scholarly journals Loss of Regulatory Protein RfaH Attenuates Virulence of Uropathogenic Escherichia coli

2002 ◽  
Vol 70 (8) ◽  
pp. 4406-4413 ◽  
Author(s):  
Gábor Nagy ◽  
Ulrich Dobrindt ◽  
György Schneider ◽  
A. Salam Khan ◽  
Jörg Hacker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Mouse Model ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Protein ◽  
Wild Type ◽  
Serovar Typhimurium

ABSTRACT RfaH is a regulatory protein in Escherichia coli and Salmonella enterica serovar Typhimurium. Although it enhances expression of different factors that are proposed to play a role in bacterial virulence, a direct effect of RfaH on virulence has not been investigated so far. We report that inactivation of rfaH dramatically decreases the virulence of uropathogenic E. coli strain 536 in an ascending mouse model of urinary tract infection. The mortality rate caused by the wild-type strain in this assay is 100%, whereas that of its isogenic rfaH mutant does not exceed 18%. In the case of coinfection, the wild-type strain 536 shows higher potential to colonize the urinary tract even when it is outnumbered 100-fold by its rfaH mutant in the inoculum. In contrast to the wild-type strain, serum resistance of strain 536rfaH::cat is fully abolished. Furthermore, we give evidence that, besides a major decrease in the amount of hemin receptor ChuA (G. Nagy, U. Dobrindt, M. Kupfer, L. Emody, H. Karch, and J. Hacker, Infect. Immun. 69:1924-1928, 2001), loss of the RfaH protein results in an altered lipopolysaccharide phenotype as well as decreased expression of K15 capsule and alpha-hemolysin, whereas levels of other pathogenicity factors such as siderophores, flagella, Prf, and S fimbriae appear to be unaltered in strain 536rfaH::cat in comparison to the wild-type strain. trans complementation of the mutant strain with the rfaH gene restores wild-type levels of the affected virulence factors and consequently restitutes virulence in the mouse model of ascending urinary tract infection.

scholarly journals PhoU enhances the ability of extraintestinal pathogenic Escherichia coli strain CFT073 to colonize the murine urinary tract

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Eric L. Buckles ◽  
Xiaolin Wang ◽  
C. Virginia Lockatell ◽  
David E. Johnson ◽  
Michael S. Donnenberg
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Mutant Strain ◽  
Human Urine ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Strain Cft073

The phoU gene is the last cistron in the pstSCAB–phoU operon and functions as a negative regulator of the Pho regulon. The authors previously identified a phoU mutant of extraintestinal pathogenic Escherichia coli strain CFT073 and demonstrated that this mutant was attenuated for survival in the murine model of ascending urinary tract infection. It is hypothesized that the PhoU protein might serve as a urovirulence factor by indirectly affecting the expression of virulence-related genes. In this study, the phoU mutant was further characterized and PhoU was confirmed as a virulence factor. Western blot analysis demonstrated that insertion of the transposon in the phoU gene disrupted the expression of PhoU. The phoU mutant had derepressed alkaline phosphatase activity under phosphate-excess and -limiting conditions. In single-challenge murine ascending urinary tract infection experiments, quantitative cultures of urine, bladder and kidney revealed no significant differences between the phoU mutant strain and the wild-type strain CFT073. However, in competitive colonization experiments, the phoU mutant strain was significantly out-competed by the wild-type strain in the kidneys and urine and recovered in lower amount in the bladder. Complementation of the phoU mutant with a plasmid containing the wild-type phoU gene restored the expression of PhoU and alkaline phosphate activity to wild-type levels and no significant difference in colonization was observed between the phoU mutant containing the complementing plasmid and wild-type in competitive colonization experiments. In human urine, the phoU mutant and wild-type grew comparably when inoculated independently, indicating that the attenuation observed was not due to a general growth defect. However, as observed in vivo, the wild-type out-competed the phoU mutant in competition growth experiments in human urine. These data indicate that PhoU contributes to efficient colonization of the murine urinary tract and add PhoU to a short list of confirmed urovirulence factors.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

scholarly journals Uropathogenic Escherichia coli CFT073 Is Adapted to Acetatogenic Growth but Does Not Require Acetate during Murine Urinary Tract Infection

