Loss of resistance to ingestion and phagocytic killing by O(-) and K(-) mutants of a uropathogenic Escherichia coli O75:K5 strain.

To determine the importance of the O75 O antigen and the K5 capsular antigen in resistance to phagocytosis and phagocytic killing, we used previously described O75(-) and K5(-) mutants from an O75(+) K5(+) wild-type uropathogenic Escherichia coli strain in phagocytosis assays with polymorphonuclear leukocytes (PMNs) and monocytes. At a 10-to-1 ratio of bacteria to phagocytes and in the presence of 10% serum, the parental strain GR-12 was resistant to both PMNs and monocytes over a 2-h incubation period. The O75(-) and K5(-) mutants were similar in sensitivity to killing by both PMNs and monocytes, decreasing in viability by 80% in the first hour. Yet, a significant difference in killing between the O75(-) and K5(-) mutants was observed in the first 15 min of incubation. The K5(-) mutant decreased in numbers by almost 60%, while the O75(-) mutant increased in numbers similarly to GR-12 in the first 15 min. The difference in killing was found not to be due to the rate of opsonization. To further determine the mechanism of resistance, a fluorescence assay was used to differentiate attached and internalized bacteria. The K5 capsule hindered the association of both the wild-type strain and the O75(-) mutant in the initial incubation time with PMNs. In conclusion, both the K5 capsule and O75 O antigen play crucial roles in resistance to phagocytosis over time.

Comparison of loss of serum resistance by defined lipopolysaccharide mutants and an acapsular mutant of uropathogenic Escherichia coli O75:K5.

In order to determine the importance of the O75 O antigen versus the K5 capsular antigen and the bimodal distribution of lipopolysaccharides (LPSs) in protection from complement-mediated lysis, mutants were made by insertion of a cat or an aphA gene in or in place of genes necessary for the synthesis of LPS and/or the K antigen of an O75(+) K5(+) uropathogenic Escherichia coli strain, GR-12. Mutations were made in the following genes: the rfbD gene (required for the synthesis of TDP-rhamnose), the rfbKM genes (necessary for the synthesis of GDP-mannose), the rol gene (regulating O-antigen length), the kfiC gene (encoding a putative glycosyltransferase), and the kfiC-rfbD genes. The resulting phenotypes were rough (O75(-)), core plus one partial O-antigen subunit, random distribution of O-antigen chain lengths, acapsular (K5(-)), and O75(-) K5(-), respectively. All five mutants and GR-12 were analyzed for survival in 80% serum. The GR-12 parent was resistant, exhibiting a 500% increase in numbers. The rol, rfbKM, rfbD, and kfiC-rfbD mutants were sensitive, experiencing 99%, 99.9%, 99.9%, and at least 99.999% killing, respectively, in the first hour. The kfiC mutant, however, increased in numbers in the first hour but experienced delayed sensitivity, decreasing in viability by 80% in the third hour. Single mutants were complemented with the wild-type gene in trans, showing restoration of the wild-type phenotype and serum resistance. Therefore, the O75 antigen is more important for survival in serum than the K5 antigen, and regulation of the O75 O-antigen chain length is crucial for protection of the bacteria from complement-mediated lysis.

Nutritional requirements for growth of uropathogenic Escherichia coli in human urine.

Enrichment with D-cycloserine was used to identify Escherichia coli auxotrophic mutants that exhibited limited growth in human urine. Bacterial synthesis of guanine, arginine, and glutamine was found to be required for optimal growth in urine. Mutants that required leucine, methionine, serine, phenylalanine, or proline also exhibited reduced growth in urine. Several other nutritional mutants, including nicotinamide auxotrophs, which are found frequently among cystitis isolates, exhibited normal growth in urine.

Restriction fragment length polymorphism of PCR amplified papE gene products is correlated with complete serotype among uropathogenic Escherichia coli isolates.

