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

Uropathogenic Escherichia coli

Fimbriae and Motility Studies

1. Assessment of virulence of uropathogenic E. coli type 1 fimbrial mutants in which the invertible element is phase-locked On or Off.

Expression of type 1 fimbria, a proven virulence factor of UPEC, is regulated at the transcriptional level by a promoter situated on an invertible element, which can exist in either 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 UTI with strain CFT073, the fimbriated “on” orientation peaked at 24 h. We proposed 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 (5’) inverted repeat of the invertible element of UPEC pyelonephritis strain CFT073 that locked the invertible element permanently in either the on or the off orientation. The virulence of these mutants was assessed in the CBA mouse model of ascending UTI at 4, 24, 48, and 72 h post-inoculation (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 (Fig. 1). Overall, these data demonstrate that type 1 fimbria expression contributes significantly to virulence early (24 hpi) in the course of a UTI and most profoundly influences colonization of the bladder. However, since wild-type did not outcompete both locked-on and locked-off mutants, the ability to phase vary itself cannot be considered a virulence property in this model.

FIG. 1. Competition of wild type and phase-locked type 1 fimbrial mutants of E. coli CFT073 in the mouse model of ascending UTI at 24 hpi. Mice were transurethrally cochallenged with wild-type CFT073 (wt) and its isogenic locked off mutant (OFF) (A) or wild-type CFT073 and the locked on mutant (ON) (B). Urine, bladder, and kidney samples were quantitatively cultured. Data are shown for samples taken 24 hpi; a line connects values from the same mouse. The horizontal dotted line at 10 2 CFU indicates the lower limit of detection.

Gunther IV, N.W., J.A. Snyder, V. Lockatell, I. Blomfield, D.E. Johnson, and H.L.T. Mobley. 2002. Assessment of Virulence of Uropathogenic E. coli Type 1 Fimbrial Mutants in Which the Invertible Element is Phase-Locked ON or OFF. Infect. Immun. 70:3344-3354.

2. Role of phase variation of type 1 fimbriae in a uropathogenic E. coli cystitis isolate during UTI.

In the previous study, we observed that the patterns of invertible element switching (phase-on vs. phase-off) differ between clinical E. coli cystitis and pyelonephritis isolates. To investigate these differences, we selected the representative E. coli cystitis isolate F11. Mutants that rendered the invertible element incapable of switching orientations were used to assess the role of the invertible element in virulence during a 7-day experimental UTI. Overall, the phase-locked off mutant was attenuated when compared to wild-type F11, most profoundly after 24 hours post-inoculation. Type 1 fimbriae were shown to be critical for colonization in the bladder at all monitored timepoints. In addition, the phase-locked off mutant colonized the kidneys in significantly fewer numbers than F11 late in infection. Levels of colonization between the phase-locked on mutant and the F11 parental strain demonstrated little difference between these strains in the murine urinary tract. This indicated that constitutive production of type 1 fimbriae did not provide a competitive advantage in colonization of this strain. Analysis of these results suggests that different patterns of colonization between clinical E. coli isolates may reflect the different patterns of invertible element phase switching.

Snyder, J.A., A.L. Lloyd, C.V. Lockatell, D.E. Johnson, and H.L.T. Mobley. 2006. Role of phase variation of type 1 fimbriae in a uropathogenic Escherichia coli cysititis isolate during UTI. Infect. Immun. 74:1387-1393.

3. Coordinate expression of fimbriae in uropathogenic E. coli.

Uropathogenic E. coli often express multiple adhesins during infection that favor attachment to specific niches within the urinary tract. We recently demonstrated that type 1 fimbria, a phase-variable virulence factor involved in adherence, was the most highly expressed adhesin during UTI. We examined whether the expression of type 1 fimbriae can affect the expression of other adhesins. Type 1 fimbrial phase-locked mutants of E. coli strain CFT073, which harbor genes for numerous adhesins, were employed in this study. CFT073-specific DNA microarray analysis of these strains demonstrates that expression of type 1 fimbriae coordinately affect expression of P fimbriae in an inverse manner. This represents evidence for direct communication between genes relating to pathogenesis, perhaps to aid the sequential occupation of different urinary tract tissues. While the role of type 1 fimbriae during infection has been clear, the role of P fimbriae must be further defined to assert the relevance of coordinated regulation in vivo. Therefore, we examined the ability of P fimbrial isogenic mutants, constructed in a type 1 fimbrial-negative background, to compete in the murine urinary tract over a period of 168 hours. No difference in the colonization of these mutants was observed. However, comparison of these results with previous studies suggests that inversely coordinated expression of adhesin gene clusters does occur in vivo. Interestingly, the mutant incapable of expressing either type 1 and P fimbriae compensated by synthesizing F1C fimbriae (Fig. 2).