2008 ◽  
Vol 76 (12) ◽  
pp. 5760-5767 ◽  
Author(s):  
Andrew T. Anfora ◽  
David K. Halladin ◽  
Brian J. Haugen ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Intracellular Signaling ◽  
High Energy ◽  
Acetate Metabolism ◽  
Acetyl Phosphate ◽  
Wild Type ◽  
K 12

ABSTRACT In vivo accumulation of d-serine by Escherichia coli CFT073 leads to elevated expression of PAP fimbriae and hemolysin by an unknown mechanism. Loss of d-serine catabolism by CFT073 leads to a competitive advantage during murine urinary tract infection (UTI), but loss of both d- and l-serine catabolism results in attenuation. Serine is the first amino acid to be consumed in closed tryptone broth cultures and precedes the production of acetyl phosphate, a high-energy molecule involved in intracellular signaling, and the eventual secretion of acetate. We propose that the colonization defect associated with the loss of serine catabolism is due to perturbations of acetate metabolism. CFT073 grows more rapidly on acetogenic substrates than does E. coli K-12 isolate MG1655. As shown by transcription microarray results, d-serine is catabolized into acetate via the phosphotransacetylase (pta) and acetate kinase (ackA) genes while downregulating expression of acetyl coenzyme A synthase (acs). CFT073 acs, which is unable to reclaim secreted acetate, colonized mouse bladders and kidneys in the murine model of UTI indistinguishably from the wild type. Both pta and ackA are involved in the maintenance of intracellular acetyl phosphate. CFT073 pta and ackA mutants were screened to investigate the role of acetyl phosphate in UTI pathogenesis. Both single mutants are at a competitive disadvantage relative to the wild type in the kidneys but normally colonize the bladder. CFT073 ackA pta was attenuated in both the bladder and the kidneys. Thus, we demonstrate that CFT073 is adapted to acetate metabolism as a result of requiring a proper cycling of the acetyl phosphate pathway for colonization of the upper urinary tract.

scholarly journals Genetic Analysis of the Role ofyfiRin the Ability of Escherichia coli CFT073 To Control Cellular Cyclic Dimeric GMP Levels and To Persist in the Urinary Tract

2013 ◽  
Vol 81 (9) ◽  
pp. 3089-3098 ◽  
Author(s):  
Erica L. Raterman ◽  
Daniel D. Shapiro ◽  
Daniel J. Stevens ◽  
Kevin J. Schwartz ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Cellulose Production ◽  
Content Type ◽  
E Coli ◽  
Successful Infection ◽  
Tract Infections

ABSTRACTDuring urinary tract infections (UTIs), uropathogenicEscherichia colimust maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states inE. coli. TheyfiRNBlocus inE. coliCFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion ofyfiRyielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A doubleyfiRNmutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in theyfiRmutant. Expression ofyhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiRsuppressed the overproduction of curli fimbriae and cellulose and further verified that deletion ofyfiRresults in c-di-GMP accumulation. Additional deletion ofcsgDandbcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of theyfiRdeletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between theyfiRmutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disableE. coliin the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenicE. coli in vivo.

scholarly journals Assessment of Virulence of Uropathogenic Escherichia coli Type 1 Fimbrial Mutants in Which the Invertible Element Is Phase-Locked On or Off

2002 ◽  
Vol 70 (7) ◽  
pp. 3344-3354 ◽  
Author(s):  
Nereus W. Gunther IV ◽  
Jennifer A. Snyder ◽  
Virginia Lockatell ◽  
Ian Blomfield ◽  
David E. Johnson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Invertible Element ◽  
Transcriptional Level ◽  
Wild Type ◽  
Invertible Elements ◽  
Tract Infections