Using whole bacteria, rather than extracted, purified DNA samples, we amplified the papE gene sequences from 63 Escherichia coli isolates belonging to O serogroups O1, O2 and O6. These isolates were from collections separated temporally as well as geographically: from four cities in the US and one in Sweden. PCR amplified papE products were digested with restriction endonucleases and the relative sizes of the fragments compared for each strain. For 41 of the strains, we found a correlation between the papE restriction fragment length polymorphism (RFLP) and the complete serotype. Furthermore, we were able to detect the presence of duplicate copies of the gene in 14 of the isolates; these 14 isolates were among the 22 that did not exhibit a correlation between the RFLP of their amplified papE sequences and their complete serotype. We conclude that RFLP analysis of PCR products is a rapid and relatively simple method for examining the DNA of E. coli containing the pap gene sequence.

Identification of the O antigen polymerase (rfc) gene in Escherichia coli O4 by insertional mutagenesis using a nonpolar chloramphenicol resistance cassette.

Computer analysis of the O4 polysaccharide gene cluster of Escherichia coli revealed the presence of two open reading frames (ORFs) encoding strongly hydrophobic polypeptides. O antigen polymerase, which is encoded by the rfc gene, is a potential membrane protein and therefore should be hydrophobic. To identify the rfc gene, these two ORFs were subjected to insertional mutagenesis. A chloramphenicol resistance cassette was designed which, when properly inserted, does not cause a polar effect in downstream genes. Each of two ORFs, cloned into a plasmid vector, was inactivated with this cassette. Two types of mutants bearing chromosomal insertions of the cassettes in each ORF were constructed by homologous recombination. These mutants were characterized by PCR, Southern blotting, and transverse-alternating-field electrophoresis. Only one class of mutants exhibited the expected O polymerase-deficient phenotype; they produced O4-specific, semirough lipopolysaccharide. Therefore, this ORF was identified as the rfc gene. The chromosomal rfc mutation was complemented in trans by the rfc gene expressed from a plasmid vector.

Effect of pap copy number and receptor specificity on virulence of fimbriated Escherichia coli in a murine urinary tract colonization model.

Escherichia coli FN506 containing pap genes that encode two different P fimbriae adherence specificity types were tested for virulence in a murine urinary colonization model. Strains containing adherence genes on either high copy or low copy plasmids were compared. Bacteria that harbored the adherence genes on high copy plasmids colonized mouse kidneys less well than bacteria with the same adherence genes in low copy even though the high copy strains exhibited greater hemagglutination capacity. Bacteria with either type of P fimbriae were able to colonize but pap-2+ bacteria showed increased colonizing capacity when strains containing pap-1 or pap-2 genes on low copy plasmids were compared. Bacteria containing plasmids with both adherence specificities had a similar colonizing capacity as bacteria with either type separately.

Alanine-scanning mutagenesis reveals residues involved in binding of pap-3-encoded pili.

In order to identify functionally important residues in the pap-3-encoded adhesin, oligonucleotide-directed mutagenesis was used to substitute alanine(s) at sixteen positions in the adhesion. These alanine substitutions span nearly every domain and hydrophilic peak of the protein. The effects of these substitutions were measured by evaluating the patterns of hemagglutination exhibited by the mutant strains. It was found that strains harboring alanine substitutions at positions 88 and 89, 128 to 130, and 316 had lost the capacity to hemagglutinate. The presence of the mutated adhesin in the assembled pilus structure was verified by the reactions of purified pili with an adhesin-specific monoclonal antibody in an enzyme-linked immunosorbent assay and with a polyclonal antibody in Western blotting (immunoblotting). Alanine substitutions at positions 68, 110 and 111, and 143 to 146 had no effect upon hemagglutination, whereas substitutions at positions 203 and 204 and position 291 resulted in diminished binding. Thus, the residues necessary for hemagglutination are scattered throughout the adhesin in both the amino and carboxy regions. Delineation of these residues may prove useful in designing a preventive treatment that would cross-react with the essential binding residues from the adhesins of several different pyelonephritis-causing strains.

