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1.
mBio ; 15(7): e0104824, 2024 Jul 17.
Article in English | MEDLINE | ID: mdl-38874412

ABSTRACT

More than half of women will experience a urinary tract infection (UTI) with most cases caused by uropathogenic Escherichia coli (UPEC). Bacterial swimming motility enhances UPEC pathogenicity, resulting in more severe disease outcomes including kidney infection. Surprisingly, the connection between motility and iron limitation is mostly unexplored despite the lack of free iron available in the host. We sought to investigate a potential connection between iron restriction and regulation of motility in UPEC. We cultured E. coli CFT073, a prototypical UPEC strain, under iron limitation and observed that CFT073 had elevated fliC (flagella) promoter activity, and this iron-specific response was repressed by the addition of exogenous iron. We confirmed increased flagellar expression in CFT073 by measuring fliC transcript, FliC protein, and surface-expressed flagella under iron-limited conditions. Interestingly, known motility regulator flhDC did not have altered transcription under these conditions. To define the regulatory mechanism of this response, we constructed single knockouts of eight master regulators and found the iron-regulated response was lost in crp, arcA, and fis mutants. Thus, we focused on the five genes regulated by all three regulators. Of the five genes knocked out, the iron-regulated motility response was most strongly dysregulated in the lpdA mutant, which also resulted in significantly lowered fitness in the murine model of ascending UTI, both against the WT and a non-motile fliC mutant. Collectively, we demonstrated that iron-mediated motility in CFT073 is partially regulated by lpdA, which contributes to the understanding of how uropathogens differentially regulate motility mechanisms in the iron-restricted host. IMPORTANCE: Urinary tract infections (UTIs) are ubiquitous and responsible for over five billion dollars in associated health care costs annually. Both iron acquisition and motility are highly studied virulence factors associated with uropathogenic Escherichia coli (UPEC), the main causative agent of uncomplicated UTI. This work is innovative by providing mechanistic insight into the synergistic relationship between these two critical virulence properties. Here, we demonstrate that iron limitation has pleiotropic effects with consequences that extend beyond metabolism and impact other virulence mechanisms. Indeed, targeting iron acquisition as a therapy may lead to an undesirable enhancement of UPEC pathogenesis through increased motility. It is vital to understand the full breadth of UPEC pathogenesis to adequately respond to this common infection, especially with the increase of antibiotic-resistant pathogens.


Subject(s)
Escherichia coli Infections , Escherichia coli Proteins , Flagella , Gene Expression Regulation, Bacterial , Iron , Urinary Tract Infections , Uropathogenic Escherichia coli , Uropathogenic Escherichia coli/genetics , Uropathogenic Escherichia coli/drug effects , Uropathogenic Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Iron/metabolism , Animals , Escherichia coli Infections/microbiology , Mice , Urinary Tract Infections/microbiology , Flagella/genetics , Flagella/metabolism , Female , Virulence , Disease Models, Animal , Locomotion , Flagellin
2.
bioRxiv ; 2023 Sep 28.
Article in English | MEDLINE | ID: mdl-37808639

