Sixteen mice were challenged with 0·05 ml of a bacterial suspension in PBS containing 108 colony-forming units (c.f.u.) of Pr2921 and 108 c.f.u. of the pmfA mutant. Seven days after inoculation, mice were killed by cervical dislocation. Kidneys and bladders were removed aseptically and homogenized in 10 ml of PBS using a Stomacher 80 Lab Blender (Seward). Viable bacterial counts were done on LB with 2 % (w/v) agar and without NaCl to avoid swarming motility. LB agar and LB agar supplemented with kanamycin (40 µg ml-1) were used for differential bacterial counts.
When the haematogenous model was used (Peerbooms et al., 1982), bacterial suspensions in PBS (0·1 ml), at a concentration of 107 c.f.u., were injected into a tail vein. Seven days after infection, mice were killed by cervical dislocation, and the presence of bacteria in the kidneys was assessed by direct sampling on LB agar.
Adherence to uroepithelial cells.
This was investigated using two different in vitro assays. First, T24/83 cells, derived from an original human bladder carcinoma T24 cell line, were used (European Collection of Cell Cultures; ECAC ref. no. 85061107) (Tolson et al., 1997). For the adherence assay, bacteria from an 18 h culture at 37 °C on LB agar were inoculated into 5 ml of LB broth and grown statically at 37 °C for 48 h. Then, bacteria were harvested and resuspended in McCoy's 5a supplemented with 2 mM glutamine and 10 % (v/v) fetal calf serum to 2x108 c.f.u. ml-1. Next, the medium from the wells was aspirated, each well was inoculated with 1 ml of the bacterial suspensions and the plates were incubated at 37 °C, 5 % CO2. After 1 h, cells were washed five times with PBS to remove unattached bacteria. After the last wash, 1 ml of 0·5 % (v/v) PBS/Triton X-100 was added per well and incubated for 15 min under the same conditions to lyse eukaryotic cells. Experiments were performed in triplicate. The number of remaining attached bacteria was determined by viable plate counts.
Second, uroepithelial cells from fresh urine of healthy female volunteers were used. In this case, the assay was performed following a modified method described by Wray et al. (1986). Uroepithelial cells were isolated from the urine of three healthy female volunteers by centrifugation at 400 g for 10 min, then washed three times in buffered saline with gelatin [BSG; containing 0·15 M NaCl, 0·004 M potassium phosphate, pH 6·0, and 0·1 % (w/w) gelatin] and pellets were resuspended in BSG at 5x105 cells ml-1. The cell suspensions were mixed with an equal volume of a bacterial suspension containing 109 c.f.u. ml-1 and incubated for 1 h at 37 °C. Then, cells were washed four times in BSG, smears were prepared for light microscopy and bacteria attached to 50 uroepithelial cells were counted.
Other assays for phenotypic characterization of the pmfA mutant.
Pr2921 and the isogenic pmfA mutant P2 were tested for a variety of phenotypes, including swarming motility, growth rate, haemagglutination, and urease and haemolysin production as described previously (Zunino et al., 2001; Legnani-Fajardo et al., 1996
). Both strains were also co-cultured in LB broth at 37 °C for 7 days to assess the mutant fitness in vitro. Then, bacteria were plated on LB with 2 % (w/v) agar and without NaCl to avoid swarming motility and LB agar supplemented with kanamycin (40 µg ml-1); these media were used for differential bacterial counts.
Statistical analysis.
MannWhitney one-tailed non-parametric analysis was used to compare bacterial colonization levels in kidneys and bladders. The chi-square test with Yates correction for small sample sizes was used to compare the numbers of animals infected with the parental and mutant bacteria. To analyse the results of the experiments to determine bacterial adherence to uroepithelial cells, the Student's t-test was used. P<0·05 was considered significant.
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RESULTS |
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Genetic analysis of the pmfA mutant
Appropriate genomic rearrangements were checked by Southern hybridization. Initially, DNA isolated from wild-type and mutant strains was digested with HindIII and SspI, separately, and probed with pmfA amplified by PCR. This probe hybridized with the restriction digest bands predicted from the published sequence. The Pr2921 DNA bands, generated with SspI (0·9 kb) and HindIII (1·2 kb), hybridized with the pmfA probe as expected. P1 and P2 did not show any reactive band because almost the whole pmfA gene had been deleted. Similarly, DNA was digested separately with SspI and SalI, and probed with the KmR cassette. Pr2921 did not show reactive bands, as expected. P1 and P2 showed two bands when digested with SspI, since the KmR gene has an internal SspI site. The SalI digestion of P1 and P2 showed a single 1·3 kb band that corresponded to the released KmR cassette. One of the mutants, P2, was chosen for further experiments.
Phenotypic analysis of wild-type Pr2921 and the mutant strain P2
Typical P. mirabilis swarming motility was exhibited by both strains when grown on LB agar plates. No differences in net growth rate were observed between parental and mutant strains when growth curves were constructed, showing generation times of 28·3 and 28·1 min, respectively. Both strains were urease-positive, agglutinated fresh equine and ovine erythrocytes, and exhibited similar haemolytic activities. After the wild-type and mutant strains were co-cultured for 7 days in LB broth, and viable plate counts on LB agar and LB supplemented with kanamycin were performed, numbers of recovered c.f.u. were similar for both strains, showing that the mutant strain was not out-competed by the wild-type strain.
