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Anomalous expression of the E. coli lac operon in Proteus mirabilis. I. Effects of L8 and L8 UV5.

The lac operon shows anomalous expression in Proteus mirabilis: the maximal induced level is 10% or less of that in E. coli, while repression reduces this by a factor of only 2-5. We have sought to determine whether this effect relates in any way to CRP-mediated activation of expression, by comparing expression in P. mirabilis of lac operons (introduced for technical reasons on IncP1 plasmids) either regulatorily wild-type or bearing L8 or L8UV5. Derivatives of RP1 bearing L8UV5 were obtained by homogenotisation of pGC9114 (RP1::Tn951) in a L8UV5 background; while derivatives of RP4 bearing lac+, L8 or L8UV5 were obtained by Mu-mediated translocation of chromosomal regions bearing these alleles, following partial heat-induction of Mucts62 on pGM14 (RP4::Mucts62) in the appropriate hosts. These plasmids could be readily transferred to, and stably maintained in, the P. mirabilis strains employed. It was found that L8 reduced the maximal level of beta-galactosidase activity, and L8UV5 restored this activity to around wild-type, in P. mirabilis quantitatively very much as in E. coli. Nevertheless, the low maximal level of expression and high basal level characteristic of the former host were unchanged. The simplest explanation of these results is that P. mirabilis contains a protein that mimics the E. coli CRP protein in interacting with the appropriate DNA binding site and thereby stimulating transcription; and that the anomalous regulation of lac in this host is unconnected with the CRP system.

Escherichia coli↗

The recA-recBCD dependent recombination pathways of Serratia marcescens and Proteus mirabilis in Escherichia coli: functions of hybrid enzymes and hybrid pathways.

The physical maps of cloned recBCD gene regions of Serratia marcescens and Proteus mirabilis were correlated to genes located in this region. The genes thyA, recC, recB, recD and argA were organized as in Escherichia coli. The 3 rec genes code for the 3 different subunits of the RecBCD enzyme and produced enzymes promoting recombination and repair of UV damage in E coli. The recBCD-dependent stimulation of recombination at specific nucleotide sequences called Chi (Chi-activation) was determined in lambda red-gam-crosses. Chi-activation by the different RecBCD enzymes decreased in the order E coli greater than S marcescens greater than P mirabilis. In E coli cloned subunits genes from S marcescens and P mirabilis led to the formation of functional hybrid enzymes consisting of subunits from 2 or even 3 species. The origin of the RecC subunit present in the hybrid enzymes affected the degree of Chi-activation. Further, changes in Chi-activation occurred when the RecD subunit in the enzyme from E coli was replaced by RecD proteins from S marcescens or P mirabilis. This suggested that the RecD subunit determines not only whether or not Chi-activation is possible but also to which extent it occurs. Finally we have reconstituted recombination pathways of S marcescens and P mirabilis by combining the cloned recA and recBCD genes from these species in E coli deleted for recA and recBCD. Both pathways can efficiently promote recombination and repair. Studies are summarized which showed that levels of repair and recombination promoted by the recA-recBCD genes are mostly higher when the recA and recBCD genes came from the same species than from 2 different species (hybrid RecBCD recombination pathway). The data are interpreted to provide evidence that in vivo the RecA protein co-operates with the RecBCD enzyme in recombination and repair of UV damage.

Chromosome Mapping↗

Isolation and characterisation of dog uropathogenic Proteus mirabilis strains.

Proteus mirabilis strains isolated from the urine of dogs with urinary tract infections, were characterised with respect to the production of haemolysin and fimbriae. In contrast to healthy dogs, P. mirabilis was also isolated in high numbers from the faeces of dogs suffering from recurrent urinary tract infections. Production of fimbriae was demonstrated by electron microscopy and the presence of genes for two different types of major fimbrial subunits (MR/P-like or UCA-like) was demonstrated by Southern hybridisation. These genes were absent in the Proteus vulgaris, Providentia rettgeri and Morganella morganii strains tested. All but one P. mirabilis strains were haemolytic and most strains produced fimbriae albeit in different amounts. The UCA fimbrial subunits from dog and human isolates have identical molecular weights and N-terminal sequences and are immunologically cross reactive. It was concluded that dog uropathogenic P. mirabilis strains are very similar to human uropathogenic P. mirabilis strains.

Amino Acid Sequence↗

Structure of the O-polysaccharide of Proteus mirabilis CCUG 10705 (OF) containing an amide of D-galacturonic acid with L-alanine.

