Department of Microbiology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada1
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411, Japan2
Department of Gastrointestinal Infections, Statens Serum Institut, DK2300 Copenhagen, Denmark3
Author for correspondence: Janet M. Wood. Tel: +1 519 824 4120 Ext. 3866. Fax: +1 519 837 1802. e-mail: jwood{at}uoguelph.ca
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ABSTRACT |
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Keywords: osmoregulation, glycine betaine, trehalose, UPEC
Abbreviations: GB, glycine betaine; UPEC, uropathogenic Escherichia coli
The GenBank accession numbers for the DNA sequences of the rpoS loci in E. coli strains HU734 and CFT073 are AF275947 and AF270497, respectively.
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INTRODUCTION |
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High urea levels and fluctuating osmolality distinguish the urinary tract from most other mammalian systems. Although the osmolality of urine from humans with a normal diet and fluid intake is in the range 0·50·8 mol kg-1, urinary osmolality may vary from approximately 0·04 mol kg-1 to 1·4 mol kg-1 in humans (Altman, 1961 ; Kunin, 1987
; Ross & Neely, 1983
) and up to 3 mol kg-1 in rats and mice (Loeb & Quimby, 1989
; Schmidt-Nielsen et al., 1983
). Urea, the primary contributor to the osmolality of urine and of renal extracellular fluid, can approach concentrations of 0·5 and 1·5 M in human and rat urine, respectively. Inorganic ions are the other major contributors. E. coli strains growing in urine are believed to utilize lactate, amino acids, peptides and
as nutrients (some can also utilize citrate and urea) (Brooks & Keevil, 1997
; Gordon & Riley, 1992
). Thus uropathogens must be urea-resistant and salinity-tolerant. They must mount these stress responses in an environment of variable pH that includes a mixture of organic nutrients.
E. coli is the primary causative agent for ascending infection of the unobstructed human urinary tract (Kunin, 2000 ). Multiple osmoregulatory mechanisms with overlapping functions facilitate the survival and growth of E. coli K-12 in media with extreme and/or fluctuating osmolalities (Table 1
). While contingent on their availability, the uptake of organic osmoprotectants such as glycine betaine (GB) is more effective in promoting cellular rehydration and growth than the uptake of K+ and/or the biosynthesis of trehalose (Wood, 1999
). The genomes of natural E. coli isolates vary widely and differ extensively from that of E. coli K-12 (Bergthorsson & Ochman, 1995
, 1998
). Thus the osmoregulatory systems present in natural isolates, including UPEC, may differ from those defined using E. coli K-12 (Table 1
).
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Alternative factor RpoS mediates the expression of genes implicated in osmotolerance (e.g. otsAB, proP), acid tolerance, thermotolerance, oxidative stress and stationary-phase survival for E. coli K-12 and other bacteria (Abee & Wouters, 1999
; Hengge-Aronis, 1996
; Ishihama, 2000
; Loewen et al., 1998
). RpoS has been clearly implicated in expression of the virulence-associated spv operon of Salmonella (Spector, 1998
) and in the acid tolerance that contributes to virulence and transmission for enterohaemorrhagic E. coli (Price et al., 2000
; Waterman & Small, 1996
). The relationship between RpoS and virulence is less clear for neonatal meningitis isolates of E. coli K1 (Wang & Kim, 2000
) and disruption of rpoS enhanced long-term colonization of the murine large intestine by rat faecal E. coli isolate BJ4 (Krogfelt et al., 2000
). The rpoS loci of laboratory (Jishage & Ishihama, 1997
) and natural (Waterman & Small, 1996
) E. coli isolates are polymorphic (including rpoS defects). It is not yet clear whether RpoS defects can impair virulence pleiotropically, by limiting expression of multiple stress tolerance genes, or whether RpoS contributes to virulence only for particular bacterial species and loci of infection.
