functional roles of SAA. This has not been the case with the classical human inflammatory marker C-reactive protein (CRP), which is not expressed in mice. It was recently reported, based on measurements from 219 blood donors, that SAA concentrations in sera have a median value of 20 mg/ml as determined by ELISA [23]. SAA is induced in murine models of both E. coli-associated mastitis [24] and colitis [12,25], but there is currently no data regarding changes in localized or systemic levels of SAA during a UTI. Four functional isoforms of SAA are present in mice [26]. SAA1 and SAA2 are highly homologous isotypes produced primarily in the liver. SAA3 is evolutionarily distinct from SAA1/2 and can be synthesized extrahepatically, with abundant expression found in adipose tissue and the mouse colon [12,25]. SAA3 has also been identified as the most up-regulated message following E. coli infection of primary mammary epithelial cells [27]. In addition, in vitro experiments showed that a 10-mer peptide derived from human mammary-associated serum amyloid A3 (M-SAA3) can interfere with the adherence of enteropathogenic E. coli (EPEC) to intestinal epithelial cells [28]. Results presented here demonstrate that SAA1/2, and to a lesser extent SAA3, are induced both locally within the bladder and systemically in response to infection with UPEC. Furthermore, we show that UPEC is resistant to any bactericidal or bacteriostatic effects associated with SAA, although this acute phase protein can effectively inhibit biofilm formation by the uropathogens. Materials and Methods Ethics Statement Mice were used for this study in accordance with European guidelines and the Slovenian legislation. The study was approved by the Veterinary Board of Slovenia Ethics Committee with the permit number 34401-8/2009/6. Serum Amyloid A Induction by UPEC Animals and bacterial strains Adult 8-week-old female C57BL/6JOLaHsd mice were housed at room temperature (22–24uC) with 45–65% humidity and a 12- hr light/12-hr dark cycle and received food and water ad libitum. Animals were euthanized by CO2 inhalation. The K-12 strain MG1655 and the UPEC isolates UTI89 and F11 have been described previously [29,30,31]. SAA Lyophilized human recombinant serum amyloid A (SAA, endotoxin-tested) (Peprotech, London, UK) was spun down, reconstituted according to manufacturer’s instructions in cell culture grade sterile water to a stock concentration of 1 mg/ml, and stored at 220uC until used. This recombinant SAA is a hybrid between SAA1 and SAA2, being identical to human SAA1a except for the presence of an N-terminal methionine and SAA2b- like substitutions of asparagine to aspartic acid and arginine to histidine at positions 60 and 71, respectively. Mouse UTI model Mice were anesthetized with ketamine HCl (100 mg/kg i.p.) and xylazine (10 mg/kg i.p.) prior to inoculation with a 100 ml suspension of UTI89 (108 CFU) in phosphate-buffered saline (PBS) via transurethral catheterization using a sterile polyethylene catheter with 0.28 mm inside diameter (Intramedic, Becton Dickinson, Sparks, MD, USA). Before catheterization, the bladder of each animal was emptied by gentle pressure on the abdomen. Infusions were performed gradually and at a slow rate to avoid injury or vesicoureteral reflux. The catheter, sheathed over a 30 G needle connected to a 1 ml syringe, was retained in the urinary bladder for 30 min. After removal of the catheter, mice were allowed to void normally. To check if this infection protocol resulted in pyelonephritis, we tested the kidneys of infected mice. Paraffin sections of kidneys, which were recovered 24 h, 4 days Figure 1. Induction and localization of SAA1/2 following inoculation of the bladder with UPEC. Immunofluorescence miscroscopy of bladder sections showing SAA1/2 (red) levels and distribution in sham-infected (PBS-treated) mice (A) and mice that were infected for either one (B) or four days (C) via intravesical instillation of the UPEC isolate UTI89. (D and E) Localization of UPEC (green) with host cell nuclei (blue) and SAA1/2 at 4 d post - inoculation of the bladder with UTI89. Large arrows point to the apical membrane of urothelium and bars mark the border between urothelium and lamina propria. Small arrows indicate a few bacteria that do not co-localize with SAA1/2. doi:10.1371/journal.pone.0032933.g001 PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e32933