ABSTRACT

More than half of all women will experience a urinary tract infection (UTI) in their lifetime with most cases caused by uropathogenic Escherichia coli (UPEC). Bacterial motility enhances UPEC pathogenicity, resulting in more severe disease outcomes including kidney infection. Surprisingly, the connection between motility and iron limitation is mostly unexplored, despite the lack of free iron available in the host. Therefore, we sought to explore the potential connection between iron restriction and regulation of motility in UPEC. We cultured E. coli CFT073, a prototypical UPEC strain, in media containing an iron chelator. Under iron limitation, CFT073 had elevated fliC (flagella) promoter activity, driving motility on the leading edge of the colony. Furthermore, this iron-specific response was repressed by the addition of exogenous iron. We confirmed increased flagella expression in CFT073 by measuring fliC transcript, FliC protein, and surface-expressed flagella under iron-limited conditions. To define the regulatory mechanism, we constructed single knockouts of eight master regulators. The iron-regulated response was lost in crp, arcA, and fis mutants. Thus, we focused on the five genes regulated by all three transcription factors. Of the five genes knocked out, the iron-regulated motility response was most strongly dysregulated in an lpdA mutant, which also resulted in significantly lowered fitness in the murine model of ascending UTI. Collectively, we demonstrated that iron-mediated motility in CFT073 is regulated by lpdA , which contributes to the understanding of how uropathogens differentially regulate motility mechanisms in the iron-restricted host. Importance: Urinary tract infections (UTIs) are ubiquitous and responsible for over five billion dollars in associated health care costs annually. Both iron acquisition and motility are highly studied virulence factors associated with uropathogenic E. coli (UPEC), the main causative agent of uncomplicated UTI. This work is innovative by providing mechanistic insight into the synergistic relationship between these two critical virulence properties. Here, we demonstrate that iron limitation has pleiotropic effects with consequences that extend beyond metabolism, and impact other virulence mechanisms. Indeed, targeting iron acquisition as a therapy may lead to an undesirable enhancement of UPEC pathogenesis through increased motility. It is vital to understand the full breadth of UPEC pathogenesis to adequately respond to this common infection, especially with the increase of antibiotic resistant pathogens.

3.
mSystems ; 7(6): e0082722, 2022 12 20.
Article in English | MEDLINE | ID: mdl-36445110

ABSTRACT

For women in the United States, urinary tract infections (UTIs) are the most frequent diagnosis in emergency departments, comprising 21.3% of total visits. Uropathogenic Escherichia coli (UPEC) causes ~80% of uncomplicated UTIs. To combat this public health issue, it is vital to characterize UPEC strains as well as to differentiate them from commensal strains to reduce the overuse of antibiotics. It has been challenging to determine a consistent genetic signature that clearly distinguishes UPEC from other E. coli strains. Therefore, we examined whether phenotypic data could be predictive of uropathogenic potential. We screened 13 clinical strains of UPEC, isolated from cases of uncomplicated UTI in young otherwise healthy women, in a series of microbiological phenotypic assays using UPEC prototype strain CFT073 and nonpathogenic E. coli strain MG1655 K-12 as controls. Phenotypes included adherence, iron acquisition, biofilm formation, human serum resistance, motility, and stress resistance. By use of a well-established experimental mouse model of UTI, these data were able to predict the severity of the bacterial burden in both the urine and bladders. Multiple linear regression using three different phenotypic assays, i.e., growth in minimal medium, siderophore production, and type 1 fimbrial expression, was predictive of bladder colonization (adjusted R2 = 0.6411). Growth in ex vivo human urine, hemagglutination of red blood cells, and motility modeled urine colonization (adjusted R2 = 0.4821). These results showcase the utility of phenotypic characterization to predict the severity of infection that these strains may cause. We predict that these methods will also be applicable to other complex, genetically redundant, pathogens. IMPORTANCE Urinary tract infections are the second leading infectious disease worldwide, occurring in over half of the female population during their lifetime. Most infections are caused by uropathogenic Escherichia coli (UPEC) strains. These strains can establish a reservoir in the gut, in which they do not cause disease but, upon introduction to the urinary tract, can infect the host and elicit pathogenesis. Clinically, it would be beneficial to screen patient E. coli strains to understand their pathogenic potential, which may lead to the administration of prophylactic antibiotic treatment for those with increased risk. Others have proposed the use of PCR-based genetic screening methods to detect UPEC strains and differentiate them from other E. coli pathotypes; however, this method has not yielded a consistent uropathogenic genetic signature. Here, we used phenotypic characteristics such as growth rate, siderophore production, and expression of fimbriae to better predict uropathogenic potential.