This report addresses the roles of ProP, ProU and RpoS in osmotolerance and virulence for two pyelonephritis isolates of E. coli. Strain HU734, used to establish the murine model for ascending urinary tract infection, is a lac- strain isolated from pyelonephritis isolate GR12 by chemical mutagenesis (Hagberg et al., 1983a , b
). Introduction of genetic lesions affecting P pili to E. coli HU734 (Hagberg et al., 1983b
) and E. coli DS17 (Roberts et al., 1994
) reduced their virulence as indicated by the murine and primate models, respectively. Although these and other data clearly establish an important role for P pili as virulence determinants in pyelonephritis, the elimination of P pili did not reduce the virulence of pyelonephritis isolate CFT073 as determined by the murine model (Mobley et al., 1993
). These and other observations suggest that E. coli CFT073 is more virulent than E. coli HU734 (Mobley et al., 1993
). E. coli strains HU734 and CFT073 have thus been compared during this study of osmoregulation and urovirulence.
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METHODS |
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Bacteria, plasmids and molecular biological manipulations.
The E. coli K-12 derivatives used for this study are listed in Table 2. The genotypes of the clinical E. coli isolates and their derivatives are described below and in Table 4
. E. coli BJ4, a faecal isolate from a healthy Wistar rat, is rough:K-:H-. It produces type 1 fimbriae and possesses two unidentified, high molecular mass plasmids (Krogfelt et al., 1993
). Strain BJ4 rpoS was isolated by bacteriophage P1-mediated transduction of BJ4 to tetracycline resistance from MC4100 rpoS::Tn10 (Krogfelt et al., 2000
). Strain HU734, a lac- derivative of acute pyelonephritis isolate GR12, has the following properties: streptomycin and spectinomycin resistance and cysteine auxotrophy, serotype O75:K5, possession of type 1 and P pili (the latter encoded by a single pap operon), carriage of ColV plasmid, resistance to killing by human and mouse serum, and failure to produce haemolysin (Hagberg et al., 1983a
; Mamelak, 1994
). Strain CFT073 was isolated from the blood of a patient with pyelonephritis. It has no antibiotic resistance or auxotrophy, is O non-typable and non-motile, expresses type 1, S and P pili (the latter encoded by two pap operons), produces haemolysin and is cytotoxic for cultured human renal epithelial cells (Mobley et al., 1993
). Isolation of strains WG541 [HU734
(putPA)566] and WG671 [WG541
(proP)218] was described previously (Culham et al., 1998
). Deletion
(proP)218 eliminates a DNA fragment that extends from 59 bp upstream to 44 bp downstream of proP.
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Deletions (proVproX)567 and
(rpoS)2062 were created in vector pCVD442 (Donnenberg & Kaper, 1991
) and introduced to the chromosomes of strains WG541, CFT073 and their derivatives by allelic replacement. Deletion
(proVproX)567 was created as follows. The 5·2 kb EcoRI fragment of pOS59 (Haardt & Bremer, 1996
) was inserted into the EcoRI site of vector pGEM7Z to create plasmid pDC70. The 4·6 kb AccIMscI fragment of pDC70 was treated with Klenow fragment of DNA polymerase I and religated to create deletion
(proVproX)567 in plasmid pDC72. The 1·5 kb EcoRVSmaI fragment of pDC72 was then ligated into the SmaI site of vector pCVD442 to create plasmid pDC76, which contains 686 bp upstream of proV, plus the first 146 bp of proV and 652 bp downstream of proX.
The in-frame rpoS deletion was created using crossover PCR essentially as described by Link et al. (1997) . PCR was used to amplify a 593 bp product (including sequence upstream of rpoS) using primers rpoS-No and rpoS-Ni (Table 3
) and a 581 bp product (including sequence downstream of rpoS) using primers rpoS-Ci and rpoS-Co2 (Table 3
) with chromosomal DNA from E. coli CFT073 as template (PCR conditions essentially as described by Brown & Wood, 1992
). These two products were gel-purified, mixed and PCR-amplified using primers rpoS-No and rpoS-Co2. Since 21 bp complementary sequences were included in primers rpoS-Ni and rpoS-Ci, this mixture supported amplification of a 1·1 kb product including an in-frame deletion of rpoS [
(rpoS)2062] encoding a protein composed of the first 6 aa of RpoS, 7 aa added as a result of the 21 bp complementary region and the last 11 aa of RpoS. The 1·1 kb product was digested with SstI, gel-purified and ligated into the SstI site of vector pCVD442 to create plasmid pDC96.