FIG. 2. F1C fimbriae expressed in static cultures of E. coli CFT073 fim pap. SDS-polyacrylamide gel electrophoresis was used to analyze the protein profiles of fimbrial preparations from concentrated static and aerated (exponential phase) Luria broth cultures of E. coli strain CFT073, UPEC76, and CFT073 fim pap. The protein ladder is shown on the left (kDa). The result of BLASTP analysis of the N-terminal sequence of the protein of small size (~10 kDa) observed in the fimbrial preparation from a static culture of CFT073 fim pap is shown below the gel. This protein was absent in the fimbrial preparations from static cultures of wild-type CFT073 and UPEC76.

Snyder, J.A., B.J. Haugen, C.V. Lockatell, N. Maroncle, E.C. Hagan, D.E. Johnson, R.A. Welch, and H.L.T. Mobley. 2005. Coordinated Expression of Fimbriae in Uropathogenic Escherichia coli. Infect. Immun. 73:7588-7596.

4. Role of motility in the colonization of uropathogenic E. coli in the urinary tract.

Flagella-mediated motility and chemotaxis have been suggested to contribute to virulence by enabling UPEC to escape host immune responses and disperse to new sites within the urinary tract. To evaluate their contribution to virulence, six separate flagellar mutations were constructed in UPEC strain CFT073. The mutants constructed were shown to have four different flagellar phenotypes: fliA and fliC mutants do not produce flagella; the flgM mutant has similar levels of extracellular flagellin as wild-type, but exhibits less motility than wild-type; the motAB mutant is non-motile; and the cheW and cheY mutants are motile but non-chemotactic. Virulence was assessed by transurethral independent challenges and cochallenges of CBA mice with wild-type and each mutant. CFU/ml of urine or CFU/g bladder and kidney were determined 3 days post inoculation for the independent challenges and at 6, 16, 48, 60, and 72 hours post inoculation for the co-challenges. While these mutants colonized the urinary tract during independent challenge, each of the mutants was outcompeted by the wild-type strain to various degrees at specific time points during co-challenge. These results suggest that flagella and flagella-mediated motility/chemotaxis contribute to the fitness of UPEC and therefore significantly enhance the virulence of UPEC.

Lane, M.C., C. V. Lockatell, G. Monterosso, D. Lampheir, J. Weinert, R. Hebel, D.E. Johnson, and H.L.T. Mobley. 2005. Role of motility in the colonization of uropathogenic Escherichia coli in the urinary tract. Infect. Immunity 73:7644-7656.

FIG. 3. Chemotaxis of uropathogenic, fecal-commensal, and diarrheagenic E. coli strains to human urine, amino acids, saccharides, and dipeptide. Numerous pyelonephritis (blue), cystitis (red), fecal-commensal (green), and diarrheagenic (magenta) E. coli strains were assayed for their ability to respond chemotactically to human urine (A), L-serine (B), L-aspartate (C), D-ribose (D), D-galactose (E), and L-Gly-Leu (F). Strains were cultured from motility agar and grown to mid-exponential phase (optical density at 600 nm of 0.3 to 0.4) in tryptone broth, pelleted, and resuspended in chemotaxis buffer to starve at 30°C for 1 h. Starved bacterial suspensions (500 µl of ~10 10 total CFU) were added to wells of chemotaxis chambers along with microcapillaries filled with attractant (undiluted or diluted in chemotaxis buffer) or chemotaxis buffer alone as a negative control. Chemotaxis chambers were incubated at 30°C for 90 min before the capillaries were removed and the contents were diluted and plated. Data are represented in a box-and-whiskers format, with the box extending from the 25th percentile to the 75th percentile, with a line at the median or 50th percentile. Whiskers extend from the box to include the highest and lowest data points. Filled boxes indicate capillaries filled with attractant, while open boxes indicate capillaries filled with chemotaxis buffer only. All strain names are indicated along the y axis. Figure 2, P < 0.05.