Subject(s)
Escherichia coli Infections , Escherichia coli Proteins , Urinary Tract Infections , Female , Humans , Animals , Mice , Escherichia coli/genetics , Escherichia coli Infections/diagnosis , Siderophores , Virulence Factors/genetics , Escherichia coli Proteins/genetics , Urinary Tract Infections/diagnosis , Anti-Bacterial Agents , Phenotype
4.
Microbiology (Reading) ; 155(Pt 5): 1523-1535, 2009 May.
Article in English | MEDLINE | ID: mdl-19372157

ABSTRACT

Proteus mirabilis causes urinary tract infections (UTIs) in individuals requiring long-term indwelling catheterization. The pathogenesis of this uropathogen is mediated by a number of virulence factors and the formation of crystalline biofilms. In addition, micro-organisms have evolved complex systems for the acquisition of nutrients, including the phosphate-specific transport system, which has been shown to be important in biofilm formation and pathogenesis. A functional Pst system is important during UTIs caused by P. mirabilis HI4320, since transposon mutants in the PstS periplasmic binding protein and the PstA permease protein were attenuated in the CBA mouse model of UTI. These mutants displayed a defect in biofilm formation when grown in human urine. This study focuses on a comparison of the proteomes during biofilm and planktonic growth in phosphate-rich medium and human urine, and microscopic investigations of biofilms formed by the pst mutants. Our data suggest that (i) the Deltapst mutants, and particularly the DeltapstS mutant, are defective in biofilm formation, and (ii) the proteomes of these mutants differ significantly from that of the wild-type. Therefore, since the Pst system of P. mirabilis HI4320 negatively regulates biofilm formation, this system is important for the pathogenesis of these organisms during complicated UTIs.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/metabolism , Biofilms , Phosphate-Binding Proteins/metabolism , Proteus Infections/microbiology , Proteus mirabilis/physiology , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Electrophoresis, Gel, Two-Dimensional , Humans , Mutation , Phosphate Transport Proteins , Phosphate-Binding Proteins/chemistry , Phosphate-Binding Proteins/genetics , Phosphates/metabolism , Proteomics , Proteus mirabilis/chemistry , Proteus mirabilis/genetics
5.
Clin Microbiol Rev ; 21(1): 26-59, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18202436

ABSTRACT

Catheter-associated urinary tract infections (CAUTIs) represent the most common type of nosocomial infection and are a major health concern due to the complications and frequent recurrence. These infections are often caused by Escherichia coli and Proteus mirabilis. Gram-negative bacterial species that cause CAUTIs express a number of virulence factors associated with adhesion, motility, biofilm formation, immunoavoidance, and nutrient acquisition as well as factors that cause damage to the host. These infections can be reduced by limiting catheter usage and ensuring that health care professionals correctly use closed-system Foley catheters. A number of novel approaches such as condom and suprapubic catheters, intermittent catheterization, new surfaces, catheters with antimicrobial agents, and probiotics have thus far met with limited success. While the diagnosis of symptomatic versus asymptomatic CAUTIs may be a contentious issue, it is generally agreed that once a catheterized patient is believed to have a symptomatic urinary tract infection, the catheter is removed if possible due to the high rate of relapse. Research focusing on the pathogenesis of CAUTIs will lead to a better understanding of the disease process and will subsequently lead to the development of new diagnosis, prevention, and treatment options.


Subject(s)
Cross Infection , Escherichia coli Infections , Escherichia coli/physiology , Proteus Infections , Proteus mirabilis/physiology , Urinary Catheterization/adverse effects , Urinary Tract Infections , Adaptation, Physiological , Adhesins, Bacterial , Biofilms/growth & development , Catheterization/trends , Cross Infection/diagnosis , Cross Infection/etiology , Cross Infection/therapy , Escherichia coli Infections/diagnosis , Escherichia coli Infections/etiology , Escherichia coli Infections/therapy , Humans , Locomotion , Prognosis , Proteus Infections/diagnosis , Proteus Infections/etiology , Proteus Infections/therapy , Technology , Urinary Catheterization/standards , Urinary Tract Infections/diagnosis , Urinary Tract Infections/etiology , Urinary Tract Infections/therapy
6.
Kidney Int ; 72(1): 19-25, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17396114