The structure of the O-polysaccharide of Proteus mirabilis CCUG 10705 (OF) was determined by chemical analyses along with one- and two-dimensional (1)H and (13)C NMR spectroscopy. The polysaccharide was found to contain an amide of D-galacturonic acid with L-alanine and based on the uniqueness of the O-polysaccharide structure and serological data, it was suggested to classify P. mirabilis OF into a new separate Proteus serogroup, O74. A weak cross-reactivity of P. mirabilis OF and P. mirabilis O5 was observed and accounted for by a similarity of their O-repeating units. The following structure of the polysaccharide of P. mirabilis OF was established: [chemical structure: see text]

Acetylation↗

Triclosan loaded ureteral stents decrease proteus mirabilis 296 infection in a rabbit urinary tract infection model.

PURPOSE: Infection and encrustation remain major limitations of ureteral stent use and to our knowledge no device has completely overcome these obstacles to date. Triclosan is a biocide currently used in a plethora of consumer and medical products that has recently been loaded into a ureteral stent. Using a rabbit model of UTI we examined the effects of triclosan impregnated stent segments on the growth and survival of Proteus mirabilis, a uropathogen commonly associated with device related UTI and encrustation. MATERIALS AND METHODS: A total of 48 male New Zealand White rabbits were instilled transurethrally with 1 x 10(6) P. mirabilis 296. A stent curl from a triclosan eluting, Percuflex Plus or Optima ureteral stent was placed intravesically. Urine was cultured on days 1, 3 and 7. On day 7 the stents were assessed for encrustation and viable organisms, while the bladders were scored for the degree of inflammation. RESULTS: Throughout the study urine isolated from the triclosan group contained significantly fewer viable organisms than controls with 7 of 13 animals completely clearing the infection by day 7. Similarly 9 of 13 triclosan eluting stents showed no viable organisms upon recovery and the remaining 4 showed significantly fewer organisms than controls. Urine and stents in all controls were positive for P. mirabilis at all time points. Although there was no significant difference in encrustation among the groups, bladders harvested from the triclosan group demonstrated significantly less inflammation. CONCLUSIONS: Triclosan eluting stents greatly decreased P. mirabilis growth and survival in a rabbit UTI model compared to controls. These stents may prove useful for decreasing device related P. mirabilis UTIs.

Animals↗

The XerC recombinase of Proteus mirabilis: characterization and interaction with other tyrosine recombinases.

XerC and XerD are two site-specific recombinases, which act on different sites to maintain replicons in a monomeric state. This system, which was first discovered and studied in Escherichia coli, is present in several species including Proteus mirabilis, where the XerD recombinase was previously characterized by our laboratory. In this paper, we report the presence of the xerC gene in P. mirabilis. Using in vitro reactions, we show that the two P. mirabilis recombinases display binding and cleavage activity on the E. coli dif site and the ColE1 cer site, together or in collaboration with E. coli recombinases. In vivo, P. mirabilis XerC and XerD are able to resolve and monomerize a plasmid containing two cer sites, increasing its stability. However, P. mirabilis XerC, in combination with E. coli XerD, is unable to perform these functions.

Amino Acid Sequence↗

Vaccines for Proteus mirabilis in urinary tract infection.

Proteus mirabilis is a documented cause of urinary tract infection (UTI) in the complicated urinary tract. Urease-mediated urea hydrolysis is responsible for both virulence of the organism and the ability to cause urolithiasis. A urease-negative mutant of P. mirabilis is unable to initiate stone formation and colonizes the kidney at a significantly lower rate. The considerable pathology caused by P. mirabilis warrants the development of a vaccine. We have initiated the advancement of vaccine studies and have determined that the MR/P fimbria, a surface adhesin of P. mirabilis, is a promising vaccine candidate. Successful vaccination would be expected both to prevent colonization by P. mirabilis and urolithiasis.

Bacterial Vaccines↗

Structural and serological studies on the O-antigen of Proteus mirabilis O14, a new polysaccharide containing 2-[(R)-1-carboxyethylamino]ethyl phosphate.