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Enzyme assays.
The GB uptake activities of bacteria cultivated in defined media or human urine were measured as described by Culham et al. (1998) , using uniformly labelled 14C-GB (American Radiolabelled Chemicals) (0·2 mM; 5 µCi µmol-1). Bacteria cultivated in urine (0·92 mol kg-1, 0·44 M urea) were harvested, washed and resuspended in unsupplemented MOPS medium rendered isotonic by adding 0·44 M urea and 0·174 M NaCl. The transport assay media also contained 0·44 M urea and were rendered isotonic by adjusting the NaCl concentration.
The catalase contents of pyelonephritis isolates HU734 and CFT073 and of E. coli K-12 derivatives GC4468 (katE+ katG+), GC202 (katE+ katG::Tn10), NC4468 (katE::lacZ katG+) and NC202 (katE::lacZ katG::Tn10) were determined as follows. Bacteria were grown to stationary phase in 75 ml LB medium, harvested by centrifugation and resuspended in 7·5 ml 20% (w/v) sucrose. The cell suspensions were sonicated, clarified by centrifugation at 13000 r.p.m. for 3 min and 20 µl of each supernatant was analysed by native gel electrophoresis. Bands with catalase activity were visualized with reagent mixtures, including horseradish peroxidase (Sigma) and hydrogen peroxide, then diaminobenzidine (Sigma) as described by Clare et al. (1984) . The catalase contents of the E. coli K-12 derivatives were as predicted by their genotypes (data not shown).
RpoS detection.
RpoS was detected in selected bacterial strains as follows. A few colonies from an overnight culture on LB medium were inoculated into 5 ml fresh liquid LB medium. At a cell density of 30 Klett units, the culture was diluted 40-fold by adding 200 ml fresh LB medium and incubated at 37 °C with shaking to a cell density of 30 Klett units (exponential phase) or 200 Klett units (stationary phase). Cells were collected by centrifugation and resuspended at 4 °C in 40 mM Tris/HCl (pH 8·1) containing 25% sucrose. After treatment with 1 mM EDTA and 0·5 mg lysozyme ml-1 at 0 °C for 10 min, cells were lysed by adding 0·5% Brij-58. The Brij lysate was supplemented with 0·01 M MgCl2 and 0·2 M KCl, and digested at 37 °C for 10 min with 20 mg RNase A ml-1 and 100 mg DNase I ml-1 in the presence of 1 mM PMSF, followed by sonication for 1 min with a Cosmo Bio Bioruptor. The supernatant after centrifugation for 30 min at 15000 r.p.m. was used as the cell lysate for the experiments. The protein concentration of cell lysates was determined using the Bio-Rad Protein Assay Kit.
For the measurement of subunits, a quantitative Western blot analysis was employed using monospecific anti-
antibodies. Anti-RpoS antibodies were raised in rabbits against purified RpoS protein (Jishage & Ishihama, 1995
). In brief, cell lysates were treated with SDS sample buffer (50 mM Tris/HCl, pH 6·8, 2% SDS, 1% 2-mercaptoethanol, 10% glycerol and 0·025% bromophenol blue) and separated on SDS polyacrylamide gels (7·5 or 10%). Proteins in the gels were directly electroblotted onto polyvinylidene difluoride membranes (Nippon Genetics). Blots were blocked overnight at 4 °C in 3% BSA in PBS, probed with monospecific antibodies against each
subunit, washed with 0·5% Tween in PBS and incubated with goat anti-rabbit IgG conjugated with hydroperoxidase (Cappel). The blots were developed with 3,3'-diaminobenzidine tetrahydrochloride (Dojindo).