ABSTRACT

P fimbria, a mannose-resistant adhesin of uropathogenic Escherichia coli (UPEC), has been shown to be associated with acute pyelonephritis. The pap gene cluster encodes the proteins required for P-fimbrial biogenesis, including papG, which encodes the tip adhesin. The three most studied PapG molecular variants, which are shown to bind distinct isoreceptors, are PapGI, -II, and -III. PapGII preferentially binds globoside, or GbO4, a glycolipid isoreceptor of the human kidney. Studies using different animal models of ascending urinary tract infection (UTI) have demonstrated a variable role for P fimbriae, and specifically PapGII-mediated adherence, in renal colonization. The disparities in the results obtained from those studies are likely to be attributed to the differences in animal models and UPEC strains utilized. One explanation that is discussed in detail is the contribution of multiple fimbriae of UPEC that potentially mediate adherence to the mammalian kidney. Overall, P fimbriae appear to play some role in mediating adherence to uroepithelial cells in vivo and establishing an inflammatory response during renal colonization, thus contributing to kidney damage during acute pyelonephritis. To verify that P fimbriae contribute to the pathogenesis of UPEC during ascending UTI (and in particular acute pyelonephritis), future studies should be conducted to satisfy fully all three tenets of the molecular Koch's postulates, including complementation of a mutated allele.


Subject(s)
Adhesins, Escherichia coli/physiology , Fimbriae Proteins/physiology , Kidney/microbiology , Pyelonephritis/physiopathology , Adhesins, Escherichia coli/analysis , Amino Acid Sequence , Animals , Escherichia coli/pathogenicity , Escherichia coli Infections/pathology , Escherichia coli Infections/physiopathology , Fimbriae Proteins/analysis , Fimbriae, Bacterial/physiology , Humans , Kidney/pathology , Kidney/physiopathology , Molecular Sequence Data
7.
Proc Natl Acad Sci U S A ; 99(26): 17020-4, 2002 Dec 24.
Article in English | MEDLINE | ID: mdl-12471157

ABSTRACT

We present the complete genome sequence of uropathogenic Escherichia coli, strain CFT073. A three-way genome comparison of the CFT073, enterohemorrhagic E. coli EDL933, and laboratory strain MG1655 reveals that, amazingly, only 39.2% of their combined (nonredundant) set of proteins actually are common to all three strains. The pathogen genomes are as different from each other as each pathogen is from the benign strain. The difference in disease potential between O157:H7 and CFT073 is reflected in the absence of genes for type III secretion system or phage- and plasmid-encoded toxins found in some classes of diarrheagenic E. coli. The CFT073 genome is particularly rich in genes that encode potential fimbrial adhesins, autotransporters, iron-sequestration systems, and phase-switch recombinases. Striking differences exist between the large pathogenicity islands of CFT073 and two other well-studied uropathogenic E. coli strains, J96 and 536. Comparisons indicate that extraintestinal pathogenic E. coli arose independently from multiple clonal lineages. The different E. coli pathotypes have maintained a remarkable synteny of common, vertically evolved genes, whereas many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain.


Subject(s)
Escherichia coli/genetics , Genome, Bacterial , Pyelonephritis/microbiology , Acute Disease , Base Sequence , Escherichia coli/pathogenicity , Female , Genetic Structures , Humans , Molecular Sequence Data , Open Reading Frames
8.
Proc Natl Acad Sci U S A ; 98(24): 13844-9, 2001 Nov 20.
Article in English | MEDLINE | ID: mdl-11717441

ABSTRACT

The antimicrobial effect of nitric oxide (NO) is an essential part of innate immunity. The vigorous host response to the human gastric pathogen Helicobacter pylori fails to eradicate the organism, despite up-regulation of inducible NO synthase (iNOS) in the gastric mucosa. Here we report that wild-type strains of H. pylori inhibit NO production by activated macrophages at physiologic concentrations of l-arginine, the common substrate for iNOS and arginase. Inactivation of the gene rocF, encoding constitutively expressed arginase in H. pylori, restored high-output NO production by macrophages. By using HPLC analysis, we show that l-arginine is effectively consumed in the culture medium by wild-type but not arginase-deficient H. pylori. The substantially higher levels of NO generated by macrophages cocultured with rocF-deficient H. pylori resulted in efficient killing of the bacteria, whereas wild-type H. pylori exhibited no loss of survival under these conditions. Killing of the arginase-deficient H. pylori was NO-dependent, because peritoneal macrophages from iNOS(-/-) mice failed to affect the survival of the rocF mutant. Thus, bacterial arginase allows H. pylori to evade the immune response by down-regulating eukaryotic NO production.


