Mobley Research Laboratory: Department of Microbiology & Immunology: University of Michigan Medical School

Uropathogenic Escherichia coli

Genomic Studies on Uropathogenic E. coli

10. Determination of the complete genome sequence of uropathogenic E. coli.

The complete genome sequence of uropathogenic E. coli, strain CFT073 was determined in collaboration with Rod Welch (Univ. Wisconsin). 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 (non-redundant) set of proteins are actually common to all three strains. 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, while many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain.

Welch, R.A., V. Burland, G. Plunkett III, P. Redford, P. Roesch, D. Rasko, E.L. Buckles, S.-R. Liou, A. Boutin, J. Hackett, D. Stroud, G.F. Mayhew, D.J. Rose, S. Zhou, D.C. Schwartz, N.T. Perna, H.L.T. Mobley, M.S. Donnenberg, F.R. Blattner. 2002. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci, USA 99:17020-17024.

11. Transcriptome analysis of uropathogenic E. coli during UTI.

Based on the sequence of UPEC strain CFT073, a pathogen-specific DNA microarray was used to analyze the transcriptome of this strain isolated directly from the urine of infected CBA/J mice. In vivo expression profiles were compared to 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. The most highly expressed genes overall 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 (Figure 5). Indeed, after ribosomal protein genes, fimA, the gene encoding the type 1 fimbrial major structural subunit, was the most highly expressed gene in vivo. Five iron acquisition systems were all highly upregulated during UTI (Figure 6), as well as genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, many genes involved in motility and chemotaxis were downregulated during growth 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. We also predict that the urinary tract is 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.

Figure 5. Relative expression of virulence factor genes in vivo (above the x-axis) as compared to in vitro (below the x-axis). A bar above the x-axis is upreguated in vivo; a bar below the x-axis is downregulated in vivo.

Figure 6. Expression of iron acquisition systems in E. coli CFT073. The signal intensity, corresponding to the relative expression of a gene, is shown for selected iron acquisition systems in vivo (growth during UTI) or in vitro (growth in static LB culture). Genes upregulated ( Å ) in vivo relative to in vitro are indicated.

Snyder, Jennifer A., Brian J. Haugen, Eric L. Buckles , C. Virginia Lockatell, David E. Johnson, Michael S. Donnenberg, Rodney A. Welch, and Harry L. T. Mobley.2004. The Transcriptome of Uropathogenic Escherichia coli During UTI. Infect. Immun. 72:6373-6381.

12. UPEC proteins induced in urine that are expressed in outer membrane fractions (surface-DIGE).

It is critical that vaccine candidate proteins are both synthesized by bacteria growing in human urine and surface-exposed. We will identify these proteins by culturing E. coli CFT073 in pooled fresh human urine.

To conduct these studies, mid-stream urine were collected and filter-sterilized. UPEC isolate CFT073 were cultured statically to mid-exponential phase in pre-warmed Luria-Bertani (LB) broth (OD 600=0.65) or human urine at 37ºC (OD 600=0.25). Bacteria, harvested from 500 ml cultures by centrifugation, were lysed in a French pressure cell at 20,000 psi. Outer membrane proteins were prepared by carbonate extraction and ultracentrifugation as described previously [19] . For fluorescence difference gel electrophoresis (2D-DIGE) [20] , bacterial proteins were minimally labeled with cyanine-derived fluors (CyDyes) containing an NHS ester-reactive group.

To determine quantitative differences within the UPEC outer membrane proteome during growth in human urine, outer membrane proteins prepared from human urine cultures were labeled with Cy3, from LB broth with Cy5, and a pooled internal standard representing equal amounts of both urine and LB preparations with Cy2. Labeled proteins were subjected to isoelectric focusing for 40,000 V .h and second dimension SDS-10 % PAGE (with 2.6% crosslinking) run at a constant current of 55 mA at 4ºC for 4 hr [19] . Following SDS-PAGE, image acquisition and pixel intensity were obtained using a Typhoon TM scanner and differential in-gel analysis were performed using DeCyder software (GE Healthcare).

A preliminary experiment was conducted and is shown in Figure 7A. Green spots represent proteins synthesized preferentially in urine. Red spots indicate protein synthesized preferentially in LB medium. Blue (white, if saturated) spots are synthesized equally under both culture conditions.

Figure 7. Identification of differentially expressed outer membrane proteins using 2D-DIGE. (A) 2D gel depicts proteins prepared from CFT073 cultured in human urine labeled with Cy3 (green), from LB with Cy5 (red), and the pooled internal standard with Cy2 (blue). (B) Three-dimensional representation of spot volumes and Western blots for select proteins demonstrate quantitative differences between proteins expressed in LB (L) and urine (U).