ABSTRACT Type 1 fimbria is a proven virulence factor of uropathogenic Escherichia coli (UPEC), causing urinary tract infections. Expression of the fimbria is regulated at the transcriptional level by a promoter situated on an invertible element, which can exist in one of two different orientations. The orientation of the invertible element that allows the expression of type 1 fimbriae is defined as “on,” and the opposite orientation, in which no transcription occurs, is defined as “off.” During the course of a urinary tract infection, we have observed that the infecting E. coli population alternates between fimbriated and nonfimbriated states, with the fimbriated on orientation peaking at 24 h. We propose that the ability of the invertible element to switch orientations during infection is itself a virulence trait. To test this hypothesis, nucleotide sequence changes were introduced in the left inverted repeat of the invertible element of UPEC pyelonephritis strain CFT073 that locked the invertible elements permanently in either the on or the off orientation. The virulence of these mutants was assessed in the CBA mouse model of ascending urinary tract infection at 4, 24, 48, and 72 h postinoculation (hpi). We conducted independent challenges, in which bladders of mice were inoculated with either a single mutant or the wild type, and cochallenges, in which a mutant and the wild type were inoculated together to allow direct competition in the urinary tract. In both sets of experimental infections, the locked-off mutant was recovered from the urine, bladder, and kidneys in significantly lower numbers than the wild type at 24 hpi (P ≤ 0.05), demonstrating its attenuation. Conversely, the locked-on mutant was recovered in higher numbers than the wild type at 24 hpi (P ≤ 0.05), showing enhanced virulence of this mutant. No significant differences were seen between the mutants and wild type in the urine or the bladder at 48 or 72 hpi. However, the wild type outcompeted the locked-off mutant in the kidneys during the cochallenge experiment at 72 hpi (P = 0.009). Overall, these data suggest that the ability of the invertible element controlling type 1 fimbria expression to phase vary contributes significantly to virulence early (24 hpi) in the course of a urinary tract infection by UPEC and most profoundly influences colonization of the bladder.

scholarly journals Antimicrobial Activity Evaluation of Tebipenem (SPR859), an Orally Available Carbapenem, against a Global Set of Enterobacteriaceae Isolates, Including a Challenge Set of Organisms

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
S. J. Ryan Arends ◽  
Paul R. Rhomberg ◽  
Nicole Cotroneo ◽  
Aileen Rubio ◽  
Robert K. Flamm ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Broth Microdilution ◽  
Wild Type ◽  
Content Type ◽  
E Coli

ABSTRACT The antimicrobial activity of tebipenem and other carbapenem agents were tested in vitro against a set of recent clinical isolates responsible for urinary tract infection (UTI), as well as against a challenge set. Isolates were tested by reference broth microdilution and included Escherichia coli (101 isolates), Klebsiella pneumoniae (208 isolates), and Proteus mirabilis (103 isolates) species. Within each species tested, tebipenem showed equivalent MIC50/90 values to those of meropenem (E. coli MIC50/90, ≤0.015/0.03 mg/liter; K. pneumoniae MIC50/90, 0.03/0.06 mg/liter; and P. mirabilis MIC50/90, 0.06/0.12 mg/liter) and consistently displayed MIC90 values 8-fold lower than imipenem. Tebipenem and meropenem (MIC50, 0.03 mg/liter) showed equivalent MIC50 results against wild-type, AmpC-, and/or extended-spectrum β-lactamase (ESBL)-producing isolates. Tebipenem also displayed MIC50/90 values 4- to 8-fold lower than imipenem against the challenge set. All carbapenem agents were less active (MIC50, ≥8 mg/liter) against isolates carrying carbapenemase genes. These data confirm the in vitro activity of the orally available agent tebipenem against prevalent UTI Enterobacteriaceae species, including those producing ESBLs and/or plasmid AmpC enzymes.