Trehalose assay.
Intracellular trehalose levels were determined by NMR spectroscopy as described by MacMillan et al. (1999) . Extracts were prepared from cells cultured in MOPS medium supplemented with glycerol as carbon source and 0·4 M NaCl in the absence of organic osmoprotectants.
Colonization of the murine urinary tract.
The ability of selected E. coli strains to colonize the murine urinary tract was determined by enumeration of bacteria in the animals urine, bladders and kidneys 1 and 3 d after transurethral inoculation with 50 µl bacterial suspensions (approx. 109 bacteria ml-1) (Hvidberg et al., 2000 ). Bladders and kidneys were homogenized on ice for 30 s using an IKA Laborteknik homogenizer before plating on relevant media. The genotypes of selected isolates from the bladders of infected mice were verified by PCR amplification using the primers listed in Table 3
.
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RESULTS |
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E. coli HU734 expressed RpoS, as indicated by Western blot analysis, but that protein was lower in electrophoretic mobility than RpoS from E. coli K-12, its derivative CSH4 or E. coli CFT073 (Fig. 2), as expected on the basis of the DNA sequence. The quantity of RpoS detected in both clinical isolates was much higher than that detected in E. coli K-12. Furthermore, RpoS was present at high levels in bacteria from both exponential-and stationary-phase cultures of the clinical isolates, whereas its expression was growth-phase-dependent in E. coli K-12, as previously observed (Jishage & Ishihama, 1995
). Both pyelonephritis isolates expressed RpoN and neither expressed RpoF under these conditions (data not shown).
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To further assess whether a trehalose biosynthetic lesion or an RpoS defect limited trehalose biosynthesis by E. coli HU734, the stationary-phase thermotolerance of strain HU734 was compared with that of strains MC4100 (rpoS+) and RH90 (rpoS359::Tn10). Using E. coli K-12 derivatives MC4100, RH90 and RO22 (otsA::Tn10 otsB::lac), Hengge-Aronis et al. (1991) demonstrated that the disruption of rpoS much more dramatically reduced stationary-phase thermotolerance than did disruption of otsA and otsB. The very poor stationary-phase thermotolerance of E. coli HU734 (comparable to that of E. coli RH90) (Fig. 4
) suggested that the limited trehalose accumulation in E. coli HU734 (Fig. 3
) resulted from an rpoS lesion with pleiotropic effects on thermotolerance. The catalase contents of the pyelonephritis isolates were also assessed in comparison with those of selected E. coli K-12 derivatives (see Methods). Expression of KatE by E. coli K-12 is RpoS-dependent, whereas expression of KatG is not (Loewen et al., 1998
). Strains HU734 and CFT073 expressed KatG but not KatE (data not shown), so this test did not further indicate the status of RpoS in these bacteria.
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Impact of rpoS, proP and proU lesions on osmoregulatory solute accumulation and osmotolerance for E. coli strains HU734 and CFT073
The rpoS locus was deleted from E. coli CFT073 (see Methods). The in-frame rpoS deletion in strain WG745 [CFT073 (rpoS)2062] eliminated trehalose accumulation (Fig. 3
) and rendered the stationary-phase thermotolerance of strain WG745 similar to that of E. coli RH90 (rpoS::Tn10) (Fig. 4
), as expected. No compound other than MOPS replaced trehalose in E. coli WG745 (Fig. 3
). The rpoS defect rendered the growth rate of CFT073 in very high-osmolality, NaCl-supplemented minimal medium (1·4 mol kg-1) comparable to that of HU734, but this defect did not account for their different abilities to tolerate lower osmolalities (e.g. 0·4 mol kg-1) (Fig. 1
).