Structural analysis of O4-reactive polysaccharides from recombinant Escherichia coli. Changes in the O-specific polysaccharide induced by cloning of the rfb genes.

In previous studies it had been shown that lipopolysaccharide from O4-specific recombinant Escherichia coli, had serological reactivities and a chemical composition that differed from wildtype O4 LPS [Haraguchi, G.E., Zähringer, U., Jann, B., Jann, K., Hull, R.A. & Hull, S.I. (1991) Microb. Pathog. 10, 351-361]. Here we present the structural elucidation of the O-specific moieties from lipopolysaccharides of some of the recombinant strains obtained in previous studies. Compositional analysis, methylation, chemical reactions and NMR spectroscopy showed that, during genetic manipulations (recombination, cosmid cloning, plasmid subcloning), a gradual structural change in the O-specific polysaccharides was observed in the recombinant strains. These changes comprised of an alteration in the position of glucose (side chain) substitution, a change in the anomeric configuration of the main-chain N-acetylglucosamine and an exchange of alpha-L-rhamnopyranose for beta-D-galactofuranose. The relevance of these results for lipopolysaccharide cloning and lipopolysaccharide biosynthesis are discussed.

Sequences of the genes encoding the minor tip components of Pap-3 pili of Escherichia coli.

We report the sequence of the papE, papF and papG genes from the O75:K5 uropathogenic P pili variant, Pap-3. Comparison of the deduced amino acid sequences with those of other P pili variants reveals regions of complete homology, as well as regions of variation. Analysis of the variations in the hydrophilic domains of these proteins will help elucidate the residues which determine binding specificity.

Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation.

In this article, we report on the nucleotide sequences of the rol genes of Escherichia coli O75 and Salmonella typhimurium LT2. The rol gene in E. coli was previously shown to encode a 36-kDa protein that regulates size distribution of the O-antigen moiety of lipopolysaccharide. The E. coli and S. typhimurium rol gene sequences consist of 978 and 984 nucleotides, respectively. The homology between the nucleotide sequences of these two genes was found to be 68.9%. Both the E. coli rol and S. typhimurium rol genes are transcribed counter to the histidine operon and code for deduced polypeptides of 325 and 327 amino acids, respectively. The S. typhimurium rol gene was previously identified to encode a protein of unknown function and to share a transcription termination region with his. The homology between these deduced polypeptide sequences was observed to be 72%. A complementation test was performed in which the S. typhimurium rol gene was placed in trans with an E. coli plasmid (pRAB3) which encodes the O75 rfb gene cluster and not rol. The protein expressed from the S. typhimurium rol gene was found to regulate the distribution of the O75 O polysaccharide on the lipopolysaccharide of the host strain, E. coli S phi 874. The mechanism of Rol action may be independent of O antigen subunit structure, and its presence may be conserved in members of the family Enterobacteriaceae and other gram-negative bacilli that express O polysaccharides on their surface membrane.

Escherichia coli virulence factors and 99mTc-dimercaptosuccinic acid renal scan in children with febrile urinary tract infection.

Correlation of virulence factors of Escherichia coli with renal inflammation documented by 99mTc-dimercaptosuccinic acid renal scan was undertaken in 59 children with febrile urinary tract infections to identify more accurately the role of bacterial virulence factors in the development of pyelonephritis. P fimbriae were present in 63% of isolates from the positive scan group and 83% of those from the negative scan group (P = 0.126). Multivariate regression analysis showed no significant role for established E. coli virulence factors in the development of pyelonephritis. The pap genome was independently associated with negative scan (P less than 0.007) and with the absence of reflux (P = 0.031). E. coli pyelonephritogenic clone O16:K1:H6 was isolated from negative scan patients and did not produce hemolysin. We conclude that P fimbriae are important in the development of febrile urinary tract infection regardless of the level of infection. Virulent E. coli clones described in prior Scandinavian urinary tract infection studies were not common causes of pyelonephritis in our patient population.

Molecular cloning and expression of the 01 rfb region from a pyelonephritic Escherichia coli 01:H1:K7.