An O-specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O14 lipopolysaccharide (LPS) and found to contain D-galactose, 2-acetamido-2-deoxy-D-glalactose, phosphate, N-(2-hydroxyethyl)-D-alanine (D-AlaEtn), and O-acetyl groups. Studies of the initial and O-deacetylated polysaccharides using one- and two-dimensional 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, NOESY, H-detected 1H,13C heteronuclear multiple-quantum coherence, and heteronuclear multiple-bond correlation experiments, demonstrated the following structure of the repeating unit: [equation: see text] This is the second bacterial polysaccharide reported to contain alpha-D-Galp6PAlaEtn, whereas the first one was the O-antigen of P. mirabilis EU313 taken erroneously as strain PrK 6/57 from the O3 serogroup [Vinogradov, E. V., Kaca, W., Shashkov, A.S., Krajewska-Pietrasik, D., Rozalski, A., Knirel, Y.A. & Kochetkov, N.K. (1990) Eur. J. Biochem., 188, 645-651]. Anti-(P. mirabilis O14) serum cross-reacted with LPS of P. mirabilis EU313 and vice versa in passive hemolysis and ELISA. Absorption of both O-antisera with the heterologous LPS decreased markedly but did not abolish the reaction with the homologous LPS. These and chemical data indicated that both strains have similar but not identical O-antigens. Therefore, we propose that P. mirabilis EU313 should belong to a new subgroup of the O14 serogroup.

Carbohydrate Conformation↗

Swarming of Proteus mirabilis over ureteral stents: a comparative assessment.

BACKGROUND AND PURPOSE: Encrustation on indwelling ureteral stents is commonly related to the presence of urease-producing bacteria that elevate the pH of the urine through the hydrolysis of urea, resulting in the precipitation of calcium and magnesium salts. Using a model previously shown to measure accurately the ability of Proteus mirabilis to swarm over catheter surfaces (Eur J Clin Microbiol Infect Dis 1999;18:206), we investigated the ability of this organism to swarm over three ureteral stents with potential encrustation-resistance properties. MATERIALS AND METHODS: Three commercially available ureteral stents were selected for evaluation: a low surface-energy stent, a hydrogel-coated stent, and a silicone stent. Ten-microliter aliquots of a 4-hour culture of P. mirabilis 296 in Trypticase soya (TSA) broth was inoculated 5 mm from a 1-cm channel cut out from TSA plates. Ten-millimeter stent sections were placed as bridges across the central channel adjacent to the inocula. Time to pathogen crossing was measured. RESULTS: The mean time (+/- SD) to pathogen migration across the three test materials was 15.9 +/- 6.1, 19.8 +/- 9.5, and 29.7 +/- 14.3 hours for the low surface-energy, hydrogel-coated, and silicone stents, respectively. Statistical analysis revealed significant differences between the crossing times of the low surface-energy (P = 0.001) and hydrogel-coated (P = 0.034) stents compared with silicone but not between the low surface-energy and hydrogel-coated stents (P = 0.387). CONCLUSION: Migration of P. mirabilis 296 across silicone stents was significantly reduced compared with low surface-energy and hydrogel-coated stents. These findings suggest that P. mirabilis may have a lower affinity for silicone stents, which may translate into a reduced risk of infection with P. mirabilis and associated stent encrustation.

Equipment Contamination↗

Genotyping demonstrates that the strains of Proteus mirabilis from bladder stones and catheter encrustations of patients undergoing long-term bladder catheterization are identical.

PURPOSE: We established the incidence of bladder stones in patients who experienced recurrent encrustation and blockage of indwelling bladder catheters and examined the relationship between isolates of Proteus mirabilis from the stones and from the crystalline biofilms on the catheters. MATERIALS AND METHODS: The first 100 patients attending a clinic for patients experiencing problems with the management of long-term bladder catheters were studied. Flexible cystoscopy was used to detect bladder stones. Catheter encrustation was assessed visually and by electron microscopy. Bacteriological analysis was performed on the stones and catheter biofilms. P. mirabilis isolates were genotyped by pulsed field gel electrophoresis of restriction enzyme digests of bacterial DNA. RESULTS: Most patients (85%) had been referred because of catheter blockage and in 61 (72%) the catheters were encrusted. P. mirabilis was recovered from 37 of 47 encrusted catheters (79%) that were examined but not from any nonencrusted catheters. Of the 61 patients with encrusted catheters 38 (62%) had bladder stones. Pairs of isolates of P. mirabilis from the stones and the catheter biofilms from 6 patients were genotyped. The DNA profiles of each pair of isolates were identical. CONCLUSIONS: The majority of patients (62%) with recurrent catheter encrustation had bladder stones. The stones harbored the strains of P. mirabilis that rapidly colonize replacement catheters with crystalline biofilm. Flexible cystoscopy to detect and remove stones might help resolve the problem of recurrent catheter encrustation.

Adult↗

Proteus morgani is less frequently associated with urinary tract infections than Proteus mirabilis--an explanation.