The osmotolerance of both pyelonephritis isolates was enhanced by osmoprotectant GB (1 mM) (Culham et al., 1998 and Fig. 1
). This was anticipated on the basis of earlier evidence that each possessed genetic loci proP and proU (Culham et al., 1994
and unpublished data). Loci proP and proU were deleted from each strain, both singly and together, in an effort to assess whether ProP and ProU were the only contributors to osmoprotection for the clinical isolates and whether those systems contributed to virulence (see Methods). Initial rates of GB uptake were measured for bacteria cultivated in MOPS minimal medium supplemented with or without 0·4 M NaCl, the concentration at which the largest stimulation of bacterial growth by GB had been observed for E. coli HU734 (Culham et al., 1998
). The osmolalities of these media were 1·0 and 0·2 mol kg-1, respectively. As for E. coli K-12 (Wood, 1999
), GB uptake activity was dramatically elevated when osmotic stress was imposed (Table 4
).
The GB uptake activities attained in strain CFT073 were similar to those observed with E. coli K-12 (Grothe et al., 1986 ). For these late-exponential-phase bacteria, deletion of rpoS reduced the GB uptake activity attained in the absence of osmotic stress but not in its presence (Table 4
, compare strains CFT073 and WG745). All the GB uptake could be attributed to transporters ProP and ProU, since strain WG696 [
(proP)218
(proVproX)567] was devoid of activity. The GB uptake activities observed in strain HU734 were three- to fourfold higher than those of CFT073 and residual GB uptake activity was observed after transporters ProP and ProU were eliminated (Table 4
, see strain WG695). Analysis of the initial rate of GB uptake as a function of GB concentration for strain WG695 [HU734
(putPA)101
(proP)218
(proVproX)567] yielded a Km for the residual activity of 22 µM. The GB concentrations at which betaine uptake rates were measured included 7·3, 17·3, 27·3, 57·3, 87·3 and 147·3 µM. This Km value clearly distinguished BetU from transporters ProP [Km (GB) 100 µM] and ProU [Km (GB) 1 µM]. The newly identified transporter, designated BetU, was capable of mediating osmoprotection by GB or proline betaine but not proline as indicated by an osmoprotection assay (see Methods), again distinguishing it from systems ProP and ProU. This transporter is not present or not expressed in E. coli K-12 and E. coli CFT073.
Contribution of osmoprotectant uptake to bacterial growth in human urine
To assess their responses to growth in human urine, the GB uptake activities of the pyelonephritis isolates were examined after cultivation in high-osmolality human urine and various iso-osmolal minimal salts media. Human urine contains both GB (78±65 µM in normal humans) and proline betaine (298±687 µM in normal humans) (Lever et al., 1994a , b
). The urea content of the urine used for these experiments was 0·44 M (see Methods).
The GB uptake activities of bacteria cultivated in this urine were more similar to those of bacteria cultivated in MOPS medium of similar osmolality and urea content (compare data columns 3 and 4 of Table 4) than to those of bacteria cultivated in iso-osmolal urea-free MOPS medium (compare data columns 3 and 2 of Table 4
). The activities of bacteria cultivated in urine were most similar to those of bacteria grown in GB-supplemented MOPS medium of similar osmolality and urea content (compare data columns 3 and 5 of Table 4
). Thus the factors controlling expression and activity of the osmoregulatory GB transporters in the pyelonephritis isolates during growth in human urine were consistent with those identified during studies of E. coli K-12 (see Discussion). Osmolality due to solutes other than urea was the primary modulator of GB uptake activity. Although all three GB transporters (ProP, ProU and BetU) were active in urine-grown bacteria, most activity could be attributed to ProP (Table 4
).