Subject(s)
Arginase/metabolism , Bacterial Proteins , Helicobacter pylori/enzymology , Nitric Oxide/biosynthesis , Animals , Arginase/genetics , Arginase/physiology , Arginine/metabolism , Cell Line , Eukaryotic Cells/metabolism , Gene Expression , Helicobacter pylori/growth & development , Helicobacter pylori/immunology , Helicobacter pylori/physiology , Interferon-gamma/pharmacology , Macrophage Activation , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Knockout , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Nitrogen Dioxide/metabolism , RNA, Messenger
9.
J Clin Microbiol ; 39(11): 3842-50, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11682496

ABSTRACT

Obstacles continue to hinder in vitro studies of the gastric human pathogen Helicobacter pylori, including difficulty culturing the organism in the absence of serum or blood, rapid loss of viability following exponential growth due to autolysis, and the necessity for using high starting inocula. We demonstrate that H. pylori grows in the chemically defined broth medium Ham's F-12 nutrient mixture (F-12) in the absence of fetal bovine serum (FBS); this represents a breakthrough for studies in which serum components or proteins interfere with interpretation of results. Cultures can be continually passaged in fresh, FBS-free F-12 medium at an initial inoculum of only approximately 10(3) CFU/ml. All H. pylori strains (n = 21), including fresh clinical isolates, grew in serum-free F-12. H. pylori grew poorly in the related medium, F-10, unless additional zinc was supplied. Enhanced growth of H. pylori in F-12 broth was obtained by addition of bovine serum albumin (BSA) (1 mg/ml), beta-cyclodextrin (200 microg/ml), or cholesterol (50 microg/ml). H. pylori also grew in several simplified versions of F-12 broth lacking glucose and most vitamins but containing hypoxanthine, pyruvate, and all 20 amino acids. On F-12 medium solidified with agar, H. pylori only grew when BSA (98% pure; 1 mg/ml), cholesterol (50 microg/ml), beta-cyclodextrin (200 microg/ml), or FBS (2 to 4%) was added; addition of urea and phenol allowed colorimetric detection of urease activity. Thus, F-12 agar plus cholesterol or beta-cyclodextrin represents the first transparent chemically defined agar and the first urease indicator agar for H. pylori. Several lines of evidence suggested that BSA itself is not responsible for H. pylori growth enhancement in F-12 containing BSA or FBS. Taken together, these innovations represent significant advances in the cultivation and recovery of H. pylori using chemically defined media. Use of F-12 or its derivatives may lead to improved understanding of H. pylori metabolism, virulence factors, and transmission, and result in improved recovery and identification of H. pylori from clinical specimens.


Subject(s)
Helicobacter pylori/growth & development , Urease/metabolism , beta-Cyclodextrins , Animals , Cholesterol/metabolism , Colony Count, Microbial , Culture Media/chemistry , Cyclodextrins/metabolism , Feces/microbiology , Gerbillinae , Helicobacter pylori/enzymology , Helicobacter pylori/genetics , Humans , Serum Albumin, Bovine/metabolism , Transformation, Bacterial
10.
J Infect Dis ; 184(8): 1041-9, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11574920

ABSTRACT

Uropathogenic Escherichia coli is the leading cause of urinary tract infection and hospital visits in North America. Cystitis and acute pyelonephritis, infection of the bladder and kidney, respectively, are the two most common syndromes encountered in patients with urinary tract infection. We sequenced and annotated 71,684 bases of a previously unidentified pathogenicity-associated island (PAI) from E. coli strain CFT073. This PAI contained 89 open-reading frames encoding a pap operon, iron-regulated genes, mobile genetic elements, and a large proportion of unknown or unidentified open-reading frames. Dot blot analysis with 11 DNA sequences from this PAI demonstrated that 7 sequences were more prevalent among uropathogens: 2 probes were more prevalent among cystitis and pyelonephritis isolates, 2 among pyelonephritis isolates only, and 3 among cystitis isolates only than among fecal isolates. These data suggest that groups of uropathogens have genetic differences that may be responsible for the different clinical outcomes.