The metabolic activities of faecal and urinary strains of Proteus morgani and P. mirabilis were compared. Regardless of origin, the generation time of P. morgani strains in urine was approximately twice as long as that of the P. mirabilis strains. Urease synthesis was constitutive in P. morgani strains but required induction with urea in the P. mirabilis strains. In the presence of urea, the P. mirabilis strains liberated ammonia more rapidly and produced alkaline conditions more quickly than P. morgani strains, although they synthesized much less urease. These characteristics may place P. morgani strains at a disadvantage in comparison with P. mirabilis strains in their ability to cause urinary tract infections.

Ammonia↗

Role of RsmA in the regulation of swarming motility and virulence factor expression in Proteus mirabilis.

Swarming by Proteus mirabilis involves differentiation of typical short vegetative rods into filamentous hyper-flagellated swarm cells that undergo cycles of rapid and co-ordinated population migration across surfaces and exhibit high levels of virulence gene expression. RsmA (repressor of secondary metabolites) and CsrA, its homologue in Escherichia coli, control many phenotypic traits, such as motility and pathogenesis in Erwinia species, glycogen biosynthesis, cell size and biofilm formation in Escherichia coli and swarming motility in Serratia marcescens. To investigate the role of RsmA in Proteus mirabilis, the rsmA gene from Proteus mirabilis (hereafter referred to as rsmA(Pm)) was cloned. RsmA(Pm) showed high sequence similarity to Escherichia coli CsrA and RsmA cloned from Erwinia carotovora subsp. carotovora, Serratia marcescens, Haemophilus influenzae and Bacillus subtilis and could complement an Escherichia coli csrA mutant in glycogen synthesis. A low-copy-number plasmid carrying rsmA(Pm) expressed from its native promoter caused suppression of swarming motility and expression of virulence factors in Proteus mirabilis. mRNA stability assays suggested that RsmA(Pm) inhibited virulence factor expression through promoting mRNA degradation. RsmA homologues cloned from Serratia marcescens and Erwinia carotovora subsp. carotovora could also inhibit swarming and virulence factor expression in Proteus mirabilis.

Amino Acid Sequence↗

Proteus mirabilis fimbriae (PMF) are important for both bladder and kidney colonization in mice.

Proteus mirabilis expresses different types of fimbriae simultaneously. Several fimbrial types have been described and their role in the colonization of the urinary tract is under study. Previously, P. mirabilis fimbriae (PMF) have been shown to be associated with bacterial colonization of the lower urinary tract but not of the kidneys. In this study, a pmfA mutant was generated and used in several in vivo and in vitro studies. Two different urinary tract infection models in the mouse and two in vitro assays of bacterial adhesion to uroepithelial cells were performed. Expression of PmfA in a collection of P. mirabilis strains of different sources was also assessed. The results shown here indicate that PMF are involved in both bladder and kidney colonization by P. mirabilis and that these fimbriae are widely distributed among P. mirabilis isolates from different origins since all strains tested expressed PmfA.

Animals↗

Proteus mirabilis bloodstream infections: risk factors and treatment outcome related to the expression of extended-spectrum beta-lactamases.

Bloodstream infection (BSI) due to Proteus mirabilis strains is a relatively uncommon clinical entity, and its significance has received little attention. This study was initiated to evaluate risk factors and treatment outcome of BSI episodes due to P. mirabilis producing extended-spectrum beta-lactamases (ESBLs). Twenty-five BSI episodes caused by P. mirabilis occurred at our hospital (Ospedale di Circolo e Fondazione Macchi, Varese, Italy) over a 7.5-year period. Phenotypic and molecular methods were used to assess ESBL production. Clinical records of BSI patients were examined retrospectively. Demographic data, underlying diseases (according to McCabe and Jackson classification and Charlson weighted index), risk factors, and treatment outcome were investigated by comparing cases due to ESBL-positive strains to cases due to ESBL-negative strains. Eleven isolates were found to express ESBLs (TEM-52 or TEM-92). The remaining 14 isolates were ESBL negative and were uniformly susceptible to extended-spectrum cephalosporins and monobactams. Comparison of the two groups showed that previous hospitalization in a nursing home (P = 0.04) and use of bladder catheter (P = 0.01) were significant risk factors for infections due to ESBL-positive strains. In addition, cases due to ESBL-positive strains showed a significantly higher mortality attributable to BSI (P = 0.04). BSI cases due to ESBL-negative isolates uniformly responded to therapy, whereas 5/11 cases due to ESBL-positive isolates failed to respond (P < 0.01). Use of carbapenems was associated with complete response independently of ESBL production. Therapeutic failure and mortality may occur in BSI episodes caused by ESBL-positive P. mirabilis isolates. Thus, recognition of ESBL-positive strains appears to be critical for the clinical management of patients with systemic P. mirabilis infections.

