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PubMed1978

Cross-protection of mice provided by active and passive immunization against experimental infections with virulent Proteus rettgeri and Providencia bacteria.

Immunization with Providencia and Proteus rettgeri Formalin-treated bacterial suspensions produced high levels of protection in mice against homologous and heterologous challenge. Mice were also cross-protected, but less effectively, by passive administration of rabbit type-specific antisera. The protective activity appeared to be due to an antigen common to strains of different O-serotypes. It was not detectable in agglutination reactions, and preliminary results indicate that it is thermostable, not being inactivated in its antibody binding capacity at 121 degrees C for 1 h.

Animals↗
PubMed1982

Urinary virulence of Proteus mirabilis in two experimental mouse models.

Two experimental mouse models were tested for their suitability in measuring urinary virulence of Proteus mirabilis. In the first model, the kidney-infecting dose and lethal dose were measured. In the second model, the kinetics of the numbers of bacteria in the kidneys and other organs of the mouse were monitored for 13 h after injection.

Animals↗
PubMed1984

Vero cell invasiveness of Proteus mirabilis.

Vero cell invasiveness was studied for a group of Proteus mirabilis strains isolated from the urinary tract and feces and for a limited group of urinary isolates of Escherichia coli. Experimental conditions affecting this invasiveness were studied. All of the P. mirabilis strains tested were capable of cell invasion, whereas none of the E. coli strains was. Correlation between the hemolytic activity of the P. mirabilis strains and their invasive ability suggested that the bacterial hemolysin may be involved in the invasion process. Other experimental evidence supporting this hypothesis is discussed. The differences in the invasive capacities of P. mirabilis and of E. coli may be important for the apparent differences in the pathogenesis of urinary tract infection by both species.

Animals↗
PubMed1992

Ability of Proteus mirabilis to invade human urothelial cells is coupled to motility and swarming differentiation.

Proteus mirabilis causes serious kidney infections which can involve invasion of host urothelial cells. We present data showing that the ability to invade host urothelial cells is closely coupled to swarming, a form of cyclical multicellular behavior in which vegetative bacteria differentiate into hyperflagellated, filamentous swarm cells capable of coordinated and rapid population migration. Entry into the human urothelial cell line EJ/28 by P. mirabilis U6450 isolated at different stages throughout the swarming cycle was measured by the antibiotic protection assay method and confirmed by electron microscopy. Differentiated filaments entered urothelial cells within 30 min and were 15-fold more invasive (ca. 0.18% entry in 2 h) than an equivalent dry weight of vegetative cells isolated before differentiation, which attained only ca. 0.012% entry in the 2-h assay. The invasive ability of P. mirabilis was modulated in parallel with flagellin levels throughout two cycles of swarming. Septation and division of intracellular swarm cells produced between 50 and 300 vegetative bacteria per human cell, compared with 4 to 12 intracellular bacteria after incubation with vegetative cells. Transposon (Tn5) mutants of P. mirabilis with specific defects in motility and multicellular behavior were compared with the wild-type for the ability to invade. Mutants which lacked flagella (nonmotile nonswarming) were entirely noninvasive, and those which were motile but defective in swarm cell formation (motile nonswarming) were 25-fold less invasive than wild-type vegetative cells. Mutants with defects in the coordination of multicellular migration and the temporal control of consolidation (cyclical reversion of swarm cells to vegetative cells) were reduced ca. 3- to 12-fold in the ability to enter urothelial cells. In contrast, a nonhemolytic transposon mutant which swarmed normally retained over 80% of wild-type invasive ability. Swarm cells and early consolidation cells were at least 10-fold more cytolytic than vegetative cells as a result of their high-level production of hemolysin.