Subject(s)
Escherichia coli/classification , Bacterial Proteins/genetics , Base Composition , DNA, Bacterial/genetics , Enzymes/genetics , Escherichia coli/genetics , Escherichia coli/pathogenicity , Escherichia coli Infections/microbiology , Humans , Open Reading Frames , Urinary Tract Infections/microbiology
11.
EMBO J ; 20(17): 4854-62, 2001 Sep 03.
Article in English | MEDLINE | ID: mdl-11532949

ABSTRACT

Proteus mirabilis is a common uropathogen in patients with long-term catheterization or with structural or functional abnormalities in the urinary tract. The mannose-resistant, Proteus-like (MR/P) fimbriae and flagellum are among virulence factors of P.mirabilis that contribute to its colonization in a murine model of ascending urinary tract infection. mrpJ, the last of nine genes of the mrp operon, encodes a 107 amino acid protein that contains a putative helix-turn-helix domain. Using transcriptional lacZ fusions integrated into the chromosome and mutagenesis studies, we demonstrate that MrpJ represses transcription of the flagellar regulon and thus reduces flagella synthesis when MR/P fimbriae are produced. The repression of flagella synthesis by MrpJ is confirmed by electron microscopy. However, a gel mobility shift assay indicates that MrpJ does not bind directly to the regulatory region of the flhDC operon. The isogenic mrpJ null mutant of wild-type P.mirabilis strain HI4320 is attenuated in the murine model. Our data also indicate that PapX encoded by a pap (pyelonephritis- associated pilus) operon of uropathogenic Escherichia coli is a functional homolog of MrpJ.


Subject(s)
Fimbriae, Bacterial/genetics , Flagellin/genetics , Operon , Proteus mirabilis/physiology , Repressor Proteins/genetics , Repressor Proteins/metabolism , Suppression, Genetic , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cloning, Molecular , Disease Models, Animal , Fimbriae, Bacterial/physiology , Fimbriae, Bacterial/ultrastructure , Flagella/genetics , Flagella/physiology , Flagella/ultrastructure , Helix-Loop-Helix Motifs , Kinetics , Mice , Mice, Inbred CBA , Microscopy, Electron , Movement/physiology , Mutagenesis , Mutagenesis, Insertional , Proteus Infections/microbiology , Proteus mirabilis/genetics , Proteus mirabilis/pathogenicity , Recombinant Fusion Proteins/metabolism , Repressor Proteins/chemistry , Sequence Deletion , Transcription, Genetic , Urinary Tract Infections/microbiology , Virulence/genetics
12.
Infect Immun ; 69(9): 5914-20, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11500473

ABSTRACT

Helicobacter hepaticus causes disease in the liver and lower intestinal tract of mice. It is strongly urease positive, although it does not live in an acidic environment. The H. hepaticus urease gene cluster was expressed in Escherichia coli with and without coexpression of the Helicobacter pylori nickel transporter NixA. As for H. pylori, it was difficult to obtain enzymatic activity from recombinant H. hepaticus urease; special conditions including NiCl2 supplementation were required. The H. hepaticus urease cluster contains a homolog of each gene in the H. pylori urease cluster, including the urea transporter gene ureI. Downstream genes were homologs of the nik nickel transport operon of E. coli. Nongastric H. hepaticus produces urease similar to that of H. pylori.


Subject(s)
Cloning, Molecular , Genes, Bacterial , Helicobacter/enzymology , Urease/genetics , Urease/metabolism , Amino Acid Sequence , Animals , Escherichia coli/enzymology , Escherichia coli/genetics , Helicobacter/genetics , Mice , Molecular Sequence Data , Multigene Family , Sequence Analysis, DNA , Urease/chemistry
13.
J Bacteriol ; 183(15): 4526-35, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11443087