5. Uropathogenic E. coli generally lack functional Trg and Tap chemoreceptors found in the majority of E. coli strictly residing in the gut.

Chemotaxis is a behavior that bacteria use to sense and respond to external chemical signals. In E.coli, four methyl-accepting chemotaxis protein (MCP) receptors are necessary for chemotaxis to amino acids (Tar and Tsr), saccharides (Trg) and dipeptides (Tap). In this study, the prevalence of each MCP was determined for 21 uropathogenic, 14 fecal-commensal and 9 diarrheagenic E. coli isolates by Southern blot, PCR or DNA sequence analysis. Of those, six uropathogenic, four fecal-commensal and four diarrheagenic strains were tested for their ability to chemotax to multiple chemoattractants characterized for each MCP using a capillary chemotaxis assay (Fig. 3). tar and tsr were present or functional in 100% and 98% of all motile isolates (n = 34), respectively. However, trg and tap were significantly less prevalent or functional among uropathogenic isolates than fecal-commensal and diarrheagenic E. coli isolates (trg and tap: 6% and 6% uropathogenic (n = 16); 50% and 60% fecal-commensal (n = 10); and 88% and 88% diarrheagenic strains (n = 8)). These data suggest uropathogenic E. coli have evolved under different selective pressures than the intestinal E. coli, reflective of the transition between the urinary tract and gut environments. Retention of the two amino acid MCP receptors emphasizes the likely importance of chemotaxis to amino acids rather than saccharides and dipeptides by uropathogenic E. coli.

Lane, M. Chelsea, Amanda L. Lloyd, Tiffany A. Markyvech, Erin C. Hagan, and Harry L.T. Mobley. 2006. Uropathogenic Escherichia coli Generally Lack Functional Trg and Tap Chemoreceptors Found in the Majority of E. coli Strictly Residing in the Gut. J. Bacteriol. 188:5618-5625.

6. Expression of flagella is coincident with uropathogenic E. coli ascension to the upper urinary tract.

Since UTIs are considered to occur in an ascending manner, flagellum-mediated motility has been suggested to contribute to virulence by enabling UPEC to disseminate to the upper urinary tract. Biophotonic imaging of CFT073 gene-specific expression at 24 hpi.Studies from our laboratory and others have shown that flagella contribute to the efficient colonization of the urinary tract.

Here, we describe the use of biophotonic imaging to monitor UPEC infection and tissue specific flagellin gene expression in real-time in vivo using a stable plasmid-based bioluminescent reporter. To ensure that the infection was occurring in its naturally ascending route, we imaged mice immediately following inoculation and found the luminescent signal exclusively localized to the bladder, indicating a lack of vesicoureteral reflux.

Fig. 4. Biophotonic imaging of CFT073 gene-specific expression at 24 hpi. Dorsal and ventral side of a representative mice infected with CFT073 containing either the (A-B)P em7-lux reporter, (C-D) P entC-lux reporter, (E-F) P fliC-lux reporter, or the (G-H)P gadA-lux reporter construct. Approximately 10 9 CFU of each reporter strain were transurethrally inoculated into 8-10 CBA/J mice. Mice were imaged at 24 hpi using an IVIS and luminescence was quantified in the bladder and kidneys using Living Image software (Xenogen Corp.).

To validate our bioluminescent reporter system, we constructed transcriptional fusions with luxCDABE to measure gadA (glutamate decarboxylase) and entC (isochorismate synthase), which are downregulated and upregulated during murine UTI, respectively. While entC was highly expressed during iron limitation in vitro and during UTI, gadA was expressed during stationary phase in vitro, but only modestly expressed in vivo (panels G&H). Using a fliC-lux transcriptional fusion, we show that transient flagellin expression by UPEC coincides with ascension of the ureters and colonization of the kidney (Fig. 4). This represents the first study in which gene-specific expression has been imaged within the internal organs of an experimentally infected intact animal.

Lane, M. Chelsea, Christopher J. Alteri, and Harry L.T. Mobley. Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract. (submitted)