Animals↗
PubMed1994

Construction of an MR/P fimbrial mutant of Proteus mirabilis: role in virulence in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a cause of acute pyelonephritis, produces at least four types of fimbriae, including MR/P (mannose-resistant/Proteus-like) fimbriae. To investigate the contribution of MR/P fimbriae to colonization of the urinary tract, we constructed an MR/P fimbrial mutant by allelic exchange. A 4.2-kb BamHI fragment carrying the mrpA gene was subcloned into a mobilizable plasmid, pSUP202. A 1.3-kb Kanr cassette was inserted into the mrpA open reading frame, and the construct was transferred to the parent P. mirabilis strain by conjugation. Following passage on nonselective medium, 1 of 500 transconjugants screened was found to have undergone allelic exchange as demonstrated by Southern blot. Colony immunoblot, Western immunoblot, and immunogold labeling with a monoclonal antibody to MR/P fimbriae revealed that MrpA was not expressed. Complementation with cloned mrpA restored MR/P expression as shown by hemagglutination, Western blot, and immunogold electron microscopy. To assess virulence, we challenged 40 CBA mice transurethrally with 10(7) CFU of wild-type or mutant strains. After 1 week, geometric means of log10 CFU per milliliter of urine or per gram of bladder or kidney for the wild-type and mutant strains were as follows: urine, 7.79 (wild type) versus 7.02 (mutant) (P = 0.035); bladder, 6.22 versus 4.78 (P = 0.019); left kidney, 5.02 versus 3.31 (P = 0.009); and right kidney, 5.28 versus 4.46 (P = 0.039). Mice challenged with the wild-type strain showed significantly more severe renal damage than did mice challenged with the MR/P-negative mutant (P = 0.007). We conclude that MR/P fimbriae contribute significantly to colonization of the urinary tract and increase the risk of development of acute pyelonephritis.

Animals↗
PubMed1996

Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection.

To examine the role of flagella in pathogenesis of urinary tract infection caused by Proteus mirabilis, we constructed a nonmotile, nonswarming flagellum mutant of strain WPM111 (an hpmA hemolysin mutant of strain BA6163, chosen because of its lack of in vitro cytotoxicity in renal epithelial cell internalization studies). A nonpolar mutation was introduced into the flaD gene, which encodes the flagellar cap protein. This mutation does not affect the synthesis of flagellin but rather prevents the assembly of an intact flagellar filament. In in vitro assays, the genetically characterized nonmotile mutant was found to be internalized by cultured human renal proximal tubular epithelial cells in numbers less than 1% of those of the flagellated parent strain. Internalization of the nonmotile mutant was increased significantly (14- to 21-fold) by centrifugation onto the monolayer. To assess virulence in vivo, CBA mice were challenged transurethrally with 10(7) CFU of P. mirabilis BA6163 (wild type) (n = 16), WPM111 (hpmA mutant) (n = 46), or BB2401 (hmpA flaD mutant) (n = 46). Differences in quantitative cultures between the parent strain and the hemolysin-negative mutant were not significant. However, the hpmA flaD mutant was recovered in numbers approximately 100-fold lower than those of the hmpA mutant or the wild-type parent strain and thus was clearly attenuated. We conclude that while hemolysin does not significantly influence virulence, flagella contribute significantly to the ability of P. mirabilis to colonize the urinary tract and cause acute pyelonephritis in an experimental model of ascending urinary tract infection.

Animals↗
PubMed1998

Use of green fluorescent protein to assess urease gene expression by uropathogenic Proteus mirabilis during experimental ascending urinary tract infection.

Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made with gfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33-38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5alpha and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression of P. mirabilis virulence genes in experimental urinary tract infection.

Animals↗
PubMed1999

Serum immunoglobulin response and protection from homologous challenge by Proteus mirabilis in a mouse model of ascending urinary tract infection.