The genes responsible for the biosynthesis of the O1 polysaccharide from a human pyelonephritic Escherichia coli were cloned and expressed in a rfb-deleted E. coli K-12 strain. Deletion analysis of the clone demonstrated that a DNA fragment size larger than 6.7 kb and smaller than 10 kb is responsible for O1-antigen biosynthesis.

Regulation by a novel protein of the bimodal distribution of lipopolysaccharide in the outer membrane of Escherichia coli.

We report on the cloning and characterization of the rfb gene cluster of the O75 lipopolysaccharide from a urinary tract isolate of Escherichia coli. Deletion cloning defined the minimum region of DNA that expressed the O75 antigen in E. coli host strains to be on a 12.4-kb insert. However, the E. coli strain expressing this region did not produce a polymerized O chain as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. A slightly larger DNA clone of 13.4 kb produced a polymerized O chain in E. coli S phi 874 but was found to be abnormal in its distribution over the surface membrane. Normal wild-type E. coli, as with Salmonella spp., has a bimodal distribution of the lipopolysaccharide on the surface which is seen as an abundance of long and short O chains attached to the lipid A-core structure. We found in a region adjacent to the cloned rfb region, and on the opposite side from where the putative polymerase (rfc) is encoded, a novel protein of 35.5 kDa expressed from a 1.75-kb DNA fragment. This protein was shown to complement in trans the E. coli strains carrying plasmids that expressed abnormal, unregulated lipopolysaccharides. The expression of these complemented strains was bimodal in distribution. Mutation of the gene encoding this protein destroyed its ability to regulate O-chain distribution. We propose to call this regulator gene rol, for regulator of O length.

Genetic characterization of the O4 polysaccharide gene cluster from Escherichia coli.

The Escherichia coli O4 serotype is among those commonly isolated from urinary tract infections. In order to study the genetics of the O-antigen, the O4 biosynthesis genes from a uropathogenic E. coli have previously been cloned into E. coli K-12. A subclone, GH58, has been identified which reacts with antisera against the O4 serotype. In contrast to the wild-type parental strain, lipopolysaccharide (LPS) from this clone is devoid of rhamnose and does not cross-react with O18 antisera. The recombinant plasmid from GH58, pGH58, was used to transform the rfb deletion strain HU1190. The resultant strain agglutinates in O4 antisera, but produces unpolymerized LPS. Escherichia coli K-12 strains HB101 and RC712 containing pGH58 produce polymerized LPS, indicating that the genetic background of the host can influence the LPS encoded by recombinant molecules. A cosmid, pGH84, has been identified which encompasses the entire pGH58 gene sequences and includes an additional 34 kilobases of DNA. HU1190 containing this cosmid agglutinates in O4 antisera and produces a polymerized LPS. By constructing several deletion subclones of pGH84, we have localized the genes necessary for polymerized LPS to a 5.5 kb ClaI-BamHI fragment. P1 transductants that make polymerized and unpolymerized O4 LPS have also been identified.

pap-2-encoded fimbriae adhere to the P blood group-related glycosphingolipid stage-specific embryonic antigen 4 in the human kidney.

A subtype of P fimbriae, encoded by the pap-2 gene cluster, has been analyzed for agglutination of erythrocytes and for binding to cryostat sections of the human kidney. We have demonstrated that pap-2-encoded fimbriae are capable of binding to erythrocytes from some animal species and to human erythrocytes which express globoside and the LKE (stage-specific embryonic antigen 4 [SSEA-4]) antigen. The pap-2 fimbriae bind to Bowman's capsule in the human kidney. Monoclonal antibodies directed against glycosphingolipids were used for the detection of specific P blood group-related antigens in the human kidney and on erythrocytes. Preincubation of kidney sections with monoclonal antibody MC813-70, which binds to the SSEA-4 antigen, inhibited adherence of purified pap-2-encoded fimbriae to Bowman's capsule. We suggest that one receptor for pap-2-encoded fimbriae is the antigen known as LKE (Luke) on human erythrocytes or SSEA-4 in the tissues.