Betaine uptake via transporters ProP and ProU was expected to stimulate the growth of E. coli and deletion of the transporter genes was expected to impair it in high-osmolality human urine (Chambers & Lever, 1996 ). Elimination of transporters ProP and ProU impaired but did not eliminate the growth of E. coli HU734 in human urine (0·86 mol kg-1; Fig. 5a
). Elimination of system ProU had no effect unless ProP was also absent. This was consistent with the larger contribution from ProP than from ProU to GB uptake by the urine-grown bacteria (Table 4
). As observed previously (Culham et al., 1998
), the relationship between the log(bacterial c.f.u. ml-1) and log(optical density) differed for bacteria retaining and lacking transporter ProP (inset, Fig. 5a
). The higher absorbance per unit concentration for bacteria lacking ProP probably resulted from their lower hydration and higher refractive index, although differences in cell size and/or shape could also alter this relationship. The activities of transporters ProU and BetU neither counteracted this effect nor stimulated bacterial growth to the level attained in the presence of ProP. Nevertheless, the growth rate of strain WG695 (
proP
proU) in urine (measured as 0·29 h-1) was much higher than that of strain HU734 (proP+ proU+) in urea-free minimal medium of comparable osmolality, without or with GB (approx. 0·15 and 0·24 h-1, respectively; Fig. 1
and Culham et al., 1998
). System BetU may have contributed to this unexpectedly rapid growth.
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Contributions of RpoS and osmoprotectant uptake to bacterial colonization of the murine urinary tract
The virulence of the two pyelonephritis isolates and their derivatives was assessed with the murine model of ascending urinary tract infection using rat faecal isolate E. coli BJ4 (Krogfelt et al., 2000 ) as a control. For these experiments, mice were inoculated transurethrally with a dense bacterial suspension (approx. 109 ml-1), obviating requirements for ascent of the urethra and proliferation within the urethra and bladder. All three strains persisted in the murine urinary tract post-inoculation (Table 5
, Fig. 6
). The numbers of bacteria recovered from day 1 urine samples and day 1 and 3 bladder homogenates of animals inoculated with the pyelonephritis isolates were significantly higher than those recovered after inoculation with faecal isolate BJ4 (numbers labelled A in Table 5
). Colonization of the urinary tract by strains CFT073 and HU734 was similar despite their differences in osmotolerance. Significant decreases in numbers of bacteria recovered from urine and bladder homogenates, but not from kidney homogenates, were observed from day 1 to day 3 for the pyelonephritis isolates. The numbers of bacteria recovered from the kidneys were low and did not vary significantly among the infecting strains. Deletion of proP, proU and/or rpoS had little impact on colonization of the murine urinary tract for the pyelonephritis isolates (Fig. 6
and data not shown). Lesion rpoS::Tn10 was also without effect on urinary tract colonization by faecal isolate BJ4 (data not shown).
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DISCUSSION |
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As it ascends the urinary tract and invades host tissues, E. coli must adapt to stresses imposed by that environment. The high osmolality, high urea concentration, low pH and organic acid content of urine are widely believed to challenge the growth and survival of E. coli within mammalian urinary tracts (Donnenberg & Welch, 1996 ; Kunin, 2000
; Mulvey et al., 2000
). Urea is a major contributor to urinary osmolality but, unlike other urinary osmolytes, it readily crosses the cytoplasmic membrane of E. coli (Zeidel et al., 1992
; K. I. Racher & J. M. Wood, unpublished data). Data reported here support the view that urea and other urinary osmolytes impose different stresses on E. coli (Randall et al., 1996
). The GB uptake activity of bacteria cultivated in urine with an osmolality of 0·92 mol kg-1 and a urea content of 0·44 M was much lower than that of bacteria cultivated in iso-osmolal NaCl-supplemented MOPS medium, more similar to that of bacteria cultivated in iso-osmolal NaCl- and urea-supplemented MOPS medium, and very similar to that of bacteria cultivated in iso-osmolal NaCl-, urea- and GB-supplemented MOPS medium (Table 4
). Only cytoplasmic-membrane-impermeant osmolytes induce transcription of the genes encoding ProP and ProU and activate the transporters themselves (Wood, 1999
). Growth in the presence of GB attenuates the osmotically induced transcription of both proP and proU, perhaps because GB uptake attenuates the signal to which these systems respond (Cairney et al., 1985
; Sutherland et al., 1986
). Thus the GB uptake activity of the pyelonephritis isolates correlates with the medium osmolality due to membrane-impermeant osmolytes and the availability of GB as an osmoprotectant during growth, phenomena consistent with the behaviour of E. coli K-12 (Wood, 1999
). Despite the failure of urea to stimulate GB uptake, betaine accumulation invoked by osmotic stress can attenuate the growth-inhibitory effects of urea (Randall et al., 1996
). Thus, osmotic-stress-induced betaine accumulation may be significant as an antidote to urea toxicity for urinary tract bacteria.