The ability of strains Pr2921 and P2 to synthesize PmfA was assessed by SDS-PAGE and Western blotting. For Western blotting, we used the anti-PmfA serum produced after successful expression and purification of the recombinant protein (Fig. 1A) and rabbit immunization. Total protein extracts from cultures of Pr2921 grown in LB broth (48 h at 37 °C) showed a clear band of about 20 kDa corresponding to PmfA when analysed by Western blot. Total protein extracts of strain P2 grown under the same conditions exhibited no reactive bands under the same conditions (Fig. 1B
), confirming that P2 is deficient in PmfA expression as expected. As a further control we were able to detect PmfA expression from a P. mirabilis mutant deleted for mrpA, a gene required for expression of the unrelated MrpA fimbriae.
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PmfA expression by P. mirabilis strains of different origins
To evaluate PmfA expression by non-uropathogenic P. mirabilis isolates, strains from several clinical and non-clinical sources were examined by Western blot. All strains showed a reactive band that corresponded to PmfA (Fig. 2).
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For the co-challenge experiment, bacteria were recovered from bladder and kidneys 7 days after transurethral challenge and were enumerated on normal non-swarming 2 % (w/v) LB agar (lacking NaCl), on which Pr2921 and P2 could grow, and on non-swarming 2 % (w/v) LB agar plates containing kanamycin (40 µg ml-1), on which only the mutant strain could grow. The number of viable bacteria counted from bladder and kidneys showed that the mutant strain was significantly out-competed by the wild-type strain in colonizing the kidneys (P=0·0054) and the bladders (P=0·0111) (Fig. 3).
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DISCUSSION |
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There is only one report related to the potential role of PMF in P. mirabilis-associated UTIs (Massad et al., 1994). In that study, the authors constructed a pmfA isogenic mutant that was tested only in an independent challenge using the ascending UTI model in the mouse. They concluded that PMF play a role in colonization of the bladder but not in colonization of kidney tissue, since they could not detect a significant difference in bacterial numbers recovered from the kidneys between mice infected with the wild-type strain and those infected with the PMF mutant (Massad et al., 1994
).
In the present work, we generated a pmfA allelic replacement isogenic mutant that was used in different in vivo and in vitro assays. We also determined the expression of PMF by several clinical and non-clinical isolates of P. mirabilis.
The process of mutating pmfA did not apparently affect other bacterial virulence attributes, including growth rate, swimming or swarming motility, urease and haemolysin production or haemagglutination. Fitness of the mutant was also confirmed when the parental and mutant strains were recovered in similar numbers after co-culturing in LB broth and subsequent plating on LB agar and LB agar supplemented with kanamycin.
The insertion of a KmR gene for mutagenesis allowed us to perform a co-challenge ascending UTI assay. We had previously used this approach to evaluate the role of ATF and MR/P fimbriae in P. mirabilis-associated UTIs (Zunino et al., 2000, 2001
). This assay allowed us to assess the ability of the wild-type strain to out-compete the mutant in the initial stages of UTI. Using this infection model we established that the mutant was unable to compete with the wild-type not only in the bladder but also in the kidneys of the infected mice.
Although UTIs are ascending rather than haematogenous, the haematogenous UTI model can provide useful data about colonization of the kidneys. When the mutant (P2) was used in this model, it was found to be significantly decreased in infectivity. This suggests that PMF play a role in localization of uropathogenic P. mirabilis to the kidneys. In both models, infectivity of the mutant was clearly impaired, including its ability to colonize the kidneys. These results are in disagreement with those reported by Massad et al. (1994), since these authors suggested that PMF are not related to kidney colonization.
Mutant strain P2 was used in two different in vitro bacterial adherence assays. In the first model, we used transformed, cultured T24/83 cells. We had used this technique in previous work to evaluate the role of MR/P in P. mirabilis adherence to uroepithelial cells (Zunino et al., 2001). When we assessed the adherent ability of Pr2921 and P2 to T24/83 cells, we noted a significant decrease in the number of adherent pmfA mutant bacteria compared to the number of adhered wild-type bacteria. We also performed another bacterial adherence test using exfoliated uroepithelial cells isolated from fresh urine, initially used by Wray et al. (1986)
to assess adherence properties of P. mirabilis uroepithelial cell adhesin fimbriae, and a significant decrease in attached mutant bacteria was also noted. These results are also different from those obtained by Massad et al. (1994)
, since these authors could not detect significant differences between adherent wild-type and mutant bacteria to uroepithelial cells. Our in vitro adherence assays to both transformed and desquamated uroepithelial cells strongly suggest that PMF play a role in bacterial adherence and colonization of the urinary tract at least during the initial stages of infection. It is interesting to note that different cell types were used by both groups.
The expression of PmfA was assessed in numerous clinical and non-clinical strains using a rabbit polyclonal antiserum for Western blotting. It is interesting to note that all strains of different origins expressed PmfA. This result confirms that P. mirabilis is a very homogeneous species (Li & Mobley, 2002) and could also indicate that P. mirabilis strains cannot be separated into clonal pathogenic groups and that the distribution of virulence factors between strains of different origins reflects the opportunistic nature of the organism. We are currently assessing the distribution of other virulence factors among numerous P. mirabilis strains from different clinical and non-clinical sources. As a complementary result, we observed that PmfA expression was also qualitatively detected in a total protein extract of the P. mirabilis mutant MSD2, generated in a previous study (Zunino et al., 2001
). This may suggest that MR/P fimbriae and PMF expression are unrelated, since PMF biosynthesis was not apparently affected by the MR/P mutation.
It is interesting to note that, as in our previous study using a mutant lacking MR/P, in vitro bacterial adherence of the pmfA mutant to uroepithelial cells was not totally suppressed. Moreover, infectivity was not abolished when mice were challenged with either mrpA or pmfA mutants. These findings suggest that redundancy of effects between different fimbriae is very likely.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 2 June 2003;
revised 6 August 2003;
accepted 18 August 2003.
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