We tested the hypothesis that experimental Proteus mirabilis urinary tract infection in mice would protect against homologous bladder rechallenge. Despite production of serum immunoglobulin G (IgG) and IgM (median titers of 1:320 and 1:80, respectively), vaccinated (infected and antibiotic-cured) mice did not show a decrease in mortality upon rechallenge; the survivors experienced only modest protection from infection (mean log(10) number of CFU of P. mirabilis Nal(r) HI4320 per milliliter or gram in vaccinated mice versus sham-vaccinated mice: urine, 100-fold less [3.5 versus 5.5; P = 0.13]; bladder, 100-fold less [3.1 versus 5.1; P = 0.066]; kidneys, 40-fold less [2.7 versus 4.3; P = 0.016]). Western blots using protein from the wild-type strain and isogenic mutants demonstrated antibody responses to MR/P and PMF fimbriae and flagella. There was no correlation between serum IgG or IgM levels and protection from mortality or infection. There was a trend toward elevated serum IgA titers and protection from subsequent challenge (P >/= 0.09), although only a few mice developed significant serum IgA levels. We conclude that prior infection with P. mirabilis does not protect significantly against homologous challenge.

Animals↗
PubMed2002

Visualization of Proteus mirabilis within the matrix of urease-induced bladder stones during experimental urinary tract infection.

The virulence of a urease-negative mutant of uropathogenic Proteus mirabilis and its wild-type parent strain was assessed by using a CBA mouse model of catheterized urinary tract infection. Overall, catheterized mice were significantly more susceptible than uncatheterized mice to infection by wild-type P. mirabilis. At a high inoculum, the urease-negative mutant successfully colonized bladders of catheterized mice but did not cause urolithiasis and was still severely attenuated in its ability to ascend to kidneys. Using confocal laser scanning microscopy and scanning electron microscopy, we demonstrated the presence of P. mirabilis within the urease-induced stone matrix. Alizarin red S staining was used to detect calcium-containing deposits in bladder and kidney tissues of P. mirabilis-infected mice.

Animals↗
PubMed2003

UreR, the transcriptional activator of the Proteus mirabilis urease gene cluster, is required for urease activity and virulence in experimental urinary tract infections.

Proteus mirabilis, a cause of complicated urinary tract infection, produces urease, an essential virulence factor for this species. UreR, a member of the AraC/XylS family of transcriptional regulators, positively activates expression of the ure gene cluster in the presence of urea. To specifically evaluate the contribution of UreR to urease activity and virulence in the urinary tract, a ureR mutation was introduced into P. mirabilis HI4320 by homologous recombination. The isogenic ureR::aphA mutant, deficient in UreR production, lacked measurable urease activity. Expression was not detected in the UreR-deficient strain by Western blotting with monoclonal antibodies raised against UreD. Urease activity and UreD expression were restored by complementation of the mutant strain with ureR expressed from a low-copy-number plasmid. Virulence was assessed by transurethral cochallenge of CBA mice with wild-type and mutant strains. The isogenic ureR::aphA mutant of HI4320 was outcompeted in the urine (P = 0.004), bladder (P = 0.016), and kidneys (P < or = 0.001) 7 days after inoculation. Thus, UreR is required for basal urease activity in the absence of urea, for induction of urease by urea, and for virulence of P. mirabilis in the urinary tract.

Animals↗
PubMed2004

Use of translational fusion of the MrpH fimbrial adhesin-binding domain with the cholera toxin A2 domain, coexpressed with the cholera toxin B subunit, as an intranasal vaccine to prevent experimental urinary tract infection by Proteus mirabilis.

This is a follow-up to our previous study using an intranasal vaccine composed of MrpH, the tip adhesin of the MR/P fimbria, and cholera toxin to prevent urinary tract infection by Proteus mirabilis (X. Li, C. V. Lockatell, D. E. Johnson, M. C. Lane, J. W. Warren, and H. L. Mobley, Infect. Immun. 72:66-75, 2004). Here, we have expressed a cholera toxin-like chimera in which the MrpH adhesin-binding domain (residues 23 to 157) replaces the cholera toxin A1 ADP-ribosyltransferase domain. This chimera, when administered intranasally without additional adjuvant, is sufficient to induce protective immunity in mice.

