ABSTRACT
Escherichia coli is the leading cause of urinary tract infections (UTIs), one of the most common infections in humans. P fimbria was arguably the first proposed virulence factor for uropathogenic E. coli, based on the capacity of E. coli isolated from UTIs to adhere to exfoliated epithelial cells in higher numbers than fecal strains of E. coli. Overwhelming epidemiologic evidence has been presented for involvement of P fimbriae in colonization. It has been difficult, however, to demonstrate this requirement for uropathogenic strains in animal models of infections or in humans. In this study, a signature-tagged mutagenesis screen identified a P-fimbrial gene (papC) and 18 other genes as being among those required for full fitness of cystitis isolate E. coli F11. A P-fimbrial mutant was outcompeted by the wild-type strain in cochallenge in the murine model of ascending UTI, and this colonization defect could be complemented with the cloned pap operon. To our knowledge, this study is the first to fulfill molecular Koch's postulates in which a pathogenic strain was attenuated by mutation of pap genes and then complemented to restore fitness, confirming P fimbria as a virulence factor in a pathogenic clinical isolate.
Subject(s)
Adhesins, Bacterial/metabolism , Bacterial Adhesion , Escherichia coli Infections/microbiology , Fimbriae, Bacterial/metabolism , Urinary Tract Infections/microbiology , Uropathogenic Escherichia coli/physiology , Virulence Factors/metabolism , Adhesins, Bacterial/genetics , Animals , Coinfection/microbiology , Disease Models, Animal , Fimbriae, Bacterial/genetics , Gene Deletion , Genetic Complementation Test , Genetic Testing , Mice , Mutagenesis , Operon , Uropathogenic Escherichia coli/genetics , Virulence Factors/geneticsABSTRACT
BACKGROUND: Capsule expression may be important during ascending Escherichia coli urinary tract infections (UTIs). METHODS: An isogenic ksl(k2)ABCDE mutant of extraintestinal pathogenic E. coli (ExPEC) strain CFT073 that could not synthesize the K2 capsule was compared with wild-type CFT073, to determine virulence in a murine model of ascending UTI and in vitro killing assays. RESULTS: No significant differences were observed regarding the abilities of the mutant and the wild-type CFT073 strains to colonize the murine urinary tract in single-challenge infection experiments. However, in competitive-colonization experiments, the mutant was significantly outcompeted by the wild-type strain in urine and the kidneys. The mutant strain was also more susceptible to human serum. Complementation of the mutant with a plasmid containing the ksl(k2)ABCDE genes restored capsule expression, enhanced survival in the murine urinary tract, and restored serum resistance. CONCLUSION: These results indicate that expression of the K2 capsule is important for the pathogenesis of UTI and provides protection against complement-mediated killing. To our knowledge, this is the first study in which the E. coli capsule has been proven to play a role in infection by use of isogenic mutants and genetic complementation.
Subject(s)
Bacterial Capsules/genetics , Escherichia coli Infections/genetics , Escherichia coli/genetics , Escherichia coli/pathogenicity , Urinary Tract Infections/microbiology , Animals , Cloning, Molecular , Cosmids/genetics , DNA Primers , Disease Models, Animal , Escherichia coli Infections/blood , Humans , Mice , Mice, Inbred CBA , Molecular Sequence Data , Mutation , Plasmids , Sequence DeletionABSTRACT
Lyme disease is the most common arthropod-borne disease in North America and Europe. At present, there is no commercially available vaccine for use in humans. Outer surface protein C (OspC) has antigenic and expression characteristics that make it an attractive vaccine candidate; however, sequence heterogeneity has impeded its use as a vaccinogen. Sequence analyses have identified 21 well defined OspC phyletic groups or "types" (designated A-U). In this report we have mapped the linear epitopes presented by OspC types B, K, and D during human and murine infection and exploited these epitopes (along with the previously identified type A OspC linear epitopes) in the development of a recombinant, tetravalent, chimeric vaccinogen. The construct was found to be highly immunogenic in mice and the induced antibodies surface labeled in vitro cultivated spirochetes. Importantly, vaccination induced complement-dependent bactericidal antibodies against strains expressing each of the OspC types that were incorporated into the construct. These results suggest that an effective and broadly protective polyvalent OspC-based Lyme disease vaccine can be produced as a recombinant, chimeric protein.
Subject(s)
Borrelia burgdorferi/immunology , Lyme Disease Vaccines/immunology , Lyme Disease/immunology , Lyme Disease/prevention & control , Phosphatidylcholines/immunology , Amino Acid Sequence , Animals , Base Sequence , Borrelia burgdorferi/isolation & purification , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Fluorescent Antibody Technique , Immunoblotting , Lyme Disease Vaccines/genetics , Male , Mice , Mice, Inbred C3H , Models, Molecular , Molecular Sequence Data , Phosphatidylcholines/genetics , Reverse Transcriptase Polymerase Chain Reaction , Species Specificity , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
The OspC protein of Borrelia burgdorferi is an immunodominant antigen. Here we demonstrate that the loop 5 domain of type A OspC is surface exposed, elicits bactericidal antibody in mice, and is antigenic in humans. The data suggest that loop 5 may be suitable for inclusion in a polyvalent, chimeric OspC vaccinogen.
