word count: 223 15 Text word count (including abstract and importance): 3965 16 17 2ABSTRACT 18 We characterized the surface motility of nonpathogenic and pathogenic E. coli strains 19 with respect to the appendage requirement, flagella versus fimbriae, and the glucose requirement. 20 Nonpathogenic lab strains exhibited either slow or fast surface movement. The slow strains 21 required type 1 fimbriae for movement, while the fast strains required flagella and had an 22 insertion in the flhDC promoter region. Surface movement of three uropathogenic E. coli 23 (UPEC) strains was fast and required flagella, but these strains did not have an insertion in the 24 flhDC promoter region. We assessed swimming motility as an indicator of flagella synthesis and 25 found that glucose inhibited swimming of the slow nonpathogenic strains but not of the fast 26 nonpathogenic or pathogenic strains. Fimbriae-based surface motility requires glucose, which 27 inhibits cyclic-AMP (cAMP) and flagella synthesis; therefore, we examined whether surface 28 motility required cAMP. The surface motility of a slow, fimbriae-dominant, nonpathogenic strain 29 did not require cAMP, which was expected because fimbriae synthesis does not require cAMP. 30In contrast, the surface motility of a faster, flagella-dominant, UPEC strain required cAMP, 31 which was unexpected because swarming was unaffected by the presence of glucose. Electron 32 microscopy verified the presence or absence of fimbriae or flagella. In summary, surface 33 motilities of the nonpathogenic and uropathogenic E. coli strains of this study differed in the 34 appendage used and the effects of glucose on flagella synthesis. 35 36 IMPORTANCE 37Uropathogenic Escherichia coli strains cause 80-90% of community-acquired urinary 38 tract infections, and recurrent urinary tract infections, which can last for years, and often become 39 antibiotic resistant. Urinary tract infections can be associated with intra-vesical lesions extending 40 3 from localized trigonitis/cystitis to widely distributed pancystitis: motility may be a factor that 41 distinguishes between these infection patterns. Nonpathogenic and uropathogenic E. coli were 42 shown to exhibit fimbriae-and flagella-dependent surface motility, respectively, and the 43 difference was attributed to altered control of flagella synthesis by glucose. Uropathogenic E. 44 coli strains grow more rapidly in urine than nonpathogenic strains, which implies differences in 45 metabolism. Understanding the basis for glucose-insensitive control of flagella-dependent 46 motility could provide insight into uropathogenic E. coli metabolism and virulence. 47 48 49 Escherichia coli is an extraordinarily successful pathogen which causes a variety of 50 diseases, including urinary tract infections (1). Uropathogenic E. coli (UPEC) is the predominant 51 cause of acute and recurrent urinary tract infections, which can become antibiotic resistant and 52 persist for years (2, 3). In women with recurrent urinary tract infections (rUTIs), the cla...

Uropathogenic E. coli (UPEC) is the causative pathogen for most uncomplicated urinary tract infections. Motility is likely to contribute to these infections, and E. coli possesses flagella-dependent swimming motility, flagella-dependent surface motility (often called swarming), and the recently observed pili-dependent surface motility. Surface motility has not been extensively studied, but for the strains that have been tested nonpathogenic E. coli (NPEC) lab strains use pili, NPEC hypermotile derivatives of these lab strains use flagella, and UPEC strains use flagella. Using a representative of these three types of strains, we showed differences in the nutritional and pathway requirements for surface motility with respect to the glucose concentration, the glycolytic pathway utilized, acetogenesis, and the TCA cycle. In addition, glucose controlled flagella synthesis for the NPEC strain, but not for the hypermotile NPEC variant or the UPEC strain. The requirements for surface motility are likely to reflect major metabolic differences between strains for the pathways and regulation of energy metabolism. IMPORTANCE Urinary tract infections (UTIs) are one of the most common bacterial infections and are an increasing burden on the healthcare system because of recurrence and antibiotic resistance (1, 2). The most common uropathogen is E. coli (3, 4), which is responsible for about 80-90% of community acquired UTIs and 40-50% of nosocomial acquired UTIs (2). Virulence requires both pili and flagella, and either appendage can contribute to surface motility, although surface motility of uropathogenic E. coli has not been examined. We found different appendage, nutrient and pathway requirements for surface motility of a nonpathogenic E. coli lab strain and a uropathogenic E. coli. We propose that these differences are the result of differences in the pathways and regulation of energy metabolism.

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Recurrent urinary tract infection (rUTI) is a common disease in women, but its pathogenesis is still unclear. The aim of this study is to investigate urothelial cytoskeleton and cell proliferation protein expression in patients with rUTI.METHODS: Female patients with rUTI (symptomatic UTI > 3 times in recent one year) were recruited. One month after recovery from rUTI (absence of white blood cell in urinary analysis), the patients were asked to provide bladder specimens by cold-cup endoscopic biopsy. Patients with stress urinary incontinence were asked to provide bladder specimen as control. The specimens were investigated with western blot and immunochemical staining for urothelial cytoskeleton and cell proliferation protein, including sonic hedgehog (SHH), tumor protein 63 (TP63), cytokeratin 5 (CK5), CK14, CK20, zonula occludens (ZO)-1, E-cadherin and tryptase. GAPDH was used as normalizing protein for the quantification.RESULTS: A total of 16 rUTI and 8 control bladder specimens were analyzed. The cell proliferation protein SHH and TP63 expression were significantly lower in the rUTI urothelium than that in control (Figure 1). The cytoskeleton protein expression in the rUTI urothelium was also significantly lower than that in control bladders, including CK5, CK14 and CK20. The immunochemical staining showed CK5 and TP63 expression were mainly located near the basal layer of urothelium, while the CK20 expression was main located in the umbrella cells (Figure 2). The immunochemical staining results also showed decreased immunoactivity of CK5, CK14, CK20, TP63 and SHH in the patients with rUTI (Figure 2). CONCLUSIONS: Our results showed deficits of urothelium cell proliferation and cytoskeleton protein in the patients who had clinically recovered from rUTI. This finding indicates the urothelium function is still unhealthy even though clinical bladder inflammation had been subsided.

Uropathogenic E. coli (UPEC) is the causative pathogen for most uncomplicated urinary tract infections. Flagellar-mediated motility is essential for virulence and colonization for ascending urinary tract infections. The appendage requirement for surface motility depends on the strain: nonpathogenic E. coli (NPEC) lab strains use pili, NPEC hypermotile derivatives use flagella, and UPEC strains use flagella. E. coli flagella-dependent surface motility had been previously shown to require glucose and amino acids. We examined the nutritional and pathway requirements of the NPEC strain W3110 for pili-dependent surface motility, which have not been previously examined. We then compared these requirements to those for two strains with flagella-dependent surface motility: a variant of W3110, W3110-J1, in which the synthesis of the activator of flagella synthesis has been upregulated and the UPEC strain UTI89. The glucose requirement for W3110 was higher than that for either W3110-J1 or UTI89. The pathways required for motility were also different. W3110, but not UTI89, required the Embden-Meyerhof-Parnas pathway via PfkA; conversely, UTI89, but not W3110, required the Entner-Doudoroff pathway, acetogenesis, and the TCA cycle. Glucose did not control flagella synthesis for W3110-J1 and UTI89. The differing requirements for surface motility are likely to reflect major metabolic differences between strains. The metabolic requirements for UTI89 motility suggest a specific adaptation to the urinary tract environment.