ABSTRACT

Proteus mirabilis urease catalyzes the hydrolysis of urea to CO(2) and NH(3), resulting in urinary stone formation in individuals with complicated urinary tract infections. UreR, a member of the AraC family, activates transcription of the genes encoding urease enzyme subunits and accessory proteins, ureDABCEFG, as well as its own transcription in the presence of urea. Based on sequence homology with AraC, we hypothesized that UreR contains both a dimerization domain and a DNA-binding domain. A translational fusion of the leucine zipper dimerization domain (amino acids 302 to 350) of C/EBP and the C-terminal half of UreR (amino acids 164 to 293) activated transcription from the ureD promoter (p(ureD)) and bound to a 60-bp fragment containing p(ureD), as analyzed by gel shift. These results were consistent with the DNA-binding specificity residing in the C-terminal half of UreR and dimerization being required for activity. To localize the dimerization domain of UreR, a translational fusion of the DNA-binding domain of the LexA repressor (amino acids 1 to 87) and the N-terminal half of UreR (amino acids 1 to 182) was constructed and found to repress transcription from p(sulA)-lacZ (sulA is repressed by LexA) and bind to the sulA operator site, as analyzed by gel shift. Since LexA binds this site only as a dimer, the UreR(1-182)-LexA(1-87) fusion also must dimerize to bind p(sulA). Indeed, purified UreR-Myc-His eluted from a gel filtration column as a dimer. Therefore, we conclude that the dimerization domain of UreR is located within the N-terminal half of UreR. UreR contains three leucines that mimic the leucines that contribute to dimerization of AraC. Mutagenesis of Leu147, Leu148, or L158 alone did not significantly affect UreR function. In contrast, mutagenesis of both Leu147 and Leu148 or all three Leu residues resulted in a 85 or 94% decrease, respectively, in UreR function in the presence of urea (P < 0.001). On the contrary, His102 and His175 mutations of UreR resulted in constitutive induction in the absence of urea. We conclude that a dimerization domain resides in the N-terminal half of the polypeptide, that Leu residues may contribute to this function, and that sequences within the C-terminal half of UreR are responsible for DNA binding to the urease promoter regions. Selected His residues also contribute significantly to UreR function.


Subject(s)
Multigene Family , Proteus mirabilis/metabolism , Trans-Activators/metabolism , Transcription Factors , Urease/genetics , AraC Transcription Factor , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , CCAAT-Enhancer-Binding Proteins/genetics , DNA/metabolism , Dimerization , Histidine/genetics , Histidine/metabolism , Leucine/metabolism , Mutagenesis, Site-Directed , Polymerase Chain Reaction/methods , Proteus mirabilis/genetics , Recombinant Fusion Proteins/genetics , Repressor Proteins , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Trans-Activators/genetics
14.
Infect Immun ; 69(5): 2838-46, 2001 May.
Article in English | MEDLINE | ID: mdl-11292696

ABSTRACT

Escherichia coli is the primary cause of uncomplicated infections of the urinary tract including cystitis. More serious infections, characterized as acute pyelonephritis, can also develop. Type 1 fimbriae of E. coli contribute to virulence in the urinary tract; however, only recently has the expression of the type 1 fimbriae been investigated in vivo using molecular techniques. Transcription of type 1 fimbrial genes is controlled by a promoter that resides on a 314-bp invertible element capable of two orientations. One places the promoter in the ON orientation, allowing for transcription; the other places the promoter in the OFF orientation, preventing transcription. A PCR-based assay was developed to measure the orientation of the invertible element during an experimental urinary tract infection in mice. Using this assay, it was found that the percentage of the population ON in urine samples correlated with the respective CFU per gram of bladder (P = 0.0006) but not with CFU per gram of kidney (P > 0.069). Cystitis isolates present in the urine of mice during the course of infection had a higher percentage of their invertible elements in the ON orientation than did pyelonephritis isolates (85 and 34%, respectively, at 24 h; P < 0.0001). In general, cystitis isolates, unlike pyelonephritis isolates, were more likely to maintain their invertible elements in the ON orientation for the entire period of infection. E. coli cells expressing type 1 fimbriae, expelled in urine, were shown by scanning electron microscopy to be densely packed on the surface of uroepithelial cells. These results suggest that expression of type 1 fimbriae is more critical for cystitis strains than for pyelonephritis strains in the early stages of an infection during bladder colonization.


Subject(s)
Bacterial Adhesion , Escherichia coli/pathogenicity , Fimbriae, Bacterial/physiology , Urinary Tract Infections/microbiology , Animals , Cystitis/microbiology , Mice , Mice, Inbred CBA , Microscopy, Electron, Scanning , Pyelonephritis/microbiology
15.
J Bacteriol ; 183(4): 1423-33, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11157956

ABSTRACT

Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)(3). To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.