Adhesins, Bacterial↗
PubMed2004

Proteus mirabilis genes that contribute to pathogenesis of urinary tract infection: identification of 25 signature-tagged mutants attenuated at least 100-fold.

Proteus mirabilis, a common cause of urinary tract infections (UTI) in individuals with functional or structural abnormalities or with long-term catheterization, forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis. Known virulence factors, besides urease, are hemolysin, fimbriae, metalloproteases, and flagella. In this study we utilized the CBA mouse model of ascending UTI to evaluate the colonization of mutants of P. mirabilis HI4320 that were generated by signature-tagged mutagenesis. By performing primary screening of 2088 P. mirabilis transposon mutants, we identified 502 mutants that ranged from slightly attenuated to unrecoverable. Secondary screening of these mutants revealed that 114 transposon mutants were reproducibly attenuated. Cochallenge of 84 of these single mutants with the parent strain in the mouse model resulted in identification of 37 consistently out-competed P. mirabilis transposon mutants, 25 of which were out-competed >100-fold for colonization of the bladder and/or kidneys by the parent strain. We determined the sequence flanking the site of transposon insertion in 29 attenuated mutants and identified genes affecting motility, iron acquisition, transcriptional regulation, phosphate transport, urease activity, cell surface structure, and key metabolic pathways as requirements for P. mirabilis infection of the urinary tract. Two mutations localized to a approximately 42-kb plasmid present in the parent strain, suggesting that the plasmid is important for colonization. Isolation of disrupted genes encoding proteins with homologies to known bacterial virulence factors, especially the urease accessory protein UreF and the disulfide formation protein DsbA, showed that the CBA mouse model and mutant pools are a reliable source of attenuated mutants with mutations in virulence genes.

Animals↗
PubMed1972

Positive and negative control of R-factor replication in Proteus mirabilis.

Replication of the 50 and 58 moles per cent guanine plus cytosine (%GC) components of R factor 222 in Proteus mirabilis during growth in the presence and absence of chloramphenicol and after shifting exponential- and stationary-phase cells to conditions which inhibit host protein or deoxyribonucleic acid (DNA) synthesis was examined. Chloramphenicol reduced the growth rate but increased the amount of both R-factor components; the 58% GC component showed a larger proportionate increase. This was inferred to indicate reduced synthesis of an inhibitor that acts on both R-factor components and an initiator for replication of the 50% GC component. Replicative patterns observed after shifting exponential- and stationary-phase cells grown with or without chloramphenicol to minimal medium or chloramphenicol for one generation, or puromycin for 3 hr, corroborated this interpretation. After shifts of exponential cells from either medium, replication of the 50% GC components paralleled host replication, thus indicating a requirement for protein synthesis; replication of the 58% GC component increased due to reduced inhibitor synthesis. R-factor DNA remained constant after shifting stationary cells from drug-free medium, thus indicating that the cells contained effective concentrations of the regulatory inhibitor, whereas increased replication of the 58% GC component occurred after identical shifts of chloramphenicol-grown cells of the same chronological age. Similar responses were observed after shifts to 5 C or to medium containing streptomycin or tetracycline. Absence of replication of the 50% GC component after shifting to medium containing nalidixic acid or phenethanol and its hereditary persistence during growth indicated that the 50% GC replicon was attached to the membrane. Thus, in P. mirabilis the three replicons of R factor 222 are regulated as follows: The composite and transfer portion (RTF) replicons represented by the 50% GC component require protein synthesis and membrane attachment and are negatively regulated by an inhibitor; the 58% GC or resistance-determinants replicon exists cytoplasmically and is subject only to negative control.

Alcohols↗
PubMed1972

Replication of the R factor Rts1 in Proteus mirabilis.

The replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density-gradient centrifugation. The proportion of Rts1 deoxyribonucleic acid (DNA) relative to the host chromosomal DNA (% R-DNA) was 7% in both exponential and stationary growth phases in Penassay Broth and supplemented M9 minimal medium at 30 C. The chromosomal DNA content per cell varied over a threefold range in the different growth media. In agreement with previous genetic observations, the replication of Rts1 was found to be temperature-sensitive and Rts1 DNA was diluted from the cells during exponential growth at 42 C. (14)N-(15)N medium transfer experiments have shown that individual copies of Rts1 are selected at random for replication during the duplication of the multicopy episome pool.

Ammonium Chloride↗
PubMed1972

Effect of thymine limitation of chromosomal deoxyribonucleic acid synthesis in Proteus mirabilis.

The effects of thymine limitation on the rates of growth, deoxyribonucleic acid (DNA) synthesis, and increase in viable cell number for a thymine auxotroph of Proteus mirabilis were investigated. At thymine concentrations of 1.0 mug/ml and below, these rates were markedly decreased. After a reduction in thymine concentration from 10 mug/ml to 0.2 mug/ml, mass synthesis continued at the preshift rate for several hours. In contrast, the rate of DNA synthesis immediately decreased, resulting in a decrease in the DNA to mass ratio to about one-half of its normal level. Viable counts remained constant for several hours after the reduction in thymine concentration, and enlarged cells and multicellular "snakes" were formed. The rate of DNA synthesis was reduced at thymine concentrations below approximately 1.7 mug/ml. The addition of thymine to cultures which had been completely starved for thymine increased the rate of DNA synthesis to at least twice its normal value; this suggests that extra rounds of chromosome replication can be induced in P. mirabilis as previously observed in Escherichia coli.

Chromosomes, Bacterial↗
PubMed1972

Murein and the outer penetration barrier of Escherichia coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa.

A penetration barrier operating outside the periplasmic enzyme penicillinase was studied in an ampicillin-resistant mutant of Escherichia coli K-12. Growth in the presence of lysozyme and sublethal concentrations of ampicillin partially opened the barrier. This could be recorded as an increased penetration of penicillin G, sodium cholate, and rifampin to their respective targets. Brief treatments with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid and sodium cholate effectively impaired the barrier against penicillin and also caused leakage of penicillinase. Wild-type E. coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa also showed an increased sensitivity to cholate after treatment with penicillins. Electron micrographs showed that lysis by cholate was due to a distortion of the cytoplasmic membrane causing a leakage of protein and RNA from the cells to the medium. Physiological data indicated that the increased sensitivity to cholate induced by growth in the presence of ampicillin or lysozyme was due to effects upon the murein. This was supported by measurement of the incorporation of (3)H-diaminopimelic acid. These results indicate that the murein sacculus either is a part of the penetration barrier or is responsible for holding the structure of the outer membrane together.

Ampicillin↗
PubMed1973

R-factor-mediated resistance to tetracycline in Proteus mirabilis.

The expression of R-factor-mediated resistance to tetracycline has been compared in Proteus mirabilis and Escherichia coli. Resistance to a range of concentrations of tetracycline was significantly lower in P. mirabilis than in E. coli in both induced and repressed states. Indirect evidence showed that conditions which result in a marked increase in the level of resistance of P. mirabilis harboring the R factor NR1 to chloramphenicol, streptomycin, and spectinomycin due to an amplification in the number of copies of r-determinants per cell do not detectably increase the level of resistance to tetracycline. Tetracycline resistance was inducible in early stationary-phase P. mirabilis NR1 although not after 5 h in this state. Double isotope labeling of control and tetracycline-induced P. mirabilis NR1 in early stationary phase revealed isotopic enrichment of certain peaks in extracts from induced cells subjected to polyacrylamide gel electrophoresis.

Acetyltransferases↗
PubMed1991

Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.

O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover.

Acetates↗