Purified P fimbriae from two cloned gene clusters of a single pyelonephritogenic strain adhere to unique structures in the human kidney.

We have completed immunofluorescence binding studies of purified fimbriae from two clones which express Pap or Pap-2 fimbriae. Although the two fimbrial types exhibited common binding to the uroepithelia of the bladder and renal pelvis and to occasional cells located within the glomeruli which we have termed glomerular elements, only Pap-2 fimbriae adhered to Bowman's capsule. Previous studies have demonstrated that the Gal alpha 1----4Gal disaccharide moiety is capable of inhibiting Pap hemagglutination and adherence to uroepithelial cells. Results of our experiments demonstrate that this disaccharide is not sufficient for blocking binding of Pap-2 fimbriae to Bowman's capsule but that GalNAc beta 1----3Gal completely blocks Pap-2 adherence to Bowman's capsule. These results indicate that the different hemagglutination capacities of the two clones reflect different receptor specificities and differential tissue tropisms in the urinary tract. These unique receptor specificities may provide uropathogenic strains of Escherichia coli carrying multiple chromosomal copies of pap-like gene clusters with the advantage of increased numbers of binding sites within the urinary tract. This, in turn, might improve the chances of colonization and the establishment of infection.

Distribution of the P-associated-pilus (pap) region among Escherichia coli from natural sources: evidence for horizontal gene transfer.

Variation in chromosomal DNA in Escherichia coli was studied with probes specific for the P-associated-pilus (pap) region. The presence of DNA homologous to pap was determined by dot blots. Variation in the number of copies of pap and in the organization of internal and flanking sequences was determined by Southern blot hybridization. The 229 strains studied were also classified by O:K:H serotyping and multilocus enzyme electrophoresis. There was considerable heterogeneity in the presence of pap and distribution of pap-homologous DNA in these E. coli strains from natural sources. In general, there was less variation in pap among strains of the same specific O:K:H serotype and enzyme electrophoretic type than among random isolates. There were, however, E. coli strains identified as members of the same clone by O:K:H serotyping and enzyme electrophoresis that were pap positive and pap negative or had different Southern blot patterns for the pap probes (pap type). There were also isolates of the same pap type that differed in two of three O:K:H serotype antigens and the majority of enzymes that determined their enzyme electrophoretic type. These latter two observations were interpreted as evidence for the horizontal (infectious) transfer of the pap-homologous sequences among clones of E. coli.

Diversity and sources of rapidly growing mycobacteria associated with infections following cardiac surgery.

Eighty-nine isolates of rapidly growing mycobacteria associated with cardiac bypass-related infections were characterized. Isolates from sporadic infections belonged to eight taxonomic groups and displayed numerous multilocus enzyme genotypes, plasmid profiles, and heavy metal and antibiotic resistance patterns. Compared with 449 noncardiac wound isolates, 45 sporadic cardiac isolates were more likely to be Mycobacterium fortuitum and M. smegmatis and less likely to be M. chelonae. About 80% of cardiac and noncardiac isolates were from southern coastal states. Eight outbreaks of cardiac bypass-related infections were identified. Strains from each outbreak were genotypically distinctive, and five outbreaks involved more than one strain. In two outbreaks, isolates from environmental sources and noncardiac infections were similar or identical to isolates from sternal wound infections. The heterogeneity of these isolates suggests that most isolates are unrelated and are derived from local environmental sources rather than from contaminated commercial surgical materials or devices.

Molecular cloning and expression of the O4 polysaccharide gene cluster from Escherichia coli.

The Escherichia coli O4 serotype is common among isolates from urinary tract infections. The genes responsible for the biosynthesis of the O4 polysaccharide in a human uropathogenic E. coli were cloned and expressed in E. coli K-12. The recombinant plasmid pGH60, which conferred the O4 phenotype, encoded eight proteins with apparent molecular weights of 39, 36.5, 35, 32.8, 26, 24, 20.7 and 13 kDa.