scholarly journals Effect of Inactivation of the Global Oxidative Stress Regulator oxyR on the Colonization Ability of Escherichia coli O1:K1:H7 in a Mouse Model of Ascending Urinary Tract Infection

2006 ◽  
Vol 74 (1) ◽  
pp. 461-468 ◽  
Author(s):  
James R. Johnson ◽  
Connie Clabots ◽  
Henry Rosen
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Wild Type ◽  
In Vitro Susceptibility ◽  
E Coli ◽  
C57bl Mice ◽  
The Impact

ABSTRACT To survive within the host urinary tract, Escherichia coli strains that cause urinary tract infection (UTI) presumably must overcome powerful oxidant stresses, including the oxygen-dependent killing mechanisms of neutrophils. Accordingly, we assessed the global oxygen stress regulator OxyR of Escherichia coli as a possible virulence factor in UTI by determining the impact of oxyR inactivation on experimental urovirulence in CBA/J and C57BL (both wild-type and p47phox−/−) mice. The oxyR and oxyS genes of wild-type E. coli strain Ec1a (O1:K1:H7) were replaced with a kanamycin resistance cassette to produce an oxyRS mutant. During in vitro growth in broth or human urine, the oxyRS mutant exhibited the same log-phase growth rate (broth) and plateau density (broth and urine) as Ec1a, despite its prolonged lag phase (broth) or initial decrease in concentration (urine). The mutant, and oxyRS mutants of other wild-type ExPEC strains, exhibited significantly increased in vitro susceptibility to inhibition by H2O2, which, like the altered growth kinetics observed with oxyRS inactivation, were reversed by restoration of oxyR on a multiple-copy-number plasmid. In CBA/J mice, Ec1a significantly outcompeted its oxyRS mutant (by >1 log10) in urine, bladder, and kidney cultures harvested 48 h after perurethral inoculation of mice, whereas an oxyR-complemented mutant exhibited equal or greater colonizing ability than that of the parent. Although C57BL mice were less susceptible to experimental UTI than CBA/J mice, wild-type and p47phox−/− C57BL mice were similarly susceptible, and the oxyR mutant of Ec1a was similarly attenuated in C57BL mice, regardless of the p47phox genotype, as in CBA/J mice. Within the E. coli Reference collection, 94% of strains were positive for oxyR. These findings fulfill the second and third of Koch's molecular postulates for oxyR as a candidate virulence-facilitating factor in E. coli and indicate that oxyR is a broadly prevalent potential target for future preventive interventions against UTI due to E. coli. They also suggest that neutrophil phagocyte oxidase is not critical for defense against E. coli UTI and that the major oxidative stresses against which OxyR protects E. coli within the host milieu are not phagocyte derived.

scholarly journals Sat, the Secreted Autotransporter Toxin of Uropathogenic Escherichia coli, Is a Vacuolating Cytotoxin for Bladder and Kidney Epithelial Cells

2002 ◽  
Vol 70 (8) ◽  
pp. 4539-4546 ◽  
Author(s):  
Debra M. Guyer ◽  
Suzana Radulovic ◽  
Faye-Ellen Jones ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Cell Lines ◽  
Kidney Tissue ◽  
Upper Urinary Tract ◽  
Wild Type ◽  
Vacuolating Cytotoxin ◽  
E Coli

ABSTRACT The secreted autotransporter toxin (Sat) of uropathogenic Escherichia coli exhibits cytopathic activity upon incubation with HEp-2 cells. We further investigated the effects of Sat on cell lines more relevant to the urinary tract, namely, those derived from bladder and kidney epithelium. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (CRL-1749) and kidney (CRL-1573) cell lines. This activity has been associated with only a few other known toxins. Following transurethral infection of CBA mice with a sat mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type E. coli CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type E. coli CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat, a vacuolating cytotoxin expressed by uropathogenic E. coli CFT073, elicits defined damage to kidney epithelium during upper urinary tract infection and thus contributes to pathogenesis of urinary tract infection.

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