The urinary osmolality contributed by solutes other than urea is not particularly high, even for unusually concentrated urine. For 0·9 mol kg-1 urine, contributed by volunteers after 14 h food and water deprivation, it is comparable to that of MOPS minimal medium supplemented with only approximately 0·13 M NaCl. In the absence of urea, addition of 0·13 M NaCl to MOPS medium does not inhibit the growth of E. coli CFT073 but it does reduce the growth rate of E. coli HU734 (Fig. 1). The slower growth of HU734 is not likely due to the impact of its rpoS defect on trehalose accumulation, since an rpoS deletion had no effect on the growth of strain CFT073 under these conditions (Fig. 1
, strains CFT073 and WG745). Among the other osmoregulatory systems known through studies of E. coli K-12 (Table 1
), only K+ transporter Trk would be expected to contribute to osmotolerance under these conditions (no organic osmoprotectant, K+ at approximately 75 mM). Further experimentation will determine whether E. coli HU734 is deficient in Trk and whether such a difference can account for its limited ability to tolerate low salinities.
Urine contains betaines and transporters ProP and ProU mediate betaine-based osmoprotection for E. coli K-12. It was therefore suggested that ProP and/or ProU may contribute to the virulence of UPEC (Chambers & Lever, 1996 ; Culham et al., 1994
, 1998
). ProP and ProU are widespread among E. coli isolates (Culham et al., 1994
) and they do contribute to GB uptake and osmotolerance for pyelonephritis isolates HU734 and CFT073 (Fig. 1
and Tables 4
and 5
) (Culham et al., 1998
). However, deletion of proP and proU did not eliminate osmoprotection by betaine and it only partly attenuated growth in high-osmolality human urine for E. coli HU734 (Fig. 5a
). Deletion of proP and proU eliminated osmoprotection but did not attenuate growth in high-osmolality urine for E. coli CFT073 (Fig. 5b
). We therefore sought evidence that other osmoregulatory mechanisms contribute to the growth of these bacteria in human urine.
The failure of proP and proU defects to attenuate the growth of strain CFT073 in high-osmolality human urine did not result from trehalose accumulation since a further rpoS deletion also failed to attenuate growth under those conditions [compare strains WG696 (proP
proU rpoS+) and WG746 (
proP
proU
rpoS), Fig. 5b
]. Strain HU734 is less osmotolerant than strain CFT073 in the absence of organic osmoprotectants (Fig. 1
) but it harbours a betaine transporter, BetU, which is not detected in strains K-12 or CFT073 (Table 4
). Like ProP and ProU, BetU is likely to ameliorate the effects of high osmolality and urea content on the growth of E. coli HU734 in urine. The genetic locus encoding BetU will now be identified and effects of its deletion on growth of E. coli HU734 in urine determined.
In addition to ProP and ProU, system BetTIBA mediates choline uptake and oxidation, leading to osmoprotection of E. coli K-12 by GB (Table 1). BetTIBA is functional in strains HU734 and CFT073 as indicated by PCR-based detection of the betT locus and osmoprotection by choline (D. E. Culham & J. M. Wood, unpublished data). Choline is present in human urine at levels (approx. 30 µM) (Altman, 1961
) above the Km of BetT (8 µM) (Styrvold et al., 1986
), but O2, required for the oxidation of choline to GB by BetBA, may be limiting in the urinary tract environment. Since choline can serve as an osmoprotectant in vitro for both HU734 and CFT073, it is unlikely that the different effects of proP and proU deletions on growth of strains HU734 and CFT073 in urine reflect the compensatory activity of system BetTIBA. Nevertheless, the possibility that this system contributes to growth of the pyelonephritis isolates in high-osmolality urine must be considered. To determine whether a urinary constituent other than GB provides osmoprotection and/or urea protection to E. coli CFT073 and its derivatives, we tested that strain for osmoprotection by diverse organic compounds (D. E. Culham, A. Lu & J. M. Wood, unpublished data). Only choline was growth-stimulatory.