Aged↗

Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli.

Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

Animals↗

Attachment of Proteus mirabilis to human urinary sediment epithelial cells in vitro is different from that of Escherichia coli.

The in vitro attachment of 335 Proteus mirabilis strains from various human sources to human urinary tract epithelial cells was measured. No significant difference in adhesive capacity was found between P. mirabilis strains isolated from the blood of 89 patients with bacteremia, the stools of 36 healthy subjects and 56 patients with diarrhea, and the urine of 62 adults and 92 children with bacteriuria. High mean adhesion values were observed in all groups. The P. mirabilis strains attached only to squamous cells and not to transitional epithelial cells, whereas most of the Escherichia coli strains tested attached to both cell types; strains from patients with acute pyelonephritis attached more often than those from patients with acute cystitis or asymptomatic bacteriuria. The attachment of P. mirabilis to squamous epithelial cells was high about day 15 of the menstrual cycle of the epithelial cell donor, but low at the beginning and the end of the cycle. In contrast, the attachment of E. coli to squamous and transitional epithelial cells did not vary significantly with the menstrual cycle of the cell donor. Differences in adhesion characteristics of E. coli and P. mirabilis may relate to the differences in clinical appearance of urinary tract infections produced by the two organisms.

Epithelial Cells↗

Proteus mirabilis fimbriae: identification, isolation, and characterization of a new ambient-temperature fimbria.

Urinary tract infections involving Proteus mirabilis may lead to complications including bladder and kidney stones, acute pyelonephritis, and bacteremia. This bacterium produces a number of fimbriae, two of which, MR/P fimbria and P. mirabilis fimbria, have been shown to contribute to the ability of this pathogen to colonize the bladder and kidney. We have now purified and characterized a previously undescribed fimbria of P. mirabilis, named ambient-temperature fimbria (ATF). Electron microscopy of a pure preparation and immunogold labeling of cells demonstrated that ATF was fimbrial in nature. The major fimbrial subunit of ATF has an apparent molecular weight of 24,000. The N-terminal amino acid sequence, E-X-T-G-T-P-A-P-T-E-V-T-V-D-G-G-T-I-D-F, did not show significant similarity to that of any previously described fimbrial protein. ATF was expressed by all eight P. mirabilis strains examined. Culture conditions affected expression of ATF, with optimal expression observed in static broth cultures at 23 degrees C. This fimbria was not produced by cells grown at 42 degrees C or on solid medium. Expression of ATF did not correlate with mannose-resistant/Proteus-like (MR/P) or mannose-resistant/Klebsiella-like (MR/K) hemagglutination and represents a novel fimbria of P. mirabilis.

Amino Acid Sequence↗

Unique ability of the Proteus mirabilis capsule to enhance mineral growth in infectious urinary calculi.

Struvite (MgNH4PO4.6H2O) calculi are a common complication of Proteus mirabilis urinary tract infections. Although urease is a major virulence factor in calculus formation, the polysaccharide capsule (CPS) of this organism also enhances struvite crystallization and growth in vitro (L. Clapham, R. J. C. McLean, J. C. Nickel, J. Downey, and J. W. Costerton, J. Crystal Growth 104:475-484, 1990). We obtained purified CPS, of known structure and varying anionic character, from P. mirabilis ATCC 49565 and several other organisms. Artificial urine was added to CPS, and the pH was elevated from 5.8 to 8.5 by the addition of urease or titration with 0.25 M NH4OH to induce struvite crystallization. Crystallization was measured by particle counting (Coulter counter), and the morphology (crystal habit) was examined by phase-contrast microscopy. In the presence of partially anionic P. mirabilis CPS, struvite formation occurred at a lower pH than in the absence of CPS or in the presence of other neutral, partially anionic, or anionic CPS. At pH 7.5 to 8.0, significantly more struvite crystals formed in the presence of P. mirabilis CPS than under other experimental conditions. With the exception of one polymer (curdlan) which did not bind Mg2+, enhancement of struvite formation by CPS polymers was inversely proportional to their Mg2+ binding ability. We speculate that the structure and partial anionic nature of P. mirabilis CPS enable it to enhance struvite formation by weakly concentrating Mg2+ ions during struvite crystal formation. This illustrates a new virulence aspect of bacterial CPS during infection.

Bacterial Capsules↗