Protein spots of interest were excised from the 2 nd dimension SDS-PAGE gel and subjected to enzymatic digestion with trypsin. Mass spectra were acquired on an Applied Biosystems 4700 Proteomics Analyzer (Time-of-flight/Time-of-flight) at the University of Michigan Proteome Consortium. Mass spectrometry (MS) spectra were acquired from 800–3500 Da and the eight most intense peaks in each MS spectrum were selected for MS/MS analysis. Peptide identifications were obtained using GPS Explorer (v3.0, Applied Biosystems), which utilizes the MASCOT search engine. Each MS/MS spectrum were searched against NCBInr.

Figure 8. 2D gel of E. coli CFT073 outer membrane proteins stained with Coomassie blue. Proteins that reacted with serum from mice with chronic UTI were identified by mass spectrometry and are named here.

Preliminary analysis demonstrates the power of this technique. In Figure 7B, three series of protein spots were analyzed using both the software and mass spec. analysis. Synthesis of two iron acquisition proteins, IroN and IutA are upregulated in urine (U) 12.5-fold and >80-fold, respectively, and not synthesized in LB medium (L). On the other hand, synthesis of flagellin (FliC) is downregulated (4.3-fold) in urine as compared to LB medium (consistent with our transcriptome study). The software depicts the relative spot volumes (shown above each respective gel strip).

13. Identify outer membrane proteins that elicit an immune response during UTI.

Most surface-exposed proteins are present on the outer membrane of Gram-negative bacteria including E. coli. Additionally, these proteins are exposed to the immune system during an infection and antibodies against these surface antigens may be present in the serum of infected animals. To identify antigenic surface-expressed proteins in UPEC, E. coli CFT073 were fractionated and the outer membrane fractions separated by 2D electrophoresis. The purity of these fractions were assessed by assaying for compartment-specific enzymes (esterase for outer membrane, NADH oxidase for inner membrane [21] , and glucose-6-phosphate dehydrogenase for cytosol [22] ). Western blotting using pooled antisera from 20 mice chronically infected with E. coli CFT073 were used to probe outer membrane fractions for seroreactive proteins. Coomassie blue-stained 2D gels will also be prepared from outer membrane fractions of E. coli CTF073. Spots that react with CFT073 antiserum

were excised and submitted for mass spectrometry analysis (Fig 8). Peptides were matched with putative polypeptides predicted from the E. coli CFT073 genome sequence. These antigens were compared to a list of predicted surface-exposed proteins generated by genomic analysis of CFT073, using criteria such as predicted transmembrane regions, outer membrane protein and lipoprotein signatures, and signal peptide cleavage.

Using this approach, we have identified 20 antigenic outer membrane proteins in CFT073 that react with immune sera (Figure 7; Western blot not shown). These include the porins OmpC and OmpX, siderophore receptors ChuA and IutA, adhesins Iha and antigen 43, as well as several hypothetical proteins. While a number of antigenic outer membrane proteins were identified from E. coli CFT073 grown in rich medium, it is likely that all antigens expressed in vivo cannot be detected under these conditions. To identify additional antigens that may not be expressed in rich medium, UPEC were cultured in vitro under conditions that mimic the urinary tract including iron limitation (with desferal supplementation), osmotic stress (high salt) and nitrogen limitation (low concentration of available nitrogen source).

14. Defining Genomic Islands and Uropathogen-Specific Genes in Uropathogenic E. coli.

Uropathogenic Escherichia coli (UPEC) are responsible for the majority of uncomplicated urinary tract infections, which can present clinically as cystitis or pyelonephritis. UPEC strain CFT073, isolated from the blood of a patient with acute pyelonephritis, was most cytotoxic and most virulent in mice among our strain collection. Based on the genome sequence of CFT073, microarrays were used for comparative genomic hybridization (CGH) analysis of uropathogenic and fecal/commensal E. coli isolates. Genomic DNA from seven UPEC (three pyelonephritis, four cystitis) isolates, three fecal/commensal strains including K‑12 MG1655 was hybridized to the CFT073 microarray. Microarray data were validated using annotated K‑12 and CFT073 sequences. The CFT073 genome contains 5379 genes, CGH analysis revealed that 2820 (52.4%) of these genes were common to all 11 E. coli strains, yet only 173 UPEC-specific genes were found in all UPEC strains by CGH but in none of the fecal/commensal strains. When the sequence of three additional sequenced UPEC strains (UTI89, 536, F11) and a commensal strain (HS) were added to the analysis, 132 genes present in all UPEC strains but in no fecal/commensal strains were identified. Ten novel genomic islands ( >30 kb ) were delineated by CGH in addition to the three known pathogenicity islands. These genomic islands comprise 814 kb of the 5231 kb (15.6%) genome, demonstrating the importance of horizontal transfer for UPEC. UPEC strains contain a greater number of iron acquisition systems than fecal/commensal strains, reflective of adaptation to the iron‑limiting urinary tract environment. Each strain displayed distinct differences in the number and type of known virulence factors. The large number of hypothetical genes in the CFT073 genome, especially those shown to be UPEC-specific, strongly suggest that many urovirulence factors remain uncharacterized.