Subject(s)
Apoenzymes/metabolism , Bacterial Proteins/metabolism , Proteus mirabilis/enzymology , Urease/metabolism , Antibodies, Bacterial , Apoenzymes/genetics , Apoenzymes/immunology , Bacterial Proteins/genetics , Carrier Proteins/genetics , Cloning, Molecular , DNA Primers , Gene Expression Regulation, Bacterial , Models, Chemical , Phosphate-Binding Proteins , Polymerase Chain Reaction , Precipitin Tests , Protein Binding , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Sequence Analysis, DNA , Serine Endopeptidases/metabolism , Two-Hybrid System Techniques , Urease/genetics , Urease/immunology
17.
Microbes Infect ; 2(12): 1497-505, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11099936

ABSTRACT

Proteus mirabilis is a causative agent of cystitis and pyelonephritis primarily in individuals with indwelling catheters or structural abnormalities of the urinary tract. The organism produces a variety of unique virulence factors that contribute to its pathogenicity and persistence in the human host.


Subject(s)
Proteus Infections/microbiology , Proteus mirabilis/pathogenicity , Urinary Tract Infections/microbiology , Amidohydrolases/metabolism , Female , Humans , Immunoglobulin A/metabolism , Iron/metabolism , Male , Metalloendopeptidases/physiology , Proteus Infections/immunology , Proteus Infections/metabolism , Proteus mirabilis/metabolism , Pyelonephritis/metabolism , Pyelonephritis/microbiology , Urinary Bladder/microbiology , Urinary Tract Infections/immunology , Urinary Tract Infections/metabolism
18.
Mol Microbiol ; 38(1): 53-66, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11029690

ABSTRACT

Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.


Subject(s)
Bacterial Toxins/chemistry , Escherichia coli/metabolism , Amino Acid Sequence , Animals , Bacterial Toxins/biosynthesis , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/pathogenicity , Humans , Mice , Molecular Sequence Data , Pyelonephritis/microbiology , Sequence Homology, Amino Acid , Urinary Tract/microbiology
20.
J Bacteriol ; 182(9): 2649-53, 2000 May.
Article in English | MEDLINE | ID: mdl-10762273

ABSTRACT

Expression of Proteus mirabilis urease is governed by UreR, an AraC-like positive transcriptional activator. A poly(A) tract nucleotide sequence, consisting of A(6)TA(2)CA(2)TGGTA(5)GA(6)TGA(5), is located 16 bp upstream of the sigma(70)-like ureR promoter P2. Since poly(A) tracts of DNA serve as binding sites for the gene repressor histone-like nucleoid structuring protein (H-NS), we measured beta-galactosidase activity of wild-type Escherichia coli MC4100 (H-NS(+)) and its isogenic derivative ATM121 (hns::Tn10) (H-NS(-)) harboring a ureR-lacZ operon fusion plasmid (pLC9801). beta-Galactosidase activity in the H-NS(-) host strain was constitutive and sevenfold greater (P < 0.0001) than that in the H-NS(+) host. A recombinant plasmid containing cloned P. mirabilis hns was able to complement and restore repression of the ureR promoter in the H-NS(-) host when provided in trans. Deletion of the poly(A) tract nucleotide sequence from pLC9801 resulted in an increase in beta-galactosidase activity in the H-NS(+) host to nearly the same levels as that observed for wild-type pLC9801 harbored by the H-NS(-) host. Urease activity in strains harboring the recombinant plasmid pMID1010 (encoding the entire urease gene cluster of P. mirabilis) was equivalent in both the H-NS(-) background and the H-NS(+) background in the presence of urea but was eightfold greater (P = 0.0001) in the H-NS(-) background in the absence of urea. We conclude that H-NS represses ureR expression in the absence of urea induction.


Subject(s)
Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Proteus mirabilis/metabolism , Repressor Proteins/metabolism , Trans-Activators/genetics , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Escherichia coli , Poly A , Proteus mirabilis/genetics , Repressor Proteins/genetics , Urease/biosynthesis
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