Subject(s)
Antibodies, Bacterial/biosynthesis , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Epitopes/immunology , Lyme Disease Vaccines/immunology , Lyme Disease/immunology , Amino Acid Sequence , Humans , Lyme Disease/microbiology , Lyme Disease Vaccines/chemical synthesis , Molecular Sequence Data , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
Escherichia coli Infections/microbiology , Escherichia coli Proteins/physiology , Escherichia coli/pathogenicity , Female Urogenital Diseases/microbiology , Male Urogenital Diseases , Membrane Transport Proteins/physiology , Transcription Factors/physiology , Virulence Factors/physiology , Alkaline Phosphatase/metabolism , Animals , Down-Regulation , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Humans , Kidney/microbiology , Membrane Transport Proteins/genetics , Mice , Mice, Inbred CBA , Transcription Factors/genetics , Urinary Bladder/microbiology , Urine/microbiology , Virulence/immunology , Virulence Factors/geneticsABSTRACT
Outer surface protein C (OspC) of the Lyme disease spirochetes is an important virulence factor that has potential utility for vaccine development. Of the 21 OspC types that have been identified, it has been postulated that types A, B, I, and K are specifically associated with invasive infections. Through an analysis of isolates collected from patients in Maryland we found that OspC types C, D, and N are also associated with invasive infections. This observation suggests that there is greater diversity in the group of OspC types associated with invasive infection than has been previously suggested. Detailed knowledge of the antigenic structure of OspC is essential for vaccine development. To determine if the antibody response to OspC is type specific, recombinant proteins of several different OspC types were immunoblotted and screened with sera from mice infected with isolates having known OspC types. These analyses revealed a high degree of specificity in the antibody response and suggested that the immunodominant epitopes of OspC reside in the variable domains of the protein. To localize these epitopes, OspC fragments were generated and screened with serum collected from infected mice. These analyses led to identification of previously uncharacterized epitopes that define the type specificity of the OspC antibody response. These analyses provide important insight into the antigenic structure of OspC and also provide a basis for understanding the variable nature of the antibody response to this important virulence factor of the Lyme disease spirochetes.
Subject(s)
Antibodies, Bacterial/immunology , Antibody Specificity , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Borrelia burgdorferi/immunology , Epitopes/immunology , Lyme Disease/immunology , Animals , Antigens, Bacterial/chemistry , Bacterial Outer Membrane Proteins/chemistry , Borrelia burgdorferi/isolation & purification , Borrelia burgdorferi/pathogenicity , Computer Simulation , Epitope Mapping , Epitopes/chemistry , Evolution, Molecular , Humans , Lyme Disease/microbiology , Mice , Molecular Sequence Data , Protein Conformation , VirulenceABSTRACT
A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any E. coli pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/pathogenicity , Proteome , Transcription, Genetic , Urinary Tract Infections/microbiology , Animals , Culture Media , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Humans , Mice , Mice, Inbred CBA , Oligonucleotide Array Sequence Analysis , Open Reading Frames , Species Specificity , Urine/microbiologyABSTRACT
Recently, we identified a fimbrial usher gene in uropathogenic Escherichia coli strain CFT073 that is absent from an E. coli laboratory strain. Analysis of the CFT073 genome indicates that this fimbrial usher gene is part of a novel fimbrial gene cluster, aufABCDEFG. Analysis of a collection of pathogenic and commensal strains of E. coli and related species revealed that the auf gene cluster was significantly associated with uropathogenic E. coli isolates. For in vitro expression analysis of the auf gene cluster, RNA was isolated from CFT073 bacteria grown to the exponential or stationary phase in Luria-Bertani broth and reverse transcriptase PCR (RT-PCR) with oligonucleotide primers specific to the major subunit, aufA, was performed. We found that aufA is expressed in CFT073 only during the exponential growth phase; however, no expression of AufA protein was observed by Western blotting, indicating that under these conditions, the expression of the auf gene cluster is low. To determine if the auf gene cluster is expressed in vivo, RT-PCR was performed on bacteria from urine samples of mice infected with CFT073. Out of three independent experiments, we were able to detect expression of aufA at least once at 4, 24, and 48 h of infection, indicating that the auf gene cluster is expressed in the murine urinary tract. Furthermore, antisera from mice infected with CFT073 reacted with recombinant AufA in an enzyme-linked immunosorbent assay. To identify the structure encoded by the auf gene cluster, a recombinant plasmid containing the auf gene cluster under the T7 promoter was introduced into the E. coli BL-21 (AI) strain. Immunogold labeling using AufA antiserum revealed the presence of amorphous material extending from the surface of BL-21 cells. No hemagglutination or cellular adherence properties were detected in association with expression of AufA. Deletion of the entire auf gene cluster had no effect on the ability of CFT073 to colonize the kidney, bladder, or urine of mice. In addition, no significant histological differences between the parent and aufC mutant strain were observed. Therefore, Auf is a uropathogenic E. coli-associated structure that plays an uncertain role in the pathogenesis of urinary tract infections.
Subject(s)
Escherichia coli/genetics , Fimbriae, Bacterial/genetics , Urinary Tract Infections/metabolism , Animals , Erythrocyte Aggregation , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Fimbriae, Bacterial/immunology , Fimbriae, Bacterial/metabolism , Hemagglutination Tests , Immunohistochemistry , Kidney/microbiology , Kidney/pathology , Mice , Microscopy, Immunoelectron , Multigene Family , Urinary Bladder/microbiology , Urinary Bladder/pathologyABSTRACT
Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.