Taken together, these results suggest that E. coli strains K-12 and CFT073 possess an osmoregulatory mechanism distinct from trehalose or betaine accumulation, not shared by E. coli HU734, that confers osmotolerance at the low-salinity characteristic of human urine (perhaps a K+ accumulation mechanism). In contrast, only strain HU734 contains organic osmoprotectant transporter BetU. Past research has revealed broad variations in osmotolerance among naturally occurring E. coli isolates (Kunin et al., 1992 ). Perhaps the differences between pyelonephritis isolates HU734 and CFT073 identified here represent naturally occurring variations in osmotic stress tolerance.
This research was designed to assess the contribution of betaine accumulation mediated by transporters ProP and ProU to the virulence of UPEC. The virulence of bacteria lacking ProP and/or ProU was assessed by examining bacterial growth in urine and applying the murine model for ascending urinary tract infection. In contrast to our previous report on system ProP (Culham et al., 1998 ), these defects had only minor effects on the recovery of bacteria from murine urine, bladders or kidneys, 1 or 3 d post-transurethral inoculation with either E. coli HU734 or E. coli CFT073 (Table 5
; Fig. 6
). Since ProP and ProU are the sole mediators of betaine uptake by strain CFT073, we can conclude that betaine accumulation is not essential for colonization of the urinary tract by that organism within this experimental model. The analogous conclusion cannot yet be reached for E. coli HU734 since it retains system BetU. Osmoregulatory betaine accumulation accelerates growth in high-osmolality media (Fig. 1
). Betaine accumulation is thus most likely to affect virulence by stimulating bacterial proliferation in the urethra and bladder prior to attachment and invasion of the bladder epithelium (Mulvey et al., 2000
; Struve & Krogfelt, 1999
). The requirement for this growth stimulation may be bypassed when bacteria are introduced as a concentrated suspension (109 c.f.u. ml-1) to the murine bladder.
Alternative factor RpoS was of interest during this study because it mediates the transcription of some osmoregulatory genes (Table 1
) and it is required for the virulence of some pathogens (see Introduction). E. coli HU734, which is less osmotolerant (Fig. 1
) and is considered less virulent (Mobley et al., 1993
) than E. coli CFT073, expresses a defective RpoS variant (see Figs 1
, 2
, 3
and 4
). RpoS activates transcription of 50100 genes in E. coli K-12 in response to stationary phase, osmotic and other stresses (Ishihama, 2000
). In addition, RpoS can inhibit gene expression (Dove et al., 1997
; Farewell et al., 1998
). Particularly important in the current context is the indirect, negative effect of RpoS on expression of the genes encoding type 1 pili (Dove et al., 1997
). In marked contrast to E. coli K-12, both pyelonephritis isolates examined during this study expressed high levels of RpoS during both exponential- and stationary-phase growth (Fig. 2
). In view of the contribution of type 1 pili to bladder colonization, it will be interesting to determine whether cystitis isolates of E. coli also show high RpoS levels.
The murine model was used to assess the impact of an rpoS deletion on virulence for E. coli CFT073 (Fig. 6). The rpoS lesion had no significant impact on recovery of bacteria from the murine urine, bladders or kidneys 1 or 3 d post-inoculation. The failure of this lesion to influence urinary tract colonization may reflect the net outcome of both positive and negative perturbations, since RpoS mediates the expression of many genetic loci with known or suspected relevance to urovirulence (e.g. fim). It is also possible that an effect of the rpoS lesion would be detected during competitive colonization of the urinary tract. For example, an rpoS::Tn10 insertion conferred a competitive advantage on faecal isolate BJ4 during colonization of the murine intestine (Krogfelt et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 12 October 2000;
revised 16 January 2001;
